DEDD), which is overexpressed in > 60% of TNBCs, drives a mitogen-independent G1/S cell cycle transition through cytoplasm localization. The gain of cytosolic DEDD enhances cyclin D1 expression by interacting with heat shock 71 kDa protein 8 (HSC70). Concurrently, DEDD interacts with Rb family proteins and promotes their proteasome-mediated degradation. DEDD overexpression renders TNBCs vulnerable to cell cycle inhibition. Patients with TNBC have been excluded from CDK 4/6 inhibitor clinical trials due to the perceived high frequency of Rb-loss in TNBCs. Interestingly, our study demonstrated that, irrespective of Rb status, TNBCs with DEDD overexpression exhibit a DEDD-dependent vulnerability to combinatorial treatment with CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo. Thus, our study provided a rationale for the clinical application of CDK4/6 inhibitor combinatorial regimens for patients with TNBC.Lacking targetable molecular drivers, triple-negative breast cancer (TNBC) is the most clinically challenging subtype of breast cancer. In this study, we reveal that Death Effector Domain-containing DNA-binding protein ( The use of of CDK4/6 inhibitors to treat patients with TNBC is limited by loss of Rb. Here, the authors show that a combination of CDK4/6 inhibitor and EGFR inhibitor is effective against DEDD-overexpressing TNBC, independent of Rb status. Despite the heterogeneous nature of the tumor, targeted anti-cancer therapies have achieved clinical success through the exploitation of functionally essential genomic alternations, or tumor-specific vulnerabilities3. In the past decade, a compendium of the conceptual frameworks explaining mechanisms of tumor vulnerabilities has been proposed and experimentally validated, including oncogene/non-oncogene addiction4, synthetic lethality5, collateral lethality6, and synthetic essentiality7. A successful clinical translation of cancer genome studies  project)) for effective targeted anti-cancer regimens relies on two prerequisites: (1) tumor-specific genetic or epigenetic events that confer unique vulnerabilities (the Achilles’ heel of cancer) to therapeutic targeting; (2) prognostic biomarkers coupled with an accessible clinical assay for selecting patients who will most likely respond to the targeted therapy.Cancer is a highly heterogeneous genetic disease8. Estrogen receptor (ER)-targeting agents  and human epidermal growth factor receptor 2 (HER2)-targeting therapeutics  have become first-line treatments for ER-positive breast cancer and HER2-positive breast cancer, respectively. Extensive pre-clinical and clinical studies have demonstrated that biomarker-based  patient selection for targeted therapies has led to a significant improvement in cancer treatment with reduced side effects compared to traditional chemotherapies9. Despite such clinical success, targeted therapy options for the most aggressive breast cancer subtype, triple-negative breast cancer (TNBC), remains limited. Because TNBC lack established therapeutic targets , non-specific chemotherapy remains the primary treatment option for TNBC patients. TNBC exhibits a higher level of genome instability and a distinct mutational landscape11. Despite the highly heterogeneous nature of TNBC genome landscapes, some TNBC-specific tumor vulnerabilities are emerging as clinically translatable targets for TNBC patients. For example, poly-ADP ribose polymerase (PARP) was identified to have a synthetic lethal dependency on BRCA1 mutation and has been targeted in TNBC clinical trials, ultimately resulting in recent Food and Drug Administration (FDA) approval of PARP inhibitors for TNBC12. Additionally, bromodomain and extra-terminal (BET) protein13 and the pyrimidine synthesis pathway14 have shown promise to be targetable TNBC vulnerabilities.Breast cancer targeted therapies are among the most successful examples of applying the concept of targeting tumor-specific vulnerabilitiesIn this study, we employed a pooled whole-genome RNA interference (RNAi) functional screen in TNBC cells to explore potential synthetic lethal pathways that synergize with epidermal growth factor receptor (EGFR)/HER2 inhibitor lapatinib (LAP).DEDD) as a potential vulnerability from our screen. Interestingly, DEDD is significantly upregulated in >60% of TNBC tumors. While DEDD has been known to function as a pro-apoptotic protein in the nucleus15, we found that DEDD is strongly expressed in the cytosol of tumor cells. Mechanistically, cytosolic DEDD promotes G1/S cell cycle transition through multiple mechanisms. First, DEDD interacts with heat-shock cognate 71 kDa protein (HSC70) to enhance cyclin D1 expression. Second, overexpressed cytosolic DEDD promotes the proteasome-mediated degradation of retinoblastoma (Rb) family proteins to enable G1/S transition. Addicted to an accelerated G1/S cell cycle progression, tumor cells with DEDD overexpression exhibit an increased susceptibility to the combinatorial treatment of cyclin-dependent kinases 4/6 (CDK4/6) and EGFR inhibitors. Furthermore, a combinatorial regimen of CDK4/6 and EGFR inhibitors synergistically inhibited the progression of TNBC xenografts and patient-derived xenograft (PDX) in vivo. These pre-clinical results provide a strong rationale to extend recently FDA-approved CDK4/6 inhibitors to TNBC patients.Unexpectedly, we identified death effector domain-containing protein  were potentially critical for cell survival  are dysregulated (amplification and messenger RNA (mRNA) upregulation) in more than 40% of TNBC tumors  are located close together on chromosome 1q23.3–42.1 -containing proteins. Without known enzymatic activity, DEDD executes its biological function primarily through protein–protein interactions via its DED domain18. Previous studies suggested that DEDD may interact with cyclin B1, decrease Cdk1/cyclin B1 activity, and regulate cell size during pre-mitosis phases by facilitating the G1-phase rRNA synthesis19. However, most studies have focused on the capacity of DEDD to promote apoptosis through partnering with other DED-containing proteins20. Since DEDD is involved in pro-apoptotic processes, it is thought to have tumor suppressor activities21. Paradoxically, DEDD is aberrantly overexpressed in TNBC  than TNBC cells with moderate DEDD expression (HCC1806)  incorporation assay. We found that knocking down DEDD significantly decreased EdU incorporation in HCC38 and MDA-MB-468 TNBC cells  for 18 h, and then released cell cycle inhibition by replacing nocodazole media with a fresh culture medium. We found that DEDD protein peaked in cells at the G1 phase, but not other cell cycle phases  Fig. , suggestlls Fig. , suggestion Fig. , suggestDEDD nuclear localization leads to caspase activation, which induces apoptosis, and abolishing the nuclear translocation of DEDD decreases the apoptosis-promoting potential of the protein20. Interestingly, immunohistochemistry (IHC) staining for DEDD in TNBC tissue samples showed that DEDD primarily localizes to the cytoplasm of tumor cells that have high DEDD expression   DEDD or ΔNLS-DEDD in DEDD-diploid normal cell lines including 293FT and normal human breast epithelial cells (HMECs) 22. Ectopic overexpression of WT DEDD promoted G1–S transition in 293FT cells, which could be significantly diminished by preventing DEDD nuclear-cytosol shuttling using nuclear export inhibitor leptomycin (lepto)  was sufficient to override the G1/S checkpoint and increase the proportion of cells in S phase independent of mitogen activation (serum starvation) as determined by increased EdU incorporation  Fig. 15. Then,to) Fig. . This obion Fig. . Additioion Fig. . Cyclin wal Fig. . InteresDEDD executes its functions through protein-protein interactions23. To explore interaction partners of DEDD in the cytosol, we immunoprecipitated the ΔNLS-DEDD protein complex and analyzed co-immunoprecipitated proteins by mass spectrometry. Interestingly, multiple peptides representing the HSC70 were identified  expression, which phenocopied the Rb protein expression pattern in Rb-WT TNBC cell lines  and RBL2 (p130), were reported to be CDK4/6 substratesnes Fig. . MoreoveEDD Fig. . We furtDEDD, we hypothesized that gain-of-function DEDD in TNBC promotes mitogen-independent G1/S transition and confers addiction to an accelerated cell cycle program, thereby creating a tumor-specific vulnerability for therapeutic targeting. CDK4/6 inhibitors show significant clinical efficacy in cancer treatment by targeting the central G1/S transition machinery29. Three CDK4/6 inhibitors have recently received FDA approval for ER + breast cancer, including albociclib (PALBO) , ribociclib (RIBO) , and abemaciclib (ABE) . Expression of WT Rb, the perceived CDK4/6 downstream target, is currently considered a prerequisite for the clinical anti-cancer efficacy of CDK4/6 inhibitors30.Based on the multifaceted cell cycle-promoting mechanisms of 31, TNBC patients are excluded from CDK4/6 inhibitor clinical trials. Interestingly, we found that targeting CDK4/6 in combination with LAP in DEDD-overexpressed TNBC significantly and synergistically inhibited cell proliferation in both Rb-WT  and Rb-deficient (MDA-MB-468) TNBC cells  rendered the KTB cells resistant to LAP mono treatment, confirming the proliferation-driving function of cytoplasmic DEDD. Interestingly, KTB cells with overexpressed DEDD were more responsive to CDK4/6 inhibitor treatment and Combo treatment  could further shift the sensitivity of TNBC cells to combo treatment. Overexpression of ΔNLS-DEDD in MDA-MB-436 cells, which moderately express DEDD  is a transcription factor and has been shown to be frequently upregulated in multiple cancers and linked to sustained Akt signalingnes Fig. . We nextent Fig. . Ectopicent Fig. , right. ent Fig. . This fuEDD Fig. , signifiEDD Fig. , Left, slls Fig. , right. lls Fig. . To testlls Fig. . Notablylls Fig. .Fig. 6CyDEDD expression and localization as well as the sensitivity of TNBC cells to Combo treatment in vivo. First, we examined DEDD expression and localization in xenograft breast tumors derived from two TNBC cell lines  and two TNBC PDXs that were described previously35. IHC analysis of DEDD showed heterogeneous but strong DEDD expression that was localized predominately to the cytoplasm of epithelial cells within the tumors  transformation in soft-agar assays (data not shown). Likewise, DEDD did not influence overall survival and disease-free survival of TNBC patients who received traditional chemotherapies /AKT pathway through its lipid phosphatase activity, while a nuclear pool of PTEN maintains chromosomal stability41. DEDD has been well documented to be a nuclear-targeting protein through its three nuclear localization sequences18. DEDD functions as an apoptosis facilitator in the nucleus by interacting with caspase-8 and