Journal: The Journal of Cell Biology

Article Title: The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

doi: 10.1083/jcb.201001160

Figure Lengend Snippet: Recruitment of p400 to DSBs requires mdc1. (a) H2AX −/− MEFs or H2AX −/− MEFs complemented with H2AX (H2AX wt ) were exposed to 5 µM bleomycin. 20 µM Wortmannin was added 60 min before bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones detected by Western blot. (b) 293T cells expressing vector or mdc1 shRNA (shmdc1) were exposed to 5 µM bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones detected by Western blot. (c) 293T cells expressing GFP or mdc1 shRNA were transiently transfected with vector or p84-ZFN. 18 h later, ChIP assays using p400 antibody and primer pairs located at +1.5 kb were performed. Results ± SE ( n = 3). (d) 293T cells stably expressing FLAG-RNF8 or RNF8 lacking the catalytic domain (FLAG-RNF8 δring ), which functions as a dominant-negative inhibitor of endogenous RNF8 function , were exposed to bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones detected by Western blot.

Article Snippet: Antibodies used: p400 (Bethyl Laboratories, Inc.); H2AX, 53BP1 (Cell Signaling Technology); γ-H2AX, Tip60, FK2, H2B, acetylated H4 (Millipore); H3, H4, RNF8 (Abcam); ATM, PC116, brca1 (EMD); ATM2C1 (GeneTex); pS1981-ATM (R&D Systems); RNF8 antibody for foci experiment was a generous gift from Junjie Chen (MD Anderson Cancer Center, University of Texas, Houston, TX).

Techniques: Western Blot, Expressing, Plasmid Preparation, shRNA, Transfection, Stable Transfection, Dominant Negative Mutation

Journal: The Journal of Cell Biology

Article Title: The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

doi: 10.1083/jcb.201001160

Figure Lengend Snippet: p400 is required for RNF8-dependent ubiquitination of the chromatin. (a) 293T cells expressing p400 or p400 ATPase were irradiated as indicated. Cells were fixed and processed by immunofluorescent staining using FK2 antibody. Bar, 5 µm. (b) 293T cells expressing p400 or p400 ATPase were irradiated (2Gy). Immunofluorescent staining using the FK2 antibody was used to detect proteins ubiquitinated by RNF8. Cells with >5 foci per cell were counted, with an average of 100 cells per slide. Results ± SE ( n = 100). (c) 293T cells expressing p400 or p400 ATPase were transiently transfected with vector or p84-ZFN. 18 h later, ChIP assays using the FK2 antibody and primer pairs located at −3.5 kb, −1.5 kb, −0.5 kb, 0.5 kb, 1.5 kb, and 3.5 kb were performed. Results ± SE ( n = 3). (d) 293T cells expressing p400 or p400 ATPase were irradiated (2Gy), fixed, and immunofluorescent staining to detect RNF8 and FK2 performed. Bar, 5 µm. (e) Quantitation of FK2 and RNF8 foci in p400 and p400 ATPase cells from d. Cells with >5 foci were counted, with an average of 100 cells per slide counted. Experiments represent at least three independent replicates. Results ± SE ( n = 100).

Article Snippet: Antibodies used: p400 (Bethyl Laboratories, Inc.); H2AX, 53BP1 (Cell Signaling Technology); γ-H2AX, Tip60, FK2, H2B, acetylated H4 (Millipore); H3, H4, RNF8 (Abcam); ATM, PC116, brca1 (EMD); ATM2C1 (GeneTex); pS1981-ATM (R&D Systems); RNF8 antibody for foci experiment was a generous gift from Junjie Chen (MD Anderson Cancer Center, University of Texas, Houston, TX).

Techniques: Ubiquitin Proteomics, Expressing, Irradiation, Staining, Transfection, Plasmid Preparation, Quantitation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: UBE2T-regulated H2AX monoubiquitination induces hepatocellular carcinoma radioresistance by facilitating CHK1 activation

doi: 10.1186/s13046-020-01734-4

Figure Lengend Snippet: UBE2T induces HCC cell radioresistance in coordination with RNF8. a 293 T cells were transfected with FLAG-UBE2T and treated with IR (4 Gy). IP was performed by using FLAG antibody, and the IP product was analyzed by immunoblotting. b Immunoblotting analysis of FLAG-IP derived from the irradiated 293 T cells transfected with empty vector or FLAG-RNF8. c Representative images of immunofluorescence staining for UBE2T (green) and RNF8 (red) in MHCC-97H cells treated with IR (4 Gy). d Immunoblotting analysis of the cytosolic and chromatin fractions of IR (4 Gy) treated MHCC-97H cells transfected with siRNA-RNF8 or siRNA-control. e Representative images and quantification of UBE2T overexpressing MHCC-97H cells stained for RNF8 (red) foci before and after IR (4 Gy). f Representative images and quantification of UBE2T silencing MHCC-97H cells stained for RNF8 (red) foci before and after IR (4 Gy). g UBE2T overexpressing cells or control cells were transfected with siRNA-RNF8 or siRNA-control, and harvested at the indicated timepoints after IR (4 Gy). h Cells with the same treatment as that in panel g were collected at the indicated timepoints after IR (4 Gy) to test cell cycle distribution. i Cells with the same treatment as that in panel g were collected at the indicated timepoints after IR to test γH2AX level. j Colony formation assays were conducted in UBE2T stably overexpressing MHCC-97H cells transduced with lentivirus coding control shRNA or shRNA targeting RNF8. Data represent the mean ± SD. In ( e ) and ( f ), * P < 0.05, by 2-tailed paired Student’s t test. In ( h ), ns, not significant, * P < 0.05, by one-way ANOVA

Article Snippet: The sources of antibodies against the following proteins were as follows: H2AX (D17A3; 7631), γH2AX (Ser139, 20E3; 9718), p-ATR (2853), p-ATM (13050), and p-CHK1 (Ser345, 133D3; 2348) from Cell Signaling Technology; UBE2T (10105–2-AP), CHK1 (25887–1-AP), RNF8 (14112–1-AP), TRIM21 (12108–1-AP), BRCA1 (22362–1-AP), RAD51 (14961–1-AP), BMI1 (10832–1-AP), RING2 (16031–1-AP), ATR (19787–1-AP), ATM (27156–1-AP), HSP70 (10995–1-AP) and LaminB1 (12987–1-AP) from Proteintech Group; H2AX (ab229914) from Abcam; FLAG (F1804) from Sigma-Aldrich; GAPDH (RM2002), HA (RM1004), myc (RM1003) and β-actin (RM2001) from Rui Antibody Biotech.

Techniques: Transfection, Western Blot, Derivative Assay, Irradiation, Plasmid Preparation, Immunofluorescence, Staining, Control, Stable Transfection, Transduction, shRNA

Journal: Cell reports

Article Title: Feedback repression of PPARα signaling by Let-7 microRNA

doi: 10.1016/j.celrep.2021.109506

Figure Lengend Snippet:

Article Snippet: pCMV6-mouse Rnf8 , Origene , Cat# MR207821.

Techniques: Recombinant, Protease Inhibitor, TaqMan microRNA Assay, Chromatin Immunoprecipitation, Knock-Out, Negative Control, Software