Fig. 5: PFN1 increases genome instability during replication stress.

a, b γH2AX induction in MCF-10A cells following HU treatment (5 mM, 6 h). Insoluble fractions after RIPA lysis were analyzed by Western blot. γH2AX was normalized to histone H3. c, d Metaphase spreads in HeLa cells after 6 h HU (4 mM or 2 mM) treatment, 20 h release, and 4 h nocodazole (10 μM) treatment. Aberrant chromosomes are indicated by red arrows and representative chromosomes are magnified in the insets. Scale bar, 10 µm. Around 150 metaphases per condition were analyzed from n = 3 independent experiments. e, f Micronuclei in PFN1 knockdown (e) or overexpression (f) MCF-7 cells after 6 h HU treatment (4 mM in e and 2 mM in f) with and without 50 µM Mirin and C5. Relative changes in the percentages of micronuclei-positive cells under HU vs. no HU conditions are shown as mean ± SEM of n = 2 independent experiments with around 2000 cells per condition. g–i HeLa cells with PFN1 overexpression (g, h) or knockdown (i) were labeled with 10 µM BrdU (g, h) or EdU (i) overnight, treated with 2 mM HU for 6 h (g, h) or 12 h (i), released overnight, and stained for BrdU under denaturing condition or EdU by Click reaction. Scale bar, 10 µm. Cytosolic DNA intensities were expressed as percentages of total DNA. Shown are mean ± SEM of n = 2 independent experiments with around 500 cells in each group. P-values in e, f and h, i were based on One-Way ANOVA and Dunnett’s multiple comparisons test. j, k PFN1-overexpressing HEK293T cells (j) and PFN1 knockdown MCF-7 (k) cells were treated or not with 4 mM HU for 6 h, released overnight, and analyzed by RT-qPCR for the indicated genes. Shown are HU/no HU ratios of mRNA levels of each gene. Data represent mean ± SEM of n = 4 independent experiments. Statistical significance was determined by Two-Way ANOVA and Dunnett’s multiple comparisons test. l, m Clonogenic assays using mock and HU-treated (6 h) MCF-7 cells with PFN1 overexpression (l) and knockdown (m). Data represent mean ± SEM of n = 5 (l) and n = 3 (m) independent experiments. For all statistical tests, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. Source data are provided as a Source Data file.