Figure 1. PCNA^K164R mutant cell lines have increased sensitivity to DNA damage.

(A) Western blot analyses of chromatin-associated PCNA, ubiquityl-PCNA (K164), phospho-RPA32 (S33), and γH2AX, with or without 20 and 40 J/m² UV treatment, with histone H2AX as the loading control from wild-type RPE-1 and PCNA^K164R cells. Quantified band intensities were normalized to loading controls.

(B) Western blot analyses of whole-cell extracts from wild-type RPE-1 and PCNA^K164R cells for phospho-p53 (S15), p53, and p21, with or without 20 and 40 J/m² UV treatment, with GAPDH or tubulin as the loading control. Quantified intensities of phospho-p53 (S15), p53, and p21 levels were normalized to loading controls.

(C) Comparison of drug sensitivity comparing average percentage of viability in RPE-1 wild-type and PCNA^K164R cell lines. Each drug and concentration tested is indicated. Error bars indicate standard deviation, and statistical significance was calculated using Student’s t test with *p < 0.05, **p < 0.01, and ***p < 0.001; n = 9–12 replicate wells across three biological replicates for all data points.

(D) Representative cell-cycle distributions based on DNA content (DAPI) of RPE-1 wild-type and PCNA^K164R cell lines treated with MMC (20 nM) or MMS (20 μM) for 48 h.

(E) Cell-cycle distributions of RPE-1 wild-type and PCNA^K164R cell lines treated with MMC or MMS from three biological replicates. Percentage of each population in G1 (green), S (purple), or G2/M phase (gray) is shown. Error bars indicate standard deviation, and statistical significance was calculated using Student’s t test with *p < 0.05, **p <0.01, and ***p < 0.001; n = 6 replicate wells across three biological replicates. For wild type, statistics represent the comparison of each cell-cycle phase in drug-treated (MMC, MMS) versus untreated (UT) cell lines. For PCNA^K164R cell lines, statistics represent the comparison of each cell-cycle phase versus wild type within each treatment group (UT, MMC, MMS).

(F) Histogram (left, middle) and quantification of mean fluorescent intensity (right) of EdU staining of S-phase cells from RPE-1 wild-type (blue) and PCNA^K164R cells (maroon) treated with MMC and MMS; n = 6 across three biological replicates. Error bars indicate standard deviation, and significance was calculated using two-way ANOVA with *p < 0.05, **p < 0.01, and ***p < 0.001.