Fig. 8

Cyclin A–CDK2 controls the chromatin remodeling activity of CSB through S158 phosphorylation. a Western analysis of U2OS-CSB-KO cells stably expressing the vector alone, Myc-CSB or Myc-CSB carrying a S158A mutation. Immunoblotting was performed with anti-CSB-pS158 and anti-Myc antibodies. b Western analysis of synchronized HCT116 cells. The arrow indicates the position of CSB-pS158. Asterisks indicate non-specific bands. c Western analysis. Asynchronous and synchronized HCT116 cells post release from a double thymidine block as indicated were treated with DMSO or the CDK inhibitor roscovitine. The arrow indicates the position of CSB-pS158. Asterisks indicate non-specific bands. d In vitro kinase assays with recombinant cyclin A-CDK2 and bacteria-expressed recombinant CSB fragments as indicated. 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 8 g. SDs from three independent experiments are indicated. *P < 0.05. ns: P > 0.05 (Student t test). i Clonogenic survival assays of olaparib-treated hTERT-RPE CSB-KO cells complemented with various alleles as indicated. SDs from three independent experiments are indicated. *P < 0.05 (Student t test) for comparison between CSB and S158A