caspase-10, and stimulating caspase-dependent cell death42. Surprisingly, once overexpressed in TNBC cells, DEDD primarily resides in the cytoplasm  phosphorylates Rb on Ser567 in response to genotoxic stress. Hyperphosphorylation of Rb leads to its interaction with E3 ligases Hdm248. However, the functional relevance of the above in relation to observations to tumorigenesis in vivo has not been addressed. In this study, we discovered that aberrant overexpression of DEDD greatly accelerated ubiquitination and proteasomal degradation of Rb family proteins, including Rb protein and Rb family protein RBL1 in TNBC cells  is a transcription target of FoxM1, and FoxM1 promotes breast cancer tumorigenesis by transcriptionally activating PDGFA, which led to further Akt phosphorylation in breast cancer cells54. Supporting such findings, we observed that phosphorylation and total expression of FoxM1 were effectively blocked by CDK4/6 inhibitors  and palbociclib (P-7766) were purchased from LC Laboratories. Abemaciclib (HY-16297), ribociclib (HY-15777), and nocodazole (HY-13520) were purchased from MedchemExpress Inc. Leptomycin B (9676S) was purchased from Cell Signaling Technology. MG-132 (80053-196) was purchased from VWR. CHX (01810) was purchased from Sigma-Aldrich. The 293FT (R70007) cells were purchased from Thermo Fisher Scientific Company. BT-20, BT-474, HCC1806, HCC1937, HCC38, MDA-MB-231, MDA-MB-436, and MDA-MB-468 cell lines were obtained from ATCC . The 293FT cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% FBS and 1% penicillin–streptomycin (Pen-Strep). BT-20, BT-474, MDA-MB-231, MDA-MB-436, and MDA-MB-468 cell lines were cultured in DMEM/Ham’s F-12 (1:1) supplemented with 10% FBS and 1% Pen-Strep. HCC1806, HCC1937, and HCC38 cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% Pen-Strep. The MCF-10A was originally purchased from ATCC and cultured in DMEM/F-12 medium supplemented with 5% horse serum (Invitrogen), 20 ng/mL EGF, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 100 ng/mL cholera toxin, and 1% Pen-Strep. Normal human mammary epithelial lines from Komen Tissue Bank (KTB) were obtained from our collaborator Dr. Nakshatri and cultured in DMEM/F-12 supplemented with 10% FBS, 0.4 μg/mL hydrocortisone, 5 μg/mL insulin, 20 ng/mL EGF, 24 mg/L adenine, and 5 μM ROCK  inhibitor (Y-27632). All cell lines were maintained at 37 °C in a 5% CODEDD complementary DNA (cDNA) sequence  It was obtained from the UCSC Genome Browser.Human DEDD coding sequence:5′-ATGGCGGGCCTAAAGCGGCGGGCAAGCCAGGTGTGGCCAGAAGAGCATGGTGAGCAGGAACATGGGCTGTACAGCCTGCACCGCATGTTTGACATCGTGGGCACTCATCTGACACACAGAGATGTGCGCGTGCTTTCTTTCCTCTTTGTTGATGTCATTGATGACCACGAGCGTGGACTCATCCGAAATGGACGTGACTTCTTATTGGCACTGGAGCGCCAGGGCCGCTGTGATGAAAGTAACTTTCGCCAGGTGCTGCAGCTGCTGCGCATCATCACTCGCCACGACCTGCTGCCCTACGTCACCCTCAAGAGGAGACGGGCTGTGTGCCCTGATCTTGTAGACAAGTATCTGGAGGAGACATCAATTCGCTATGTGACCCCCAGAGCCCTCAGTGATCCAGAACCAAGGCCTCCCCAGCCCTCTAAAACAGTGCCTCCCCACTATCCTGTGGTGTGTTGCCCCACTTCGGGTCCTCAGATGTGTAGCAAGCGGCCAGCCCGAGGGAGAGCCACACTTGGGAGCCAGCGAAAACGCCGGAAGTCAGTGACACCAGATCCCAAGGAGAAGCAGACATGTGACATCAGACTGCGGGTTCGGGCTGAATACTGCCAGCATGAGACTGCTCTGCAGGGCAATGTCTTCTCTAACAAGCAGGACCCACTTGAGCGCCAGTTTGAGCGCTTTAACCAGGCCAACACCATCCTCAAGTCCCGGGACCTGGGCTCCATCATCTGTGACATCAAGTTCTCTGAGCTCACCTACCTCGATGCATTCTGGCGTGACTACATCAATGGCTCTTTATTAGAGGCACTTAAAGGTGTCTTCATCACAGACTCCCTCAAGCAAGCTGTGGGCCATGAAGCCATCAAGCTGCTGGTAAATGTAGACGAGGAGGACTATGAGCTGGGCCGACAGAAACTCCTGAGGAACTTGATGCTGCAAGCATTGCCCTGA-3′.DEDD DEL-NLS coding sequence:5′-ATGGCGGGCCTAGCGGCAGCAGCAAGCCAGGTGTGGCCAGAAGAGCATGGTGAGCAGGAACATGGGCTGTACAGCCTGCACCGCATGTTTGACATCGTGGGCACTCATCTGACACACAGAGATGTGCGCGTGCTTTCTTTCCTCTTTGTTGATGTCATTGATGACCACGAGCGTGGACTCATCCGAAATGGACGTGACTTCTTATTGGCACTGGAGCGCCAGGGCCGCTGTGATGAAAGTAACTTTCGCCAGGTGCTGCAGCTGCTGCGCATCATCACTCGCCACGACCTGCTGCCCTACGTCACCCTCAAGCTGAGACTCGCTGTGTGCCCTGATCTTGTAGACAAGTATCTGGAGGAGACATCAATTCGCTATGTGACCCCCAGAGCCCTCAGTGATCCAGAACCAAGGCCTCCCCAGCCCTCTAAAACAGTGCCTCCCCACTATCCTGTGGTGTGTTGCCCCACTTCGGGTCCTCAGATGTGTAGCAAGCGGCCAGCCCGAGGGAGAGCCACACTTGGGAGCCAGCGAATCCTGCGGATCTCAGTGACACCAGATCCCAAGGAGAAGCAGACATGTGACATCAGACTGCGGGTTCGGGCTGAATACTGCCAGCATGAGACTGCTCTGCAGGGCAATGTCTTCTCTAACAAGCAGGACCCACTTGAGCGCCAGTTTGAGCGCTTTAACCAGGCCAACACCATCCTCAAGTCCCGGGACCTGGGCTCCATCATCTGTGACATCAAGTTCTCTGAGCTCACCTACCTCGATGCATTCTGGCGTGACTACATCAATGGCTCTTTATTAGAGGCACTTAAAGGTGTCTTCATCACAGACTCCCTCAAGCAAGCTGTGGGCCATGAAGCCATCAAGCTGCTGGTAAATGTAGACGAGGAGGACTATGAGCTGGGCCGACAGAAACTCCTGAGGAACTTGATGCTGCAAGCATTGCCCTGA-3′.DEDD cDNA coding sequence was cloned into lentivirus mammalian expression vector pLOVE empty (Addgene #15948) or pLenti-CMV-puro-DEST (Addgene #17452) with an N-terminal 3×-FLAG tag. DEDD mutant entry vector was assembled through GeneArt Gene Synthesis (Thermo Fisher Scientific). For lentiviral production, a lentiviral expression vector was co-transfected with second-generation lentivirus packing vectors psPAX2 and pMD2.G (Addgene) into 293FT cells using CalFectin . Forty-eight hours after transfection, breast cancer cell lines were stably infected with viral particles. pLOVE-GFP served as packaging and infection efficiency control. Lentiviral-based pLKO.1 DEDD shRNA vectors were purchased from Sigma-Aldrich Co. . The control shRNA vector (pLKO.1 scrambled shRNA) was from Addgene Inc. .Human DEDD-targeting shRNA sequences used in the lentiviral constructs were: shDEDD−493, 5′-CCGGGAGGAGACATCAATTCGCTATCTCGAGATAGCGAATTGATGTCTCCTCTTTTTTG-3′ (targeting CDs); shDEDD-867, 5′-CCGGCATCATCTGTGACATCAAGTTCTCGAGAACTTGATGTCACAGATGATGTTTTTTG-3′ (targeting CDs); shDEDD1056, 5′-CCGGGAAACTCCTGAGGAACTTGATCTCGAGATCAAGTTCCTCAGGAGTTTCTTTTTTG-3′.The Stable expression or knockdown clones were selected by culturing cells with puromycin (1–5 μg/mL) for at least 48 h. The uninfected cells were also treated with puromycin as a control. Infected cells have a multiplicity of infection (MOI) around 0.3–0.6.DEDD or 3×-FLAG-Del-NLS-DEDD:Primers for constructing 3×-FLAG-DEDD—forward primer: melting temperature (TM): 72.4 ℃N-3×-FLAG-5′-CACCATG GAC TAC AAA GAC CAC GAC GGC GAC TAC AAA GAC CAC GAC ATC GAC TAC AAA GAC GAT GAC GAC AAGATGGCGGGCCTA.DEDD—reverse primer: TM: 72.1 ℃N-3×-FLAG-5′-TCA GGG CAA TGC TTG CAG CAT CAA GTT CCT CAG GAG TTT CTG TCG GCC CAG CTC ATA GTC.Primers for detecting gene mRNA level:DEDD—forward primer: TM: 56.6 ℃5′-ATGGACGTGACTTCTTATTG-3′.DEDD—reverse primer: TM: 56.4 ℃5′-ATACTTGTCTACAAGATCAGGG-3′.RB1—forward primer: TM: 57.2 ℃5′-CAGAAATGACTTCTACTCGAAC-3′.RB1—reverse primer: TM: 58.9 ℃5′-AATGTGGCCATAAACAGAAC-3′.ABCB10—forward primer: TM: 62.1 ℃5′-CCATATTTCAGGATTTCAGCC-3′.ABCB10—reverse primer: TM: 54.8 ℃5′-GACTAATAGTTCCAGAAGCAG-3′.UAP1—forward primer: TM: 56.7 ℃5′-GATAGTCAGAATGGGAAAGAC-3′.UAP1—reverse primer: TM: 56.3℃5′-GCATAGGAGATAAGAGGAGAG-3′.17.In vitro pooled drop-out screening by infect HCC1806 cells at 0.3 MOI with DECIPHER shRNA lentiviral library , which includes about 27,000 shRNA constructs targeting 5043 genes on human signaling pathways. After 8 days of puromycin (1 µg/mL) selection, infected cells were treated with either dimethyl sulfoxide (DMSO) or LAP (2 µM) for 14 days with replacing fresh medium containing drug every 48 h. After 14 days of treatment, the cells are collected and sent for barcodes sequencing by Illumina sequencing. The row barcode counts were analyzed for the “drop-out” hits using the MAGeCK software as described beforeN, N-dimethyl formamide) , 10% SDS (sodium dodecyl sulfate) ) overnight. The 96-well plate was read at 570 nm absorbance by a microplate reader. We used the wells with cells untreated as 100% control and the wells with only medium as 0% control. Percentage of inhibition of cell proliferation was calculated as [100 − ((OD treated  − OD empty)/(OD untreated – OD empty) × 100]. For direct cell counting, cells (3000/well) were seeded in at least triplicate in 96-well plates and treated with various concentrations of the single treatment or combination treatment for designated times. Cell proliferation was determined by directly counting cell numbers in each well using hemacytometer and compared using the wells with untreated cells as 100% control and the wells with only medium as 0% control. For the clonogenic assay, cells were plated approximately 3000 cells/well in 24-well plates in 10% FBS. After 24 h, cells were treated in triplicate with various concentrations of a single or combination treatment for approximately 2 weeks with media replacement and treatment every 48 h. Cells were washed with phosphate-buffered saline (PBS), fixed, and stained with a solution containing 6% glutaraldehyde and 0.5% crystal violet. Plates were dried, and each well was photographed and quantified using ImageJ 1.43u .For MTT -2,5-diphenyltetrazolium bromide) assays, cells (3000/well) were seeded in at least triplicate in 96-well plates and treated with various concentrations of a single treatment or combination treatment for designated times. Cell proliferation was determined by incubating with MTT for 3 h and then in lysing buffer (50% DMF (® Green Supermix (Bio-Rad). Relative expression levels were calculated using the 2−ΔΔCT method.Total cell RNA will be extracted using TriZol reagent (Invitrogen) following the manufacturer’s instructions. One microgram of total RNA was subjected to reverse transcription to synthesize cDNA using the Verso cDNA Kit (Thermo Fisher Scientific). A 20 μL volume reaction consisted of 2 μL reverse transcription product and 20 nM of both forward and reverse primer. The samples were incubated with 2× iTaq™ Universal SYBR® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit or by Click-iT™ EdU Alexa Fluor™ 594 Imaging Assay. In the Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific) plus 2 μg/mL propodium podide staining (Thermo Fisher Scientific), cells were rehydrated in 1% bovine serum albumin-PBS at a density of 1 × 106 cells/mL, transferred into flow cytometry analysis tubes, and stored overnight at 4 °C prior to samples analysis by Beckman Coulter FC500 Flow Cytometer. EdU signal was detected in FL1 (515–536 nm), and PI signal was detected in FL3 (590–615 nm). Twenty thousand events will be recorded and analyzed using the FlowJo software. In the Click-iT™ EdU Alexa Fluor™ 594 Imaging Assay, cells were mounted in ProLong™ Gold Antifade Mountant for 24 h before imaging using the Leica fluorescence microscope in HCRI Tissue Core Facility . All experiments were repeated three times, and quantitative results were analyzed by one-way analysis of variance (ANOVA) or t test, where P < 0.05 was considered statistically significant.Twenty-four hours before the experiment, the culture medium was replaced with medium without FBS. This would synchronize the cells in G0. After 24-h serum starvation, the cells were treated with 1 ng/mL EGFs to induce cell cycle reentry. Cells were treated with 10 µM of EdU for 1–6 h. Cells were collected