Figure 3.

The fraction of cells in S phase is not strongly impacted by siRNA-mediated depletion of RAD51, with or without PP7-15q-TERRA inducible expression. a) Western blotting was used to evaluate siRNA-mediated knockdown efficiency of RAD51, with and without PP7-15q-TERRA inducible expression. Vinculin is shown as a loading control. Representative blots of three biologically-independent experiments are shown. b) Distribution of cells of indicated conditions in G1, S or G2/M cell cycle phases. To evaluate cell cycle distribution of cells with depleted RAD51, DNA content was assessed by flow cytometry analysis of fixed DAPI-stained cells. Briefly, 1 million cells/condition was harvested by trypsinization. Cells were washed twice with cold 1x PBS and resuspended in 1 ml cold 70% ethanol while vortexing. After 30 min, ethanol was aspirated and fixed cells were resuspended in 1x PBS containing 0.2 μg/ml RNase, DNase-free (Merck) and incubated at 37°C for 15 min. 1x PBS containing 2 μg/ml DAPI (BioChemica) was then added to stain DNA. Samples were processed with a BD LSR Fortessa. 20.000 events were acquired per condition, per biological replicate. Data are means ± s.d. of three independent biological replicates. Two-tailed unpaired t-tests were used to calculate P-values: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Flow cytometry data were analyzed with FlowJo and statistical analysis was performed using GraphPad Prism