Fig. 1: PFN1 is important for unperturbed DNA replication.

a PFN1 is detected on nascent DNA by iPOND assay. HEK293T cells were labeled or not with EdU (10 µM) for 25 min, followed or not by thymidine (10 µM) chase for 1 h. b, c PLA between HA-PFN1 and biotinylated EdU in transfected HeLa cells. Scale bar, 5 µm. Quantification (in c) represents PLA foci numbers per nucleus out of at least 250 cells per condition. Error bars are mean ± SEM. Percentages of PLA-positive cells (>5 foci per nucleus) are shown on the bottom. d Loss of PFN1 delays S phase progression. MCF-10A cells infected with the indicated shRNAs were synchronized by double thymidine and released for the indicated times. The percentages of cells in G2 phase are colored in red. Samples were stained with propidium iodide and analyzed by flow cytometry and FlowJo. Gating strategies are provided in Supplementary Fig. 10. e–g Representative images (e) and tract length analysis (f, g) of single-molecule DNA fiber assay in MCF-10A cells expressing PFN1 shRNAs or cDNAs. Cells were labeled with 25 μM IdU (1st) and 250 μM CIdU (2nd) for 30 min each. Scale bar, 10 µm. h DNA fiber analysis in PFN1 knockdown MCF-10A cells functionally rescued by WT or S137D mutant form of PFN1. i DNA fiber analysis in MCF-7 cells expressing YFP or YFP-PFN1 tagged with NLS or NES. j DNA fiber analysis in control or XPO6 knockout MCF-10A cells. For (f–j), at least 300 DNA fibers were analyzed for each experimental condition, and mean values of tract lengths are shown. All DNA fiber and PLA data were analyzed using the Kruskal–Wallis test with Dunnett’s multiple comparisons. **p < 0.01; ****p < 0.0001; ns, not significant. Results were confirmed by at least two independent experiments. Source data are provided as a Source Data file.