and processed following the protocol of either Click-iTDEDD plasmid. Forty-eight hours later, cells were treated with 1 ng/mL of EGF and then with 1 µM MG-132 for 6 h before collection. Harvested cells were re-suspended in 500 µL of M2 lysis buffer . After incubation for 1 h on ice, cell lysates were incubated with 1 µg of Rb (4H1) antibody overnight (18 h). One percent of cell lysates were saved as input control. After that, the samples were incubated with 50 µL of protein A/G agarose beads (Santa Cruz) for 2 h at 4 °C. Immunocomplexes were washed with M2 lysis buffer three times before immunoblotting. For immunoblotting, proteins were extracted using M2 lysis buffer and proteinase plus phosphatase inhibitor cocktail . Protein extracts were resolved using 4–20% gradient SDS-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (Thermo Fisher Scientific #88018). Membranes were probed with primary antibodies overnight on a 4 °C shaker and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Signals were visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific #34078).For immunoprecipitation, cells first were transfected with either empty or DEDD (Sc-271192) and HSC70 (sc-7298) antibodies were purchased from Santa Cruz Biotechnology. β-Actin (ab8226) was purchased from Abcam. FLAG M2 (F1804) was purchased from Sigma-Aldrich. Cyclin B1 (#4138), cyclin D1 (#2978), cyclin A2 (#4656), cyclin E1 (#4129), p-Erk (#4370), Erk (#4695), p-EGFR (#3777), EGFR (#4267), p-Akt (#4060), Akt (#4691), Rb (#9309), p-Rb (#9308), RBL1 (#89798), Ubi (#3933), p-FoxM1 (#14655), and FoxM1 (#5436) were purchased from Cell Signaling Technology. For immunoprecipitation assay, antibodies were diluted into 50 ng/µL. For immunoblotting assay, antibodies were diluted into 2.5 ng/µL. For immunofluorescence assay, antibodies were diluted into 25 ng/µL. All immunoblotting original uncropped images were provided as Supplementary materials (Source Data (pptx)) with the manuscript.For immunofluorescence staining, cells were serum starved overnight before stimulation with 1 ng/mL of EGF and treated with 1 µM MG-132 for 6 h before fixation. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilization in 0.2% Triton X-100 (PBS) and followed by anti-FLAG and Rb antibodies (dilution 1:50) incubation for 18 h at 4 °C. After incubation with tetramethylrhodamine-conjugated or fluorescein isothiocyanate-conjugated secondary antibody  for 1 h at 25 °C, images were captured with an Olympus FV1000 2-photon confocal microscope.DEDD plasmids for 72 h before puromycin (1 μg/mL) treatment for 48 h. The cells were further cultured for two more doublings. The harvested cells were washed with 1× PBS and re-suspended in 1:9 ratio of cell mass to extraction buffer (Dynabeads® Co-Immunoprecipitation (Co-IP) Kit, Thermo Scientific) with protease inhibitors. The cell lysates were incubated on ice for 30 min and then centrifuged at 2600 × g for 5 min at 4 °C to remove large cell debris and nuclei. BCA (bicinchoninic acid) assay was used to quantify the protein concentration in the supernatant. About 5000 μg of protein was incubated with 1 μg of anti-FLAG antibody -coupled Dynabeads (10 μg of antibody) on a roller at 4 °C for 1 h. Another 2000 μg of protein was incubated with 1 mg of anti-mouse IgG1 -coupled Dynabeads (10 μg of antibody) for non-specific control immunoprecipitation. One percent of cell lysates were saved as input control. The Dynabeads® Co-IP complexes were washed with extraction buffer for three times. The proteins bound to beads were released in 60 μL of elution buffer provided by the kit. The samples were added five volumes of cold (−20 °C) acetone overnight at −20 °C.Cells were infected with lentiviruses containing pLenti-CMV-puro-3×-FLAG-ΔNLS-41. When the tumor reached the volume of approximately 100 mm3, the mice were randomly divided into groups with an even distribution of tumor sizes (10 mice/group). LAP was given every 48 h at a dose of 100 mg/kg in the vehicle (0.5% hydroxypropylmethylcellulose with 0.1% Tween-20) via oral gavage. Abemaciclib was given every 48 h at a dose of 25 mg/kg in the vehicle (0.5% hydroxypropylmethylcellulose with 0.1% Tween-20) via oral gavage. Tumor volume was calculated as: tumor volume = 1/2 (length × width2). As designated time point post treatment, tumors were collected and fixed in 4% PFA for 24 h followed by 70% ethanol for another 24 h before formalin-fixed paraffin-embedded embedding for histology analysis.All animal experiments were performed in strict accordance with IACUC guidelines. Rag1 knockout (Rag1−/−) and nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice were obtained from Taconic Farms . For TNBC xenograft experiments, 8-week-old female Rag1−/− mice were transplanted with one million cancer cells suspended in 0.1 ml of Matrigel mixture (1:1 mixture ratio of Matrigel and DMEM/F-12 medium without FBS) orthotopically into contralateral sides of the fourth mammary fat pads of the mice. For TNBC PDXs, 3.5-week-old female NOD SCID mice have received PDX tumor pieces as previously describedDEDD, Ki-67, or cleaved caspase-3 were detected by DEDD antibody (Santa Cruz Biotechnology #sc-271192), Ki-67 antibody (Ki-67 (D2H10), rabbit monoclonal antibody (IHC-specific) , or cleaved caspase-3 antibody (cleaved caspase-3 (Asp175) antibody, Cell Signaling Technology #9661) through IHC using the Polink-2 Plus HRP Rabbit with DAB Kit (GBI Labs) after 20 min of antigen retrieval. The stained slides were scanned using the Aperio scanner in the HCRI Tissue Core Facility . Images were scored for the percentage of Ki-67 tumor cell nuclei per total tumor cell nuclei in each captured field using the ImageScope software. For DEDD and cleaved caspase-3, images were scored as the percentage of positive staining area per captured total area. All quantification was performed in a fashion that was blinded to the treatment group.PFA-fixed tumor samples were paraffin embedded and cut into 5 μm thin slices and fixed on glass slides. The raw data for drug synergism analysis were generated by the MTT assay. The cells were evenly plated 24 h before administration of drug dosages. Both single and combination treatments were completed in triplicate at each dosage (4–6 different dosages in a series dilution). Then, the absorbance reading of each well was calculated as FA by the treatment. The wells treated with DMSO served as non-affected controls. The wells with medium only served as full-affected control. The mean FAs at different concentrations were then analyzed by the CompuSyn software to generate simulation for the drug synergism analysis. The CI were plotted along with indicated FA for each cell line. CI > 1, antagonism; CI = 1, addition; CI < 1, synergism.http://www.cbioportal.org]. The oncoprint heatmap was generated by including mutations, putative copy-number changes, and mRNA expression z-scores (RNA-seq V2 RSEM with z threshold ± 2). The survival data were extracted from the same dataset for Kaplan–Meier plot in R using “Survival” and “Survminer” packages. The boxplot of the TCGA segmented copy-number changes without germline CNV (n = 1099) was generated using the University of California, Santa Cruz (UCSC) Cancer Browser. Gene expression data of commonly used cells lines were extracted from Broad Institute Cancer Cell Line Encyclopedia (CCLE). The heatmap of relative gene expression was generated in R using pHeatmap package. RNA-seq profiling of DEDD knockdown cells was conducted by standard Illumina TruSeq library preparation followed by Illumina sequencing using miSeq. The differential gene expression analysis was performed using DE-seq2 package. An adjusted P value of <0.05 was considered statistically significant. We performed GSEA analysis using GenePattern interface [http://www.broadinstitute.org/gsea/] using C6 v5.2 symbol (oncogenic signature) sub-datasets. Network analysis was conducted through the web-based bioinformatics package NetworkAnalyst [https://www.networkanalyst.ca/]-based on recommended protocol. All quantitative data were analyzed and plotted using the GraphPad Prism software. P values were generated using either two-sided t test or Fisher’s exact test, or one-way ANOVA test.TCGA breast-invasive carcinoma provisional dataset (1105 samples) was sourced from cBioportal [Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting SummarySource Data


Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and  Unlike the siRNA approach, CRISPR-Cas can permanently alter the function of genes by mutating them. CRISPR-Cas9 derived from Streptococcus pyogenes is the most widely used system because of its ease of use and effectiveness4. The system requires introducing two components: the nuclease Cas9 and a single-guide RNA (sgRNA) which targets the nuclease to a specific genomic locus based on base pairing at a 20-nt target sequence. The target sequence must be followed immediately by the motif 5′-NGG-3′, which is called PAM (protospacer adjacent motif)4. The sgRNA-guided Cas9 generates a double strand break (DSB) in the target site, which may modify the genome in two different ways. The first is random insertion/deletion (indel) mutagenesis which occurs when the DSB is repaired by the error-prone non-homologous end-joining (NHEJ) pathway, often resulting in a knockout or loss-of-function mutation4. The second is precise allele editing through the high-fidelity homology-directed repair (HDR) pathway, which may occur in a small percentage of the cells (<5%)6. More definitive than siRNA, more precise than older transgene insertion methods, and more efficient than older homologous recombination methods8, CRISPR has become one of the most widely used methods to alter a gene to interrogate its function or for medical applications10.The prokaryotic defense system CRISPR-Cas was discovered as an adaptive immunity against foreign DNA molecules and converted to a revolutionary genome editing tool16, but clonal selection and expansion would be still required unless the transduction efficiency and expression of the viral vectors are very high. Among the most widely used viral vectors, lentivirus and adeno-associated virus (AAV) are preferred for human gene therapy applications, but adenovirus (AV) has important advantages as a research tool18. AV has the highest transduction efficiency in diverse target cells20; unlike lentivirus, its genome does not integrate into the host genome, thus reducing off-target mutations; and AV has a substantially higher genome capacity than AAV21. We have used this capacity to co-package three CRISPR-Cas9 components along with their promoters: Cas9, sgRNA and a fluorescent protein to aid in sorting for transduced cells. A fluorescent protein such as GFP or mCherry is also used to monitor active viral production in packaging cell lines