Fig. 7
From: ATM and CDK2 control chromatin remodeler CSB to inhibit RIF1 in DSB repair pathway choice
ATM controls the chromatin remodeling activity of CSB through S10 phosphorylation. a Western analysis of U2OS CSB KO cells stably expressing various alleles as indicated. Cells were either treated with or without 10 Gy IR. b Western analysis of anti-CSB immunoprecipitates from HCT116 cells treated with or without 10 Gy IR. c Western analysis. Myc-CSB-expressing U2OS CSB KO cells were treated with DMSO, ATM inhibitor KU55933, ATR inhibitor VE-821 or DNA-PKcs inhibitor NU-7026 for 1 h prior to 10 Gy IR. d Western analysis of ATM-deficient GM16666A and ATM-complemented GM16667A cells. Cells were transfected with the vector alone or Myc-CSB, followed by treatment with 10 Gy IR 48 h post transfection. e Relative occupancy of histone H2A in ddI-PpoI-expressing hTERT-RPE CSB KO cells complemented with various alleles as indicated. Both x- and y-axes are as described in Fig. 5a. SEM from three independent experiments are indicated. f Relative occupancy of histone H2B in ddI-PpoI-expressing hTERT-RPE CSB KO cells complemented with various alleles as indicated. Both x- and y-axes are as described in Fig. 5a. SEM from three independent experiments are indicated. g Quantification of the percentage of cyclin A- and cyclin A+ cells with 10 or more IR-induced RIF1 foci. hTERT-RPE CSB KO cells stably expressing various alleles as indicated were treated with 2 Gy IR and fixed 1 h post IR. A total of 500–550 cells were scored for each independent experiment in a blind manner. SDs from three independent experiments are indicated. *P < 0.05. ns: P > 0.05 (Student t test). h Quantification of the percentage of cyclin A+ cells with ≥ 10 IR-induced BRCA1 foci. Scoring was done as in 7 g. SDs from three independent experiments are indicated. *P < 0.05. ns: P > 0.05 (Student t test). i Quantification of the percentage of cyclin A+ cells with 10 or more IR-induced RAD51 foci. Cells were treated with 2 Gy IR and fixed 4 h post IR. Scoring was done as in 7 g. SDs from three independent experiments are indicated. *P < 0.05. ns: P > 0.05 (Student t test)
