Fig. 4: Loss of Ser-384 RPA70 phosphorylation markedly disrupts cell viability and induces basal genomic stress response.

a Homozygous knock-in of Ser-384-Ala RPA70 phospho-dead mutant in HCT116 cells was generated using CRISPR-Cas9 editing. Cells were synchronized in mitosis using nocodazole and representative western blot depicts the loss of RPA70-Ser-384 phosphorylation in RPA ^SA/SA mutant. Asynchronous parental HCT116 cells and cells arrested in mitosis were used as controls. Blots were probed with the indicated antibodies. Blots are representative of at least three independent experiments. b MTS assay shows decreased viability of phospho-dead RPA^SA/SA mutant. Cells were assayed at 0, 24, 48, and 72 h of growth. Values corrected for background absorbance were normalized to 0 h of growth. Error=SEM. The mean of three independent experiments was plotted. Triplicate wells were assayed per time point for each experiment. Statistical significance was determined using an unpaired two-tailed t-test: p = 0.38 at 24 h, ns = not significant, *p = 0.014 at 48 h, **** p = 0.000018 at 72 h. c Caspase 3/7 activity was significantly higher in the RPA mutant cells as determined by the Caspase3/7 glo assay. Bar graph depicts data corrected for background absorbance. Error = SEM. The mean of three independent experiments was plotted. Triplicate wells were assayed per experiment. Statistical significance was determined using an unpaired two-tailed t-test: **** p = 0.00000003. d Representative western blot shows replication stress-response in cells treated with vehicle (veh, 0.1% DMSO) or 20 ng/mL SN-38 for 90 min. Arrow indicates increased basal genomic stress as shown by high levels of p53 in the RPA^SA/SA mutant. Data are representative of three independent experiments. e Caspase3/7 activity was determined in response to replication stress by treating cells with 10 ng/mL SN-38 for 21 h and by using Caspase3/7 glo assay. Bar graph displays data corrected for background absorbance. Error = SEM. The mean of three independent experiments was plotted. Triplicate wells were assayed per experiment. Statistical significance was determined using an unpaired two-tailed t-test: **** p = 0.00000002.