such as 293 A and 91121. When active adenoviral particles are made and released from the cells, they infect neighboring cells forming comet-like plaques which can be monitored under fluorescent microscopy. Others have previously demonstrated the feasibility of using AV to deliver Cas9 and sgRNA into diverse target cells, including primary and immortalized cells in vitro, and in vivo18. Most have (like us) used a replication-incompetent AV5 vector lacking E1 and E3; one group used a new AV vector devoid of all viral genes14, but with the trade-off of a more cumbersome production process and less infectable AV. In this study, we demonstrate the efficacy and versatility of an all-in-one adenovirus CRISPR-Cas9 system to target a specific biological pathway, the circadian clock.However, CRISPR has limitations. Transfection of the Cas9 and sgRNA in plasmids typically only succeeds for a minority of cells, and in certain cell lines which do not proliferate or are not easily transfected with plasmids, clonal selection and expansion from the transfected heterogenic population may not be practical or possible. Various CRISPR viral vectors have been employed to try to overcome these problems24. Adverse consequences from having a faulty clock include compromised sleep quality, poor work performance, and increased risk for accidents in the short-term, as well as metabolic diseases and cancer in the long-term. Because the circadian clock is cell autonomous, and rhythms can be measured in cells in real-time using a luciferase reporter under control of a clock promoter, clock mechanisms can be studied in cell culture models such as mouse embryonic fibroblasts (MEFs) or human cell line U2OS27. Clock gene regulation, protein rhythms, and protein-protein interactions in cultured cells are comparable to those in in vivo tissues29. The molecular backbone of the circadian clock is a transcriptional negative feedback loop with interacting positive and negative elements31. CLOCK and BMAL1 are the positive elements, activating transcription of many downstream genes, including behavior-regulating genes and the main negative elements, Period  and Cryptochrome , whose products form an auto-inhibitory complex31. Unlike other core clock genes, Bmal1 has no redundant paralog. Disruption of Bmal1 results in complete arrhythmicity while disruption of a single Cry or Per family member produces subtle circadian phenotypes34.The circadian clock is a molecular circuit that controls daily rhythms in physiology and behavior, such as metabolic oscillations and wake-sleep cycles35, and adenovirus-mediated transduction is ~100% efficient and not cytotoxic, clonal selection and expansion may not be necessary to study the phenotype of knockouts if targeting is properly designed. We showed that a complete disruption of the circadian clock can be achieved in cell culture without clonal selection and expansion by targeting a splicing site in the essential clock gene Bmal1 using the all-in-one adenoviral CRISPR-Cas9 system. We believe interrogation of gene function can be performed in diverse cell lines in a rapid manner by using our all-in-one adenoviral CRISPR-Cas9 system since it does not involve conventional cloning, clonal selection and expansion.In the current study, we developed an all-in-one adenoviral vector where sgRNA can be easily cloned by the Gateway cloning system without labor-intensive conventional cloning and selection. Our vector achieves packaging of sgRNA, the SpCas9 transgene, and a fluorescent protein transgene into the same viral particle. Since indel efficiency of CRISPR-Cas9 is very high, up to ~100%4, we adopted the Gateway cloning technology. Gateway technology was developed by Invitrogen to transfer DNA fragments between plasmids using the bacteriophage lambda site-specific recombination system36. Briefly, a DNA fragment of interest is first cloned into an Entry plasmid where the DNA fragment becomes flanked by specific recombination sequences called attL1 and attL2. The DNA fragment in the Entry plasmid can then be transferred to any Destination vector by LR recombinase. Since only positive recombinants are selected, the subcloning procedure is highly efficient and rapid.To improve upon the conventional procedure of cloning sgRNA into plasmids using enzyme digestion and ligationCbh promoter (pShuttle-Cas9-DEST)  Fig. . BrieflyST) Fig. . Recombiase Fig. . It has Bmal1 promoter27. This cell line is widely used as a model system to study clock mechanisms, and bioluminescence signal from Bmal1-dLuc (or a similar reporter) is widely used as a real-time clock reporter since it faithfully reports the activity of the endogenous clock mechanism.Before packaging the vector into adenovirus, we first tested the functionality of the all-in-one vector construct itself. We used it to edit essential clock genes—whose null phenotypes were well characterized—by transfecting it into a human cell line, U2OS, expressing a rapidly degraded luciferase (dLuc) under a Bmal1. In the mammalian system, Bmal1 is the only essential clock gene without redundancy32. Thus, if CRISPR-Cas9 generates frame-shifting mutations (via NHEJ) in early exons of both alleles of Bmal1, the C-terminal transcriptional activation domain of BMAL1 will be deleted, resulting in circadian arrhythmicity37. Aided by an in silico design tool (http://chopchop.cbu.uib.no), we identified sgRNA sequences targeting four exons of Bmal1  based on the 20 nt sequence immediately preceding SpCas9 PAM sequences in those exons  clones were detected as well as indels , and then the positive cells are expanded before subjected to functional assays. This approach can be applied to many cell lines, but it cannot be used in many primary cell lines that do not proliferate or cell lines that are very difficult to transfect. Moreover, clonal selection and expansion by FACS or drug selection may be labor-intensive and time-consuming, and demands a certain level of expertise.We thus confirmed the functionality of our all-in-one plasmid, but also confirmed the limitations of the typical paradigm of CRISPR-Cas9 mutagenesis21. Thus we hypothesized that high-titer infection of all-in-one CRISPR-Cas9 adenovirus into U2OS cells can achieve ~100% transduction efficiency and generate knockouts in the majority of cells, thus making the downstream clonal selection and expansion unnecessary. To test this possibility, we generated a new adenoviral shuttle vector expressing GFP and Cas9 from two different promoters (pAdTrack-Cas9-DEST)  Fig. . The pShncy Fig. . The BmaBmal1 exon 6 because our previous transfection experiments found that targeting exon 6 produced more arrhythmic clones (62%) than targeting other Bmal1 exons. Considering significant wt contamination (15–30%), targeting near this specific splicing site  machinery was revolutionary in editing the genome to produce precisely engineered knockout and knockin mice. The functional insights from these genetically engineered mouse models cannot be overstated. However, it is also important to recognize the mechanistic insights gained by studying mutant (and wt) cells in culture, as the clock mechanism is cell-autonomous and largely conserved across cell types42. Cell culture models will continue to be critically important for future advances in circadian biology, given their time- and cost-efficiencies. As our understanding of a biological pathway is most significantly advanced by identification of relevant new genes, a next frontier in circadian biology would be to identify new clock genes. We also have much to learn still from the hundreds of natural polymorphisms in known clock genes that lack a corresponding animal model. It has been already demonstrated that some of these polymorphisms are associated with circadian disorders such as Familial Advanced Sleep Phase Syndrome44. In the pre-CRISPR era, mutant cells were usually derived from mutant animals, as cell genomes could not be easily modified in culture. Now, however, cell culture models can be precisely edited using CRISPR-Cas9.As with most biological fields, our current understanding of the circadian clock has been shaped by genetic manipulations that have led to identification of a dozen essential clock genes to date19, but adenovirus can be easily concentrated by density gradient ultracentrifugation to achieve higher titers21. Adenoviral titer can be adjusted to maximize infection rate without cytotoxicity by visual inspection of fluorescence and cell morphology. We routinely measure robust bioluminescence rhythms from ~100% AV-infected MEFs and U2OS cells for several days, demonstrating that the physiology of these cells are not compromised45. Since AV transduction can be 100% efficient in delivering Cas9:sgRNA, whereas FACS sorting following transfection still includes non-transfected cells, AV is a more efficient way to achieve knockouts even when FACS sorting is feasible. A simple serial dilution without FACS sorting would be enough for clonal isolation and expansion for downstream assays , and not possible for all cell types. We show that CRISPR-Cas9 combined with AV can remove the need for clonal selection and expansion, thus addressing all of those issues in AV-infectible cells Fig. . We and ays Fig. .Bmal1-E7 and Per2-E15 in Fig. Bmal1-E6. As a future refinement, we propose that the efficiency can be further increased by using two different adenoviruses targeting two different early exons. We have shown that MEFs could be doubly transduced by AV26. Although AV has been tested to deliver Cas9 and/or sgRNA into cells and showed knockdown of target proteins up to 90% in some cases15, to our knowledge, our present study is the first one to demonstrate a functional knockout without clonal selection by an all-in-one AV. All AV viral genes can be removed in the AV vector to increase packaging capacity and reduce immune response in vivo14. However, when the AV vector devoid of all viral genes is used, packaging efficiency suffers, resulting in vector titers ~two orders of magnitude lower, even after multiple rounds of amplification, compared to ours14. AV can also be employed to deliver catalytically-inactive Cas9 as a synthetic transcription factor. It has been shown that a catalytically-inactive Cas9 can be targeted to promoters to activate or inhibit transcription of their downstream genes10. AV is superior to lentivirus for that purpose because expression levels of transgenes are much higher by AV than lentivirus.Even if transduction of Cas9-sgRNA is near 100% and if Cas9-sgRNA can induce indels in ~100% of cells, one may expect frame-shifting mutations to occur only in 2/3 of cases. However, based on our results, the NHEJ repair process is not random, but rather locus-specific  was PCR-amplified with primers with attL1 and attL2 sequence and subcloned