FIGURE 3. Cellular viability and ssDNA gap formation in BRCA1/2-deficient cells following loss of RAD18, REV1 and/or PRIMPOL.

A) Viability of WT, REV1 KO, RAD18 KO and REV1/RAD18 KO U2OS cells after treatment with the indicated siRNAs. Cell viability is represented as a percentage of viable cells relative to control siRNA-treated cells. Values of individual experiments are indicated as dots. Columns represent the mean ± SEM of independent biological replicates (n = 3). Statistical analysis was conducted by unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

B) Viability of UWB1.289 and MDA-MB-436 cells with and without BRCA1 re-expression after transfection of Cas9 protein in complex with the indicated sgRNAs. Cell viability is represented as a percentage of viable cells relative to control sgRNA-treated cells (n = 4). Graphical representation and statistical analysis were conducted as in (A).

C-D) Schematic of the IdU/CldU pulse-labeling protocol with S1 nuclease treatment (top). Dot plot and median of CldU tract lengths (μm) in PRIMPOL KO cells complemented or not with PRIMPOL WT or mutant cDNAs and subjected to the indicated experimental conditions (bottom). Doxycycline (DOX) induces the expression of Cas9 in cells carrying Cas9 and RAD18 sgRNA constructs. P-values were calculated by Mann-Whitney test (****p < 0.0001).

E) Viability of WT and PRIMPOL KO U2OS cells transfected with the indicated sgRNAs in complex with Cas9 after treatment with the indicated siRNAs. Cell viability is represented as a percentage of viable cells relative to control siRNA-treated cells (n = 4). Graphical representation and statistical analysis were conducted as in (A).

F) Viability of PRIMPOL KO U2OS cells with and without complementation with PRIMPOL WT or mutant cDNAs after treatments with the indicated siRNAs and DOX, as in (C) and (D). Graphical representation and statistical analysis were conducted as in (A).