Depletion of SAMHD1 induces R-loop-mediated genome instability and cellular DNA damage response.
A, Flow cytometry prolife of control U2OS and SAMHD1-depleted cells, in the absence or presence of ectopic RNaseH1-GFP expression at different time points after release from double thymidine block. Cells were subjected to PI staining followed by FACS analysis. Data represent mean ± SEM, n = 3, P values were calculated using two-way ANOVA. B, Representative images of comet assays performed under alkaline electrophoresis conditions in SAMHD1-depleted and control U2OS cells, in the absence or presence of ectopic RNaseH1-mCherry expression. C, Quantitative analysis of comet tail moment lengths for each condition described in B. Statistical significance was assessed using two-tailed Student’s t-test (n = 50). D, Immunoblots of CHK1 and CHK2 in S phase-synchronized SAMHD1-depleted and control U2OS cells, in the absence or presence of ectopic RNaseH1-mCherry (RNH1-mCherry) expression. Fold-induction was normalized to the empty vector-transfected control U2OS cells by quantifying the band intensity of the blots and calculating the ratios of p-CHK1 and p-CHK2 to total expression levels of CHK1 and CHK2 expression, respectively. E, Representative image of immunofluorescence assay of H2AX phosphorylated on S139 (γH2AX) in S phase synchronized SAMHD1-depleted and control U2OS cells, in the absence or presence of ectopic RNaseH1-mCherry (RNH1-mCherry) expression. Cells were co-stained with DAPI (blue) and anti-γH2AX antibodies (green) (n = 2). F, Quantification of γH2AX positive signal per nucleus for the immunofluorescence assay described in E. Data represent mean ± SEM, the statistical significance was assessed using the two-tailed Student’s t-test (n > 5). G, Representative images of proximity ligation assay (PLA) between PCNA and RNA polymerase II (RNAPII) in S phase synchronized SAMHD1-depleted and control U2OS cells, in the absence or presence of ectopic RNaseH1-GFP (RNH1-GFP) expression (n = 2). Cells were subjected to PLA (red) and co-stained with DAPI (blue). H, Quantification analysis of number of PLA foci per nucleus in G. The mean value for each data is shown as a red line. Statistical significance was assessed using the two-tailed Mann-Whitney U test (n = 322).