into pEAR A1A .pEntry-sgRNA was generated by Mutagenex  as follows. U6-sgRNA fragment from pU6-(BbsI)_Cbh-Cas9-T2A-mCherry plasmid (Addgene #6432421). Then, R1-CmR-ccdB-R2 fragment from the Gateway pDEST  was subcloned into the intermediate vector _Cbh-Cas9-T2A-mCherry plasmid (Addgene #64324) was subcloned into XbaI and NotI sites of pShuttle , respectively _Cbh-Cas9-T2A-mCherry plasmid (Addgene #64324), and R1-Cmet al.4 The final PCR products were purified and mixed with either pShuttle-Cas9-DEST or pAdTrack-Cas9-DEST along with LR clonase (Thermo Fisher Scientific #11791020) to generate final all-in-one vectors. Bacterial colonies transformed with the reaction mixture were screened by colony PCR that amplifies DNA region flanked by R1 and R2 (negative clones), or B1 and B2 (positive recombinants). Two different sets of primers were used for pShuttle and pAdTrack as follows.The first round of PCR was performed using two sets of primers as described in Table pShuttle-Cas9-DEST:attR1 Up Fwd: 5′-GAGCCCACTGCTTACTGGCTTATC-3′Cbh Rev: 5′-CGTACTTGGCATATGATACACTTGA-3′Size of PCR amplicons: 1,966 bp negative clone and 691 bp for positive recombinant.pAdTrack-Cas9-DEST:KpnIattR1F: 5′-ATAGGTACCCCCACTGCTTACTGGCTTATCGAAATTAATAC-3′KpnIattR2R: 5′-CCGTAAGTTATGTAACGGGTACCTCTAGATCAACCAC-3′Size of PCR amplicons: 1820 bp for negative clone and 545 bp for positive recombinant.Bmal1 promoter was described previously. For transfection with the all-in-one plasmids, cells were plated into 6 cm dishes to be approximately 60% confluent on the day of transfection. Cells were transfected with Polyfect  according to the manufacturer’s protocol and incubated for 2 days before they were subjected to trypsinization and FACS sorting using BD FACSAria SORP equipped with an Automated Cell Deposition Unit (ACDU) for sorting into 96 well plates. Cells were trypsinized with Trypsin (0.25%)-EDTA (2.21 mM) for 3 min and were filtered through mesh with 50um pores. FACS-sorted cells were collected into three groups: negative, intermediate and bright mCherry, and plated into 35 mm dishes and grown for 2 days before harvest and genomic DNA extraction. Before the cells were harvested, they were inspected for mCherry expression. Even the bright mCherry group included 10–30% of non-mCherry cells. Harvested cell pellets were homogenized in 250 ul solution A containing 0.1 M Tris-HCl pH = 9.0, 0.1 M EDTA, and 1% SDS, and incubated at 70 °C for 30 minutes. 35 ul 8 M potassium acetate was added and incubated at room temperature for 5 minutes. The samples were centrifuged at 13,000 rpm for 15 minutes and genomic DNA was purified by subjecting the supernatant to phenol-chloroform extraction and ethanol precipitation. The extracted genomic DNA pellet was dissolved in 100 ul water and used as a template to PCR amplify the target genomic locus with a set of primers . Digestion products were resolved on 8% acrylamide/bis TBE gel and visualized with EtBr.A U2OS cell line expressing luciferase under a Bmal1 exon 7 and Per2 exon 15. Wt contamination was confirmed: 4 out of 16 were wt for Bmal1 exon 6 and 2 out of 15 were wt for Per2.PCR amplicons obtained above were cloned into plasmids using the TOPO-PCR cloning kit (Invitrogen #K2800), and inserts were sequenced from 20 colonies each for Bmal1 exon 6, 8 and 9, and Per2 exon 5 were set up for bioluminescence monitoring as described previously19. For immunoblotting for BMAL1, a previously described anti-BMAL1 antibody (GP3) was used47. For human PER2, novel polyclonal anti human PER2 antibodies were generated in guinea pigs using aa 1–200 peptide by Cocalico Biologicals, Inc . hP2-GP49 was used in the current study. The antibody was validated by oscillations of human PER2 in U2OS cells and a novel monoclonal antibody against human PER2 (hP2-C6A3). The monoclonal antibody was selected from clones of antibodies raised against aa 1–200 of human PER2 by Boreda Biotech . The monoclonal antibody was also able to detect oscillations of PERs in U2OS cells similarly . When compared with 293 cells, expression forming units (efu) of the adenovirus was 5–10 fold lower for U2OS cells. However, U2OS cells could be infected with ~100% efficiency without further purification, unlike infection for MEFs19. To ensure 100% infection in U2OS cells with the all-in-one adenovirus, the cells were infected at MOI of 50 twice, two days apart.Adenoviruses expressing mutant BMAL1 lacking the DNA-binding domain or wt BMAL1 were described previouslyCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGTCATATGCCATGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCNNN: MCST7-F 5′-TAATACGACTCACTATAGGGT3-F 5′-ATTAACCCTCACTAAAGGGA1 Seq-F 5′-GATTAAGTTGGGTAACGCCAGGGA1 Seq-R 5′-CTTGAGCGTCGATTTTTGTGATGCA1 Flk-F 5′-GTAATACGACTCACTATAGGGC.Supplementary figures


Chronic exposure of pancreatic β-cells to excess free fatty acids is thought to contribute to type 2 diabetes pathogenesis in obesity by impairing β-cell function and even leading to apoptosis. In β-cells, lipid droplet-associated protein perilipin 5 (PLIN5) has been shown to enhance insulin secretion by regulating intracellular lipid metabolism; the roles of PLIN5 in response to lipotoxicity remain poorly understood.INS-1 β-cells were transfected with PLIN5-overexpression adenovirus (Ad-PLIN5) and treated with palmitate. C57BL/6 J male mice were fed with high fat diet and tail intravenous injected with adeno-associated virus overexpressing PLIN5 (AAV-PLIN5) in β-cells.Our data showed that palmitate and PPAR agonists including WY14643 (PPARα), GW501516 (PPARβ/δ), rosiglitazone (PPARγ) in vitro all induced PLIN5 expression in INS-1 cells. Under palmitate overload, although upregulating PLIN5 promoted lipid droplet storage, it alleviated lipotoxicity in INS-1 β-cells with improved cell viability, cell apoptosis and β-cell function. The protection role of PLIN5 in β-cell function observed in cell experiments were further verified in in vivo study indicated by mitigated glucose intolerance in high fat diet fed mice with β-cell-specific overexpression of PLIN5. Mechanistic experiments revealed that enhanced FAO induced by elevation of PLIN5, followed by decreased ER stress may be a major mechanism responsible for alleviation of lipotoxicity observed in the present study.Our finding substantiated the important role of PLIN5 in protection against lipotoxicity in β-cells.The online version of this article (10.1186/s12986-019-0375-2) contains supplementary material, which is available to authorized users. The prevalence of type 2 diabetes (T2DM), a chronic metabolic disorder, has been increasing steadily, partially due to the rising obesity rates. Excess adiposity and dyslipidemia commonly seen in obesity not only induce insulin resistance but also directly damage β-cell function, a fundamental defect in diabetes . Indeed,Excessive FFA are mainly stored in the form of triglyceride (TG) in lipid droplet (LD) in most of mammalian cells. LD consist of a score of neutral lipids  surrounded by a phospholipid monolayer in which are embedded proteins named LD-associated proteins . In mammThe perilipin family proteins (PLINs) were the major LD-associated proteins that encompass five members, from PLIN1 to PLIN5. In contrast to PLIN1, which is mostly highly expressed in white adipose tissue, PLIN5  is mainly expressed in tissues with high oxidative capacity such as red muscle, heart, liver and brown adipose . As a LD12 viral genome/ml. After 5-month dietary manipulation, AAV-PLIN5 or AAV-MIP was delivered to HFD mice by tail vein injection at 5 × 1011 viral genome per mouse. CHOW mice were injected with saline as the control group. 1 week after injection, fasting (12 h) and fed glucose (2 h) levels were determined from tail vein blood with a glucometer . 10–14 days after injection, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted. For GTT, mice were fasted for overnight (16 h) and then injected i.p. with D-glucose at 1.5 g/kg of body weight. For ITT, mice were performed after 6 h fasting and injected i.p. with human regular insulin  at 0.75 IU/kg of body weight. Blood glucose was measured from tail vein blood at the indicated time interval after the injection using a glucometer (FreeStyle). The areas under the curves for blood glucose in GTT (AUCg) and ITT (AUCitt) were calculated. Serum insulin at 0, 15, 30 min after GTT  and fasting serum FFA  were measured according to manufacturers’ instructions. At the end of the study, mice pancreatic islets were obtained as described previously  reacting with mitochondrial dehydrogenase to produce an orange water soluble formazan dye. Prior to the analysis, the INS-1 cells were plated in 24-well cell culture plates at a density of 5 × 10Cell apoptosis was measured by flow cytometric analysis. Briefly, after treatments, the cells were trypsinized, washed with PBS, and suspended in 195 μl binding buffer. Then, cells were incubated with 5 μl Annexin V-FITC for 15 min and 5 μl propidium iodide  for 5 min at 4 °C in the dark. The cells were then analyzed on a FAC Scan flow cytometer  to quantify apoptosis.INS-1 cells were treated with adenovirus for 24 h in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), after which 0.5 mM PA was added to the medium for another 12 h or 48 h. Cells were starved for 2 h in glucose-free RPMI 1640 and then were washed twice with glucose-free Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4). GSIS was investigated as described previously, with minor modifications . BrieflyS6K, Bcl-2, phospho-mTOR (p-mTOR), total mTOR which were all purchased from Cell Signaling and PLIN5 from Progen Biotechnik, ACO from Abcam, CHOP from Immunoway, CPT-1 and BiP from Santa Cruz. All the secondary antibodies and internal reference GAPDH were all purchased from Santa Cruz. Immunoreactive bands were visualized with an ECL reagent kit (Millipore). Optical densities of each band were calculated and analyzed by using Image J analysis software.Proteins were extracted and immunoblotted as described previously . The folCyclin D1, Cyclin D2, spliced XBP-1 (XBP-1(s)), sterol-regulatory element binding protein-1c (SREBP-1c), CHOP, BiP, and PLIN5 were detected using primers indicated in Table Total RNA from INS-1 cell line was isolated using TRIzol reagent . First-strand cDNA was synthesized with Moloney murine leukemia virus (M-MLV) reverse transcriptase and random hexamer primers . Gene expressions were analyzed using the SYBR Green PCR system, following the manufacturer’s recommendations . t-tests. To compare data sets of more than two groups, we used one-way ANOVA followed by Bonferroni’s multiple comparison tests. Two-way ANOVA determined significance for GTT and ITT. P < 0.05 (two-tailed) was considered significant.All quantitative data were presented as means ± SEM and cell experiments were performed