Fig. 2: PFN1 and SNF2H function together to promote DNA replication.

a Percentages of EdU-labeled (10 μM, 25 min) PFN1 knockdown and overexpressing MCF-10A cells in late S-phase. Data are mean ± SEM of n = 3 independent experiments containing about 3000 cells per group. P-values were based on One-Way ANOVA and Dunnett’s multiple comparisons test. b Anti-HA pulldown using transfected HEK293T cells. c Anti-GFP pulldown using MCF-7 cells expressing YFP or YFP-NLS-PFN1. Details for quantification and normalization of SNF2H intensities are described in the Methods section. d Anti-SNF2H pulldown using parental MCF-10A cells. e, f PLA between HA-PFN1 and endogenous SNF2H in HeLa cells. Scale bar, 5 µm. Around 500 nuclei were quantified as in (1b). g, h DNA fiber analysis in MCF-10A cells. i, j DNA fiber analysis in HEK293T cells. k DNA fiber analysis in MCF-7 cells. For (g–k), at least 300 fibers were analyzed per condition. l Percentages of DNA fibers representing newly fired origins (CIdU only and CIdU-IdU-CIdU) in MCF-10A cells with SNF2H or PFN1 knockdown or PFN1 overexpression. Data are mean ± SEM of n = 2 independent experiments with around 3000 fibers per group. P-values were based on One-Way ANOVA analysis and Dunnett’s multiple comparisons test. m Control and PFN1 knockdown MCF-10A cells were synchronized in late G1 by double thymidine block followed or not with 2 h release into S phase. Cells were lysed with RIPA buffer, and insoluble (chromatin-enriched) vs. soluble fractions were analyzed by Western blot. n, o MCF-10A cells were lysed with RIPA and analyzed as in m. Details for protein quantification and normalization are described in the Methods section. p PLA between endogenous SNF2H and biotinylated EdU in MCF-10A cells with PFN1 knockdown/rescue or overexpression. Scale bar, 5 µm. PLA intensities were quantified by image J⁹⁵ and shown as mean ± SEM of around 200 positive cells per condition. P-values for PLA and DNA fiber data were based on Kruskal–Wallis test with Dunnett’s multiple comparisons as in Fig. 1. For all statistical tests, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. All results were confirmed by at least two independent experiments. Source data are provided as a Source Data file.