Fig. 6

UBE2T promotes CHK1 activation, DDR and HCC radioresistance via monoubiquitinating H2AX/γH2AX. a Histone fraction from MHCC-97H cells transfected with FLAG-UBE2T was exposed to IR (4 Gy) and analyzed for H2AX/γH2AX monoubiquitination. b Immunoblotting of MHCC-97H cells expressing FLAG-UBE2T at the indicated timepoints after IR. c Histone fraction from MHCC-97H cells transfected with siRNA-UBE2T was exposed to IR and analyzed for H2AX/γH2AX monoubiquitination. d Immunoblotting of UBE2T silencing MHCC-97H cells at the indicated timepoints after IR. e Cell extracts from 293 T cells expressing FLAG-UBE2T WT or FLAG-UBE2T C86A were treated with IR and immunoprecipitated with anti-FLAG antibodies, followed by immunoblotting with indicated antibodies. f-k Cells with the same treatment as that in panel e. f Cells were stained for γH2AX foci (red) and FLAG-UBE2T (green). Scale bar: 20 μM. g Total cell lysates were analyzed by immunoblotting. h Cytosolic and chromatin fractions were analyzed by immunoblotting. i Cell cycle distribution was analyzed by flow cytometry. j Immunofluorescence staining was performed for γH2AX foci analysis. Quantification is shown. k Cell survival was assessed by CCK-8 assay. (l) Cellular extracts from 293 T cells expressing FLAG-H2AX WT or FLAG-H2AX K119/120R were treated with IR and immunoprecipitated with anti-FLAG antibodies, followed by immunoblotting with indicated antibodies. m Cells with the same treatment as that in panel l. Cells were stained for FLAG-H2AX (green) and UBE2T (red). Scale bar: 20 μM. n-r MHCC-97H cells were transfected with myc-tagged adenoviral-control, adenoviral-H2AX WT, adenoviral-H2AX K119/120R, or empty vector, treated with IR. n Total cell lysates were analyzed by immunoblotting. o Cytosolic and chromatin fractions were analyzed by immunoblotting. p Cell cycle distribution was analyzed by flow cytometry. q Immunofluorescence staining was performed for γH2AX foci analysis. r Cell survival was assessed by CCK-8 assay. Data represent the mean ± SD. In (i), (j), (p), and (q), ns, not significant, *P < 0.05 and **P < 0.01, by one-way ANOVA. In (k) and (r), ns, not significant, *P < 0.05 and **P < 0.01, by two-way ANOVA