at least three times. Differences of numeric parameters between two groups were assessed using Student’s PLIN5. Hence, the upregulation of PLIN5 by PA may dependent on PPARα activation in INS-1 cells. And the induction role of PLIN5 expression by PPARβ/δ and/or PPARγ activation is indirect which needs to be investigated in the future study.Recently, PLIN5 was confirmed to be an LD protein in both human and murine islets and altering expression level of PLIN5 can impact insulin secretion by regulating lipolysis . However. In fasted islets, elevated PLIN5 expression and high TG content was observed recently [SREBP-1c . However, upregulating PLIN5 compromised this induction effect of PA which suggesting the mitigation of ER stress induced by PA overload . Furthermore, overexpressing CPT-1A in PLIN5 deficient cells alleviated the induction of BiP, CHOP by PA incubation  and GFP, with sites for ECORI, BamHI, NdeI, SacI and ApaI. The size of AAV2/8 vector, MIP and PLIN5 was 4,492 bp, 874 bp and 1,392 bp respectively. Left: map of AAV-PLIN5; Right: map of AAV-MIP. Sequences of MIP: 5′-TTGTAGCTGGAATAGAGCATGCACTAACAGATGGAGACAGCTGGCTTTGAGCTCTGAAGCAAGTATTACATATGGAGACTTGCTGGCCTTCAGGTGCTTATCTTGTTATTGGATACTGCAGGAGGATGTACCACAGGGCTTCAGCTCAGCTGACCCCCAAGTGGGATATGGAAAGAGAGATAGAGGAGGAGGGACCATTAAGTGCCTTGCTGCCTGAATTCTGCTTTCCTTCTACCTCTGAGAGAGAGCTGGGGACTCGGCTGAGTTAAGAACCCAGCTATCAATTGGAACTGTGAAACAGTCCAAGGGACAAAGATACTAGGTCCCCAACTGCAACTTCCTGGGGAATGATGTGGAAAAATGCTCAGCCAAGGACAAAGAAAGCATCACCCACTCTGGAACAATGTCCCCTGCTGTGAACTGGTTCATCAGGCCATCAGGGCCCCTTGTTAAGACTCTAATTACCCTAGGACTAAGTAGAGGTGTTGACGTCCAATGAGCGCTTTCTGCAGACCTAGCACCAGGGAAGTGTTTGGAAACTGCAGCTTCAGCCCCTCTGGCCATCTGCTGACCTACCCCACCTGGAGCCCTTAATGGGTCAAACAGCAAAGTCCAGGGGGCAGAGAGGAGGTGCTTTGGTCTATAAAGGTAGTGGGGACCCAGTAACCACCAGCCCTAAGTGATCCGCTACAATCAAAAACCATCAGCAAGCAGGAAGGTACTCTTCTCAGTGGGCCTGGCTCCCCAGCTAAGACCTCAGGGACTTGAGGTAGGATATAGCCTCCTCTCTTACGTGAAACTTTTGCTATCCTCAACCCAGCCTATCTTCCAGGTTATTGTTTCAACA-3′ Sequences of PLIN5: 5′-ATGTCTGAAGAAGAGGCGGCTCAGATCCCCAGATCCAGTGTGTGGGAGCAGGACCAGCAGAACGTGGTGCAGCGTGTGGTGGCTCTGCCCCTGGTCAGGGCCACGTGCACCGCGGTCTGCGATGTTTACAGTGCAGCCAAGGACAGGCACCCGCTGCTGGGCTCCGCCTGCCGCCTGGCTGAGAACTGCGTGTGCGGCCTGACCACCCGTGCCCTGGACCACGCCCAGCCGCTGCTCGAGCACCTGCAGCCCCAGCTGGCCACTATGAACAGCCTCGCCTGCAGGGGCCTGGACAAGCTGGAAGAGAAGCTTCCCTTTCTCCAGCAACCTTCGGAGACGGTGGTGACCTCAGCCAAGGACGTGGTGGCCAGCAGTGTCACGGGTGTGGTGGACCTGGCCCGGAGGGGCCGGCGCTGGAGCGTGGAGCTGAAGCGCTCCGTGAGCCATGCTGTGGATGTTGTACTGGAAAAATCAGAGGAGCTGGTGGATCACTTCCTGCCCATGACGGAGGAAGAGCTCGCGGCACTGGCGGCTGAGGCTGAAGGCCCTGAAGTGGGTTCGGTGGAGGATCAGAGGAGACAGCAGGGCTACTTTGTGCGCCTCGGCTCCCTGTCAGCACGGATCCGCCACCTGGCCTACGAGCACTCTGTGGGGAAACTGAGGCAGAGCAAACACCGTGCCCAGGACACCCTGGCCCAGCTGCAGGAGACGCTGGAGCTGATAGACCACATGCAGTGTGGGGTGACCCCCACCGCCCCGGCCCGCCCTGGGAAGGTGCACGAGCTGTGGGGGGAATGGGGCCAGCGCCCTCCGGAGAGCCGCCGCCGGAGCCAGGCAGAGCTGGAGACGCTGGTGCTGTCCCGCAGCCTGACCCAGGAGCTGCAGGGCACGGTAGAGGCTCTGGAGTCCAGCGTGTGGGGCCTGCCCGCCGGCGCCCAGGAGAAGGTGGCTGAGGTGCGGCGCAGTGTGGATGCCCTGCAGACCGCCTTCGCTGATGCCCGCTGCTTCAGGGACGTGCCAGCGGCCGCGCTGGCCGAGGGCCGGGGTCGCGTGGCCCACGCGCACGCCTGCGTGGACGAGCTGCTGGAGCTGGTGGTGCAGGCCGTGCCGCTGCCCTGGCTGGTGGGACCCTTCGCGCCCATCCTTGTGGAGCGACCCGAGCCCCTGCCCGACCTGGCGGACCTGGTGGACGAGGTCATCGGGGGCCCTGACCCCCGCTGGGCGCACCTGGACTGGCCGGCCCAGCAGAGAGCCTGGGAGGCAGAGCACAGGGACGGGAGTGGGAATGGGGATGGGGACAGGATGGGTGTTGCCGGGGACATCTGCGAGCAGGAACCCGAGACCCCCAGCTGCCCGGTCAAGCACACCCTGATGCCCGAGCTGGACTTCTGA-3′ (EPS 980 kb)


Clerodendron cyrtophyllum, was isolated from root of Clerodendron cyrtophyllum Turcz, which was able to produce the highest activity of β-glucosidase up to 33.72 U/mL at 144 h during fermentation on Potato Dextrose Broth (PDB). The obtained fungus was grown on isoflavones-rich soybean extract to produce genistein and daidzein, achieving the conversion rate of 98.7%. Genistein and daidzein were isolated and purified by column chromatography using hexane/acetone (29:1/1:1), reaching purities of over 90% of total isoflavones, as identified and determined by TLC, LC-MS/MS, and 1H and 13C NMR spectroscopy. These results imply that the isolated P. citrinum is a potential fungal strain for industrial-scale production of genistein and daidzein from isoflavones-containing soybean extracts. These products may serve as potential raw materials for manufacture of functional foods that are based on aglycones.Soybeans offer an abundant source of isoflavones, which confer useful bioactivities when existing in aglycone forms. The conversion of isoflavones into aglycones via fermentation of soybean products is often realized by β-glucosidase, an enzyme produced by fungi. In this study, a filamentous fungus,  Isoflavones are polyphenolics that exert estrogen-like effects and have been widely utilized in manufacture of foods and cosmetics . IsoflavGlycine max) ; ;  with a flow rate of 1.0 mL/min. Mass spectra of Electron Spray Ionisation (ESI) were recorded on the Agilent 1100 Series mass spectrometer connected with Varian 320-MS . 1H-NMR spectra were recorded by 500 MHz  with DMSO-d6 or acetone-d6 as solvents and tetramethylsilane as an internal standard. 13C-NMR spectra were recorded at 125 MHz (Bruker XL-500) with DMSO-d6 or acetone-d6 as solvents and as internal standard.To perform TLC (thin layer chromatography) analysis, silica gel 60 Fp < 0.05 via Student’s t-test. Statistical analysis was performed in the JMP Pro 13.2 software.Experiments were carried out in triplicate for the accuracy of data. Statistical significant differences were realized at Clerodendron cyrtophyllum Turcz sp. were used as microbial sources to isolate fungi with β-glucosidase activity on PDA medium. At 10−5 dilution, colonies were isolated separately. They were then subcultured on sterilized PDA plates several times to obtain pure culture for identification and β-glucosidase enzyme assay. After several subcultures at 30 °C for 72 h, 10 fungi were isolated with different morphological characteristics of colony. Five fungi were isolated from fresh samples (designated as BC) of C. cyrtophyllum Turcz’s while five other fungi were isolated from dried plant samples (designated as BSD).Roots from For enzymatic screening and fungal selection, the ten fungal isolates were tested for their capability of enzyme production. The β-glucosidase activity of these fungi is shown in the Aspergillus species.On SA medium, colonies of BC2 grew fast and displayed a compact green or yellow basal felt enclosed by a layer of white of erect conidiophores. The diameter of colonies was approximately measured as 2.4 cm. Microscopically, conidiophore stipes are long and smooth-walled and the color of hyaline turned dark towards the vesicle. Conidial heads were biseriate, large, globose, and showed tendency to radiate, splitting into several loose columns with age. Additionally, phialides, usually in the form of septate metulae, were also observed. These morphological characteristics affirm that this fungus belongs to the Penicillium species.The shade of colonies of BSD5 growing on SA medium was green in color and a dense felt of conidiophores was observed. The average diameter of colonies after three days of incubation was 1.2 cm. Observing under a microscope, conidiophores were hyaline and smooth-walled. In addition, terminal verticils, carrying 3–5 metulae each, were observed on conidiophores. In each metula, around 3–7 phialides were recognized. Regarding conidia, they are smooth-walled and their shape appeared to be globose or subglobose. In addition, the production of conidia was basipetal from the phialides. Therefore, the described fungus belonged to the Penicillium sp. was identified to ensure that this fungus belonged to Penicillium sp. Penicillium sp. was sequenced as:After identification by morphological characteristics, genomic DNA of the “CATGCTCCGGCCGCCATGGCGGCCGCGGGAATTCGATTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGCCCCTCGGGGCCCAACCTCCCACCCGTGTTGCCCGAACCTATGTTGCCTCGGCGGGCCCCGCGCCCGCCGACGGCCCCCCTGAACGCTGTCTGAAGTTGCAGTCTGAGACCTATAACGAAATTAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCTCTGGTATTCCGGAGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCCGTCCCCCCCGCCGGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTCGTCACCCGCTCTAGTAGGCCCGGCCGGCGCCAGCCGACCCCCAACCTTTAATTATCTCAGGTTGACCT”.Penicillium citrinum existing in the database, as evidenced by the excellent bootstrap results. Therefore, the examined fungus presumably belonged to the Penicillium genus and firmly aligned with the P. citrinum species, as demonstrated by the the strong sequence similarities with the said species.To gain insights into the evolutionary relationship, two methods for creation of the phylogenetic trees, namely Neighbor Joining (NJ) and Maximum Parsimony (MP), were employed, resulting in almost identical topologies. The aligned dataset consisted of 42 taxa and 100 characters. Our sequence (marked as unknown) achieved 100% matching with those of The optimum incubation time is the time interval at which the highest β-glusosidase activity was attained. After incubation for 24, 48, 72, 96, 120, 144, and 168 h, β-glusosidase activity was 0.23, 2.56, 8.16, 21.25, 23.55, 33.37, and 23.23 U/mL , respectP. citrinum was tested to grow in fermentation medium containing various carbon sources under the temperature of 30 °C, agitation speed of 200 rpm, and for six days. Carbon source is a critical factor affecting the production of enzymes. Therefore, v/v) volumetric ratio of P. citrinum inoculum to CDN substrate, the transformation into aglycones from glucoside forms complete took place during 72 h of fermentation (v/v) of P. citrinum enzyme to CDN with fermentation time of 72 h at 30 °C.entation . This in15H10O4, daidzein) with molecular weight of 254.23 (15H10O4). Data indicates that daidzein obtained after extraction and purification from fermentation broth is of high purity. Similarly, genistein ion fragments appeared at m/z 269.0 ) appeared as yellow-brown with 5% FeCl3 and dark green with Ce(SO4)2 (data not shown). 1H-NMR : δ (ppm) 6.28 , 6.41 , 6.90 , 7.46 , 8.163 , 13.03 . 13C-NMR : δ (ppm) 94.15 , 99.52 , 105.87 , 115.65 , 122.75 , 123.76 , 130.86 , 153.98 , 163.64 , 158.76 , 158.13 , 164.70 , 181.35 .For genistein , light yellow needle crystals, Rf = 0.30 ) appeared as dark-green with Ce(SO4)2 and colorless with 5% FeCl3 (data not shown). 1H-NMR : δ (ppm) 6.89 , 6.98 , 7.47 , 8.06 , 8.14 . 13C-NMR : δ (ppm) 102.04 , 115.07 , 116.58 , 123.43 , 127.23 , 129.99 , 125.74 , 157.11 , 157.37 , 162.45 , 174.63 .For daidzein ; white needle crystals, RPenicillium funiculosum was 30–36 U/mL, achieved on 3% rice bran or defatted oil cakes after 288 h fermentation. By stark contrast, the peak activity was found at a much lower level of 2.8 U/mL with Penicillium miczynskii cultured on 3% pineapple peel within 216 h of fermentation  to their aglycones by ectively , on soy hila J18 . MoreoveIsoflavone aglycones can be derived from soybean waste by extraction with ethanol/water solvent followed by acid hydrolysis and purification , reachinP. citrinum fungus strain that produces the highest β-glucosidase activity of 33.72 U/mL, thus placing it amongst the most active fungi in this regard, has been successfully isolated from roots of Clerodendron cyrtophyllum Turcz. The fungus demonstrated a catalytic capacity to hydrolyze isoflavones-rich soybean extract into aglycones  with a hydrolysis yield of 98.7% after 72 h of fermentation at 30 °C. Purification of the hydrolyzed mixture (rich in genistein and daidzein) by column chromatography using hexane/acetone (29:1/1:1) resulted in aglycone products with purity of over 90%. These results imply that soybean extract is a promising raw material for manufacture of functional foods derived from aglycones. Our study demonstrated a potential pathway for the production of aglycones from residual soybean extract via fermentation with an isolated fungi as a biocatalyst, with applciations in the pharmaceutical and functional food industries.The


The engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology enables purification of specific genomic regions interacting with their associated molecules. In enChIP, the locus to be purified is first tagged with engineered DNA-binding molecules. An example of such engineered DNA-binding molecules to tag the locus of interest is the clustered regularly interspaced short palindromic repeats (CRISPR) system, consisting of a catalytically-inactive form of Cas9 (dCas9) and guide RNA (gRNA). Subsequently, the tagged locus is subjected to affinity purification for identification of interacting molecules. In our previous studies, we developed enChIP systems for analysis of mammalian genome functions. Here, we developed an enChIP system to analyze bacterial genome functions.Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-dCas9) in bacteria. Inducible expression of 3xFLAG-dCas9 in Escherichia coli was confirmed by immunoblot analysis. We were able to purify specific genomic regions of E. coli preserving their molecular interactions. The system is potentially useful for analysis of interactions between specific genomic regions and their associated molecules in bacterial cells to understand genome functions such as transcription, DNA repair, and DNA recombination.We generated a plasmid inducibly expressing  To understand the regulatory mechanisms underlying genome functions such as transcription, it is essential to identify the molecules associated with a genomic region of interest in vivo. The engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology we developed recently, enables specific isolation of genomic regions of interest interacting with their associated molecules , 2. ExamStreptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-dCas9) and gRNA in bacteria. The developed enChIP system isolated target genomic regions from Escherichia coli. The system might enable identification of molecules associated with a specific genomic region in bacteria, and thus help to elucidate their genome functions.Previously, we developed enChIP systems for analysis of mammalian genome functions. Here, we report development of an in-cell enChIP system for bacterial cells Fig. a. The syBglII and BstZ17I and treated with bacterial alkaline phosphatase (E. coli C75) . The double-strand DNA (dsDNA) containing the coding sequence of the 3xFLAG-tag and the N-terminal portion of dCas9 (agatctaaagaggagaaaggatctATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGCTCATGGATAAGAAATACTCAATAGGCTTAGCTATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATAC)  was synthesized (Invitrogen) and digested with BglII and BstZ17I. The cleaved pdCas9-bacteria and the synthetic DNA fragment were purified by agarose gel electrophoresis and ligated.To construct the doxycycline (Dox)-inducible 3xFLAG-dCas9 expression plasmid 3xFLAG-dCas9/p-bacteria (Addgene #64325), pdCas9-bacteria (Addgene #44249)  was digeSpeI and HindIII and treated with bacterial alkaline phosphatase (E. coli C75). The dsDNA for targeting the lacZ gene (ACTAGTGCTCTGCTACCTGCGCCAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAAGCTT) (underlined: the target sequences) was synthesized (Invitrogen) and digested with SpeI and HindIII. The cleaved pgRNA-bacteria and the synthetic DNA fragment were purified by agarose gel electrophoresis and ligated. For construction of gRNAs targeting the promoter region of the rpoH gene, oligodeoxyribonucleotides   was digeDH5α  was transformed with 3xFLAG-dCas9/p-bacteria alone or together with gRNA expression plasmids, and transformed bacteria were selected with chloramphenicol (Cam) (25 µg/ml) for 3xFLAG-dCas9/p-bacteria alone or a combination of Cam (25 µg/ml) and ampicillin (Amp) (50 µg/ml) for 3xFLAG-dCas9/p-bacteria plus gRNA expression plasmid.DH5α transformed with 3xFLAG-dCas9/p-bacteria was cultured in 2 ml of LB media containing Cam (25 µg/ml) at 37 °C overnight with shaking. One hundred microliters of the culture liquid was mixed with 900 µl of LB media containing Cam (25 µg/ml) and incubated for 1 h with shaking. Subsequently, Dox (2 µM) was added to the culture media for induction of expression of 3xFLAG-dCas9. After incubation with shaking for 4 h, 400 µl of the culture liquid was centrifuged at 5000 rpm for 1 min, and the bacterial pellets were suspended in 100 µl of 4× SDS buffer. After boiling at 100 °C for 5 min, 10 µl of the sample was subjected to SDS-PAGE with a 5–20% gradient gel followed by immunoblot analysis with anti-FLAG M2 antibody (Ab) .600 of the culture media reached 0.5, Dox (2 µM) was added for induction of 3xFLAG-dCas9 expression. After incubation at 37 °C for 4.5 h with shaking, the bacterial cells were fixed with 1% formaldehyde at 37 °C for 5 min and neutralized with glycine at room temperature for 10 min. After centrifugation, the cell pellets were suspended in 800 µl of modified lysis buffer 3 , and DNA was fragmented by sonication using Ultrasonic disruptor UD-201 (TOMY SEIKO) with conditions: Output, 3; Duty, 100% (continuous); Time, Free; 6 cycles of sonication for 10 s and cooling on ice for 20 s (the average length of chromatin fragments was 1 kbp). The sonicated chromatin was subjected to enChIP-real-time PCR analysis as described previously . First, we designed two gRNAs targeting the promoter region of the rpoH gene  was markedly lower than that of the rpoH promoter region  factor . This gee sigma 3 factor [e sigma 3 factor [e sigma 3 factor [E. coli genome while preserving their chromatin structures, and potentially contributes to the understanding of bacterial genome functions such as transcription and DNA repair.In this study, we developed an enChIP system for analysis of bacterial genomes. This system enables efficient isolation of specific genomic regions from the Further studies might be necessary to assess the utility of this system combined with MS and NGS to identify molecules associated with the target genomic regions in bacteria.


The circadian oscillator is a molecular feedback circuit whose orchestration involves posttranslational control of the activity and protein levels of its components. Although controlled proteolysis of circadian proteins is critical for oscillator function, our understanding of the underlying mechanisms remains incomplete. Here, we report that JmjC domain–containing protein 5 (JMJD5) interacts with CRYPTOCHROME 1 (CRY1) in an F-box/leucine-rich repeat protein 3 (FBXL3)-dependent manner and facilitates targeting of CRY1 to the proteasome. Genetic deletion of JMJD5 results in greater CRY1 stability, reduced CRY1 association with the proteasome, and disruption of circadian gene expression. We also report that in the absence of JMJD5, AMP-regulated protein kinase (AMPK)-induced CRY1 degradation is impaired, establishing JMJD5 as a key player in this mechanism. JMJD5 cooperates with CRY1 to repress circadian locomotor output cycles protein kaput (CLOCK)–brain and muscle ARNT-like protein 1 (BMAL1), thus linking CRY1 destabilization to repressive function. Finally, we find that ablation of JMJD5 impacts FBXL3- and CRY1-related functions beyond the oscillator. In mammals, circadian rhythms are generated by a molecular oscillator in which the circadian locomotor output cycles protein kaput (CLOCK)–brain and muscle ARNT-like protein 1 (BMAL1) transcription factors drive expression of the genes coding for their own repressors, the CRYPTOCHROME (CRY) and PERIOD (PER) proteins. A key feature of the oscillator is that the protein stability of its components is highly regulated. Previous studies had implicated the JmjC domain–containing protein 5 (JMJD5) in regulation of the circadian clock in plants and flies. Here, we show that cells and livers that lack JMJD5 exhibit dysregulation of circadian gene expression. Mechanistically, JMJD5 is required for CRY1 degradation, including its destabilization by AMP-regulated protein kinase (AMPK), by facilitating its interaction with the proteasome. We found that JMJD5 is needed for normal CRY1-mediated transcriptional repression, thereby uncovering an inverse relationship between CRY1 stability and circadian repression. Finally, we showed that JMJD5 impinges on non-clock roles of F-box/leucine-rich repeat protein 3 (FBXL3) and CRY1. Altogether, our studies demonstrate that JMJD5 is a novel link between the oscillator and other physiological processes. Bmal1 gene transcription, which contributes to robust amplitude in circadian rhythms. However, the function of the circadian oscillator involves a much larger repertoire of factors that include other transcription regulators, kinases, phosphatases, ubiquitin ligases and peptidases, and chromatin regulators. Together, this large cohort of molecules acts in concert to generate circadian rhythms, coordinate the clock with other physiological processes, and enable environmental information to be integrated into its function.Circadian rhythms are endogenous, approximately 24-hour oscillations in behavior and physiology that evolved as an adaptation to the day–night cycle. These rhythms are generated by a cell-autonomous timekeeping mechanism known as the molecular circadian oscillator. At its most basic, the oscillator is a transcription–translation circuit formed by two interlocked delayed negative feedback loops . In one β-TRCP1/2 ubiquitin ligase complex  and 300 mM NaCl) and incubated on ice for 45–60 minutes. This was then centrifuged at 3,000 rpm for 10 minutes at 4 °C. The resulting supernatant was the nuclear extract and was used in subsequent applications. Nuclear extracts from the MEFs were prepared using this protocol, and confluent 35-mm dishes of cells were used.Whole-cell lysates from cells and livers were prepared using lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 