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 Death and Differentiation

Article Title: L3MBTL2 regulates chromatin remodeling during spermatogenesis

doi: 10.1038/s41418-019-0283-z

Figure Lengend Snippet: L3MBTL2 interacts with RNF8 and L3mbtl2 deficiency leads to reduced nuclear RNF8 and ubH2A levels in elongating spermatids. a–c L3MBTL2 interacts with RNF8. HEK293 cells were transfected with HA-RNF8 in the absence or presence of L3mbtl2-Flag. Cell lysates were used for immunoprecipitation (IP) and subsequent Western blotting (WB) using indicated antibodies. Cell lysates were used for WB as indicated to show inputs (a). HEK293 cells were transfected with L3mbtl2-Flag in the absence or presence of HA-RNF8. Cell lysates were used for IP and /or WB as indicated (b). Lysates from mouse testes were immunoprecipitated with anti-RNF8 antibody or normal IgG, and the precipitates and lysates were subjected to WB as indicated (c). d mRNA levels of Rnf8 in germ cells from control and L3mbtl2 cKO mice at 8 months of age. Rpl19 is the internal control. e Western blot analyses of RNF8 on whole lysates of germ cells from control and L3mbtl2 cKO mice at 8 months of age. GAPDH is the loading control. f Western blot analyses of RNF8 on nuclear extracts of germ cells from control and L3mbtl2 cKO mice at 8 months of age (left panel). Nuclear RNF8 levels relative to LaminB1 levels were quantified by densitometry (right panel). g Immunohistochemical staining for RNF8 in the testes of control and L3mbtl2 cKO mice at 8 months of age. The signals for RNF8 in elongating spermatids were reduced in L3mbtl2 cKO compared with control testes. h Western blot analyses of ubH2A on acid extracts of germ cell chromatin from control and L3mbtl2 cKO mice at 8 months of age. ubH2A levels relative to H2A levels, and H2A levels relative to H3 levels were quantified by densitometry (right panels). i Immunohistochemical staining of ubH2A in the testes of control and L3mbtl2 cKO mice at 8 months of age. The signals for ubH2A in elongating spermatids were reduced in L3mbtl2 cKO compared with control testes while the signals in other cell types remained unchanged between the two genotypes

Article Snippet: Sections then were incubated with rabbit anti-L3MBTL2 antibody (HPA000815, Sigma-Aldrich; {"type":"entrez-protein","attrs":{"text":"A08416","term_id":"84503","term_text":"pir||A08416"}} A08416 , Boster Biological Technology) or anti-RNF8 antibody (14112-1-AP, Proteintech) at 4 °C overnight.

Techniques: Transfection, Immunoprecipitation, Western Blot, Immunohistochemical staining, Staining

Journal: Cell Death and Differentiation

Article Title: L3MBTL2 regulates chromatin remodeling during spermatogenesis

doi: 10.1038/s41418-019-0283-z

Figure Lengend Snippet: Inhibition of L3MBTL2 reduces nuclear RNF8 expression and ubH2A levels in GC2 cells. a, b Effects of siRNA-mediated L3mbtl2 knockdown on Rnf8 mRNA and protein expression. GC2 cells were transfected with control (Ctrl) or L3mbtl2 siRNAs (siL3). 45 h after transfection, cells were serum-starved for 6 h. Cells then were collected for real-time PCR analysis or Western blotting for L3mbtl2 and Rnf8 mRNA (a) and protein (b) levels. c Effects of siRNA-mediated L3mbtl2 knockdown on nuclear RNF8 protein levels. GC2 cells were transfected with control (Ctrl) or L3mbtl2 siRNAs (siL3). 45 h after transfection, cells were serum-starved for 6 h before cells were collected for nuclear protein extraction and western blotting for RNF8 (left panel). LaminB1 and GAPDH were used for loading controls for nuclear and cytosolic extracts respectively. Nuclear RNF8 levels relative to LaminB1 levels were quantified by densitometry (right panel). d Effects of siRNA-mediated L3mbtl2 knockdown on ubH2A levels. GC2 cells were transfected with control (Ctrl) or L3mbtl2 siRNAs (siL3). 45 h after transfection, cells were serum-starved for 6 h before histones were extracted using HCl (0.2 N). Acid extracts were used for western blotting for ubH2A and H2A (left panel). ubH2A levels relative to H2A levels were quantified by densitometry (right panel). *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Sections then were incubated with rabbit anti-L3MBTL2 antibody (HPA000815, Sigma-Aldrich; {"type":"entrez-protein","attrs":{"text":"A08416","term_id":"84503","term_text":"pir||A08416"}} A08416 , Boster Biological Technology) or anti-RNF8 antibody (14112-1-AP, Proteintech) at 4 °C overnight.

Techniques: Inhibition, Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Protein Extraction