0.5% TX-100, 0.5% NP-40, 0.25% Sodium Deoxycholate 0.025% SDS along with EDTA-free protease inhibitor cocktail and phosphatase inhibitors . Briefly, cells or crushed tissue was incubated with lysis buffer for 30 minutes on ice and then spun at 10,000 rpm for 10 minutes at 4 °C. To prepare the nuclear lysates, liver tissue was homogenized in a hypotonic buffer , cells were lysed using a lysis buffer containing 200 mM NaCl, 50 mM Tris-HCl, 1% TX-100, and 1% NP-40 supplemented with phosphatase and protease inhibitors. HEK293T cells were seeded out in 6-well plates and transfected the next day with a total of 2.5 μg of DNA . Forty-eight hours post transfection, the cells were lysed, incubated on ice for 30–45 minutes, and spun at 10,000 rpm for 10 minutes at 4 °C. The supernatant was incubated with the M2 beads overnight at 4 °C while tumbling. Subsequently, the beads were washed 3 times with chilled 1X TBS for 5 minutes each. The protein was eluted from the beads with equal volume of 3X flag peptide (Sigma Cat# F4799-4MG). The eluate was boiled in NuPAGE LDS Sample Buffer and reducing buffer and subjected to SDS-PAGE-immunoblot analysis.HEK293T cells were transfected with 1.5 μg V5-FBXL3, 400 ng HA-CRY1K:R, and 600 ng of V5-JMJD5. Forty-eight hours post transfection, the cells were lysed using the lysis buffer described above and immunoprecipitated with HA antibody bound to protein G beads for 1 hour at 4 °C. The beads were washed 3 times with chilled 1X TBS for 5 minutes with mild tumbling. The bound proteins were eluted by boiling in sample buffer and subjected to SDS-PAGE-immunoblot analysis.Jmjd5+/+ and Jmjd5−/− MEFs were transfected with HA-RPN1 and 48 hours later were lysed with buffer containing 400 mM NaCl, 50 mM Tris-HCl, 1% TX-100, and 0.25% Sodium Deoxycholate, supplemented with phosphatase and protease inhibitors. The lysates were incubated on ice for 30–45 minutes and spun at 10,000 rpm for 10 minutes at 4 °C. Protein G beads were prebound with anti-HA tag antibody. The lysates were incubated with the antibody–bead complexes for 1 hour at 4 °C and washed 5 times with 1X TBS containing 0.5% Triton X-100. The bound proteins were eluted by boiling in sample buffer and subjected to SDS-PAGE-immunoblot analysis to detect endogenous CRY1 levels bound to RPN1.FLAG M2 (Sigma), Anti-HA (12CA5), V5 (Abcam), and Anti-CRY1 were the antibodies used in this study. Anti-CRY1 (687) antibody was a kind gift from Satchin Panda.Jmjd5+/+ and Jmjd5−/− MEFs were seeded out in 6-well plates at 350,000 cells per well and shocked 48 hours later with 100 nM of Dexamethasone for 2 hours before supplementing with fresh medium. Thirty-six hours post shock, the cells were harvested using 1 ml TRIzol (Fisher Scientific Cat# 15596026) and stored at −80 °C every 4 hours. Total RNA was then prepared using the manufacturer’s instructions. For real-time qPCR, 1 μg of RNA was reverse transcribed to cDNA using qScript cDNA SuperMix (Quanta Biosciences Cat# 95048–025). FastStart Universal SYBR Green Master (Rox) (Roche Cat# 4913850001) was used to perform the qPCR reaction in a BIO-RAD CFX384 Touch Real-Time PCR System.mDbp-F: GAG CCT TCT GCA GGG AAA CAmDbp-R: GCC TTG CGC TCC TTT TCCmCLOCK-F: AGAACTTGGCATTGAAGAGTCTCmCLOCK-R: GTCAGACCCAGAATCTTGGCTBmal1-F: GCC CCA CCG ACC TAC TCTmBmal1-R: TGT CTG TGT CCA TAC TTT CTT GGmCry1-F: ATC GTG CGC ATT TCA CAT ACmCry1-R: TCC GCC ATT GAG TTC TAT GATmCry2-F: GCA GAG CCT GGT TCA AGCmCry2-R: GCC ACT GGA TAG TGC TCT GGmPer1-F: GCT TCG TGG ACT TGA CAC CTmPer1-R: TGC TTT AGA TCG GCA GTG GTmPer2-F: TCC GAG TAT ATC GTG AAG AAC GmPer2-R: CAG GAT CTT CCC AGA AAC CAmNr1d1-F: GGA GCT GGG CCT ATT CAC CGCmNr1d1-R: GCT GCT CCA CCG AAG CGG AA.mJmjd5-F: CGCAGTCCTCCAGACACACCmJmjd5-R: CAAGATCACAGGCCTCCCAGmmMrpl46-F: GGTCCGGTCATTTTTTTTGTCAmMrpl46-R: GGGAGCAGGCATTCCTACAGmRORA-F: GTGGAGACAAATCGTCAGGAATmRORA-R: TGGTCCGATCAATCAAACAGTTCmABCG5-F: AGG GCC TCA CAT CAA CAG AGmABCG5-R: GCT GAC GCT GTA GGA CAC ATmGSTM3-F: CCC CAA CTT TGA CCG AAG CmGSTM3-R: GGT GTC CAT AAC TTG GTT CTC CAmLPL-F: GGG AGT TTG GCT CCA GAG TTTmLPL-R: TGT GTC TTC AGG GGT CCT TAGmLIPG-F: ATG CGA AAC ACG GTT TTC CTGmLIPG -R: GTA GCT GGT ACT CCA GTG GGmPCK1-F: CTG CAT AAC GGT CTG GAC TTCmPCK1-R: CAG CAA CTG CCC GTA CTC CmANGPTL4-F: CAT CCT GGG ACG AGA TGA ACTmANGPTL4-R: TGA CAA GCG TTA CCA CAG GChJMJD5-F: GGC CCG TGA TCC TGA AAG GhJMJD5-R: GGC TCA TTC ACG ATG TAT TTG ChCRY1-F: ACA GGT GGC GAT TTT TGC TTChCRY1-R: TCC AAA GGG CTC AGA ATC ATA CThFBXL3-F: GCA GCT TGT GAT ATA CTA TCG CAhFBXL3-R: TGG TCG AGC AGT TGA AAT AAG TChHPRT1-F: CCT GGC GTC GTG ATT AGT GAThHPRT1-R: AGA CGT TCA GTC CTG TCC ATA AAll the shRNA and siRNA knockdowns were performed using transient cotransfection of the constructs along with overexpression constructs of FLAG-CRY1, HA-JMJD5, and V5-FBXL3 in HEK293T cells in 6-well plates.shRNA hFBXL3 Sigma MISSION shRNA TRCN0000369031 (5′-CCGGCTGATCAGTGTCACGGCTTAACTCGAGTTAAGCCGTGACACTGATCAGTTTTTG-3′), shRNA hCRY1 Sigma MISSION shRNA TRCN0000231065 (5′-CCGGGGAACGAGACGCAGCTATTAACTCGAGTTAATAGCTGCGTCTCGTTCCTTTTTG-3′), siRNA hJMJD5 Sigma MISSION siRNA EHU149061, siRNA universal negative control Sigma MISSION siRNA SIC001.Jmjd5LKO mice between 6 and 9 weeks old were entrained in a light tight chamber to a 12-hour light:12-hour dark cycle for 10 days and released into constant darkness for 24 hours. Mice were then killed every 4 hours for a 24-hour period.Control or C57BL/6NTac-Jmjd5tm1a(EUCOMM)Wtsi frozen embryos were obtained from the European Conditional Mouse Mutagenesis Program (EM:04155), and embryo reconstitution was performed at Charles River. Born heterozygotes were crossed with the FLP deleter strain B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ (Jackson Laboratory 009086) to excise the lacZ-neo cassette. Thereby, conditional Jmjd5flox knockouts were created, which were bred for several generations onto a C57BL/6 background. To generate liver-specific JMJD5-null animals, Jmjd5flox/flox mice were bred with a transgenic mouse line that specifically expresses CRE in the liver at high levels /Cre+ littermates were used as controls.The following gBLOCKS were ordered from IDT and cloned into pGL3 BASIC (Promega).Wild-type E-boxes: AGTGCTAGCCATCACCCACTCACCCCTTAACGACACGTGGGCCCTCAATTGCCCTTCTCTCAGGATCTGAAGGGTCAGAGGAAAGGGTTGGATTCTTTATAACAAGGCTGGGGAGAGGCCAGGGAATGTCAGTCTAGGTTTTTCTCTCTCCCACTTCCCTTGGGTAGCAGACATTTCATTCACCCGGCACCAGGACAGGTGTCTTGTTCTGCCAAGCTGGTCAGTTTAGGAAGTAGGTTTCTCTTGAGCACTTCCTGTGGCCCAGGTATCCTCCCTGAAAAGGGGTAGTTTCCCTCCCTCACTTCCCTTTCATTATTGACGGTGTGAGACATCCTGATCGCATTGGCTGACTGAGCGGTGTCTGAGGCCCTTCAGCCCAGCACCAGCACCCAAGTCCACGTGCAGGGATGTGTGTGACACAGCCCTGACCTCAGTGGGGGCCAGTAGCCAATCAGATGCCAGGAAGAGATCCTTAGCCAACCGGGGGCGGGGCCTGCGGCTCTTCGGGCAGAAGGCCAATGAGGGGCAGGGCCTGGCATTATGCAACCCGCCTCCCAGCCTCGCGGAGCTTCTGGGTTGCAAGCTTAGC.E-boxes mutated: AGTGCTAGCCATCACCCACTCACCCCTTAACGACAgGTcGGCCCTCAATTGCCCTTCTCTCAGGATCTGAAGGGTCAGAGGAAAGGGTTGGATTCTTTATAACAAGGCTGGGGAGAGGCCAGGGAATGTCAGTCTAGGTTTTTCTCTCTCCCACTTCCCTTGGGTAGCAGACATTTCATTCACCCGGCACCAGGACAGGTGTCTTGTTCTGCCAAGCTGGTCAGTTTAGGAAGTAGGTTTCTCTTGAGCACTTCCTGTGGCCCAGGTATCCTCCCTGAAAAGGGGTAGTTTCCCTCCCTCACTTCCCTTTCATTATTGACGGTGTGAGACATCCTGATCGCATTGGCTGACTGAGCGGTGTCTGAGGCCCTTCAGCCCAGCACCAGCACCCAAGTCCAgGTcCAGGGATGTGTGTGACACAGCCCTGACCTCAGTGGGGGCCAGTAGCCAATCAGATGCCAGGAAGAGATCCTTAGCCAACCGGGGGCGGGGCCTGCGGCTCTTCGGGCAGAAGGCCAATGAGGGGCAGGGCCTGGCATTATGCAACCCGCCTCCCAGCCTCGCGGAGCTTCTGGGTTGCAAGCTTAGC.S1 Fign = 3) and mice livers (mean ± SEM n = 4) were determined by qPCR analysis. JMJD5, JmjC domain–containing protein 5; MEF, mouse embryo fibroblast; qPCR, quantitative PCR.mRNA levels of JMJD5+/+ and JMJD5−/− MEFs (mean ± SEM (TIF)Click here for additional data file.S2 FigPer2-Luc promoter in Jmjd5+/+ and Jmjd5−/− MEFs . JMJD5, JmjC domain–containing protein 5; MEF, mouse embryo fibroblast.Real-time bioluminescence measurement from overexpressed (TIF)Click here for additional data file.S3 FigJmjd5LKO, JmjC domain–containing protein 5 liver knockout; loxP, locus of X-over P1.Schema of targeting vector and resulting floxed allele. Exons shown as solid boxes; Frt and loxP sites are respectively shown as green and purple triangles. Frt, flippase recognition target; (TIF)Click here for additional data file.S4 FigH321A (MUT) in the real-time luciferase assay in (A) Relative protein levels of FLAG-JMJD5 and FLAG-JMJD5(TIF)Click here for additional data file.S5 Fig(A) FLAG-CLOCK and BMAL, (B) FLAG-PER1, and (C) FLAG-PER2. BMAL, brain and muscle ARNT-like protein; CLOCK, circadian locomotor output cycles protein kaput; JMJD5, JmjC domain–containing protein 5; PER, PERIOD.(TIF)Click here for additional data file.S6 FigJmjd5LKO, JmjC domain–containing protein 5 liver knockout; WLE, whole liver extract.Total protein stain was used as loading control. CRY1, CRYPTOCHROME 1; (TIF)Click here for additional data file.S7 FigShown are the inputs for the coimmunoprecipitations of FLAG-tagged CRY1 and mutant CRY1s with (A) V5-FBXL3 and (B) HA-JMJD5. CRY1, CRYPTOCHROME 1; FBXL3, F-box/leucine-rich repeat protein 3; JMJD5, JmjC domain–containing protein 5.(TIF)Click here for additional data file.S8 FigHEK293T, human embryonic kidney 293T; HPRT1, hypoxanthine phosphoribosyltransferase 1 gene; qPCR, quantitative PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA.(TIF)Click here for additional data file.S9 FigImmunoprecipitation of FLAG-CRY2 in HEK293T cells shows no interaction with HA-JMJD5 both in the absence and presence of V5 FBXL3. FLAG-CRY1 interaction with HA-JMJD5 is shown as a positive control. * denotes an unknown band observed only when FLAG-CRY2 is transfected. CRY, CRYPTOCHROME; FBXL3, F-box/leucine-rich repeat protein 3; HEK293T, human embryonic kidney 293T; JMJD5, JmjC domain–containing protein 5.(TIF)Click here for additional data file.S10 Fig(A and B) Shown are the areas of the blot used to quantify the accumulation of CRY1. (C) Quantification of the main band (non-ubiquitylated CRY1) reveals a similar pattern of accumulation as quantifying all the forms of CRY1 as shown in (TIF)Click here for additional data file.S11 Fig71A/280A; CRY1, CRYPTOCHROME 1; JMJD5, JmjC domain–containing protein 5.Real-time luciferase assays from (TIF)Click here for additional data file.S12 FigCRY1, CRYPTOCHROME 1; MEF, mouse embryo fibroblast.(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Auxiliary subunits of the CKAMP family differentially modulate AMPA receptor properties. Published 1, December 2015In the Materials and Methods subsection "In situ hybridization" the sequences of radiolabeled oligodeoxyribonucleotide probes used for CKAMP52 and CKAMP59 were mixed up. These are the correct sequences:Ckamp52ins1 = 5’AATGTCAGCCAGAGCCCTGTGGATGTTCATCTCTCGCGGACkamp59ins1 = 5’GCGGCATAGCACGCCAGTCGAGGTTGGAGGGCTTCATGGTGTTWe apologize for the mistake.The article has been corrected accordingly.