Fig. 4: Nuclear F-actin modulates the engagement of replication fork remodellers and fork reversal.

a iPOND analysis of HEK293T cells after indicated treatments (100 nM CPT, 1 h; 100 nM LatB, 10 min prior to CPT). See Supplementary Fig. 2a for details. Proteins associated with nascent DNA were isolated by iPOND and detected with the indicated antibodies. In the control (Ctrl) experiment, the click reaction is performed using DMSO instead of biotin azide. Same representative experiment as in Fig. 2c, d. b Graph-bar depicts mean and SD of quantified RAD51 and SMARCAL1 levels at nascent DNA from three independent iPOND experiments (black dots). Values are normalized to H3 and represented as fold change over the NT sample. Statistical analysis: one-tailed t-test with Welch’s correction. c, d Electron micrographs of representative replication forks from U2OS cells: parental (P) and daughter (D) duplexes. d White arrow indicates the regressed arm (R); the four-way junction at the reversed fork is in the inset. Scale bar = 200 nm, 40 nm in the inset. e Frequency of reversed replication forks isolated from U2OS cells upon optional treatment with 100 nM CPT for 1 h. 100 nM LatB or Swi were added 10 min before CPT and retained during the genotoxic treatment. Total number of molecules analyzed per condition in brackets. f Frequency of reversed replication forks isolated from RPE-1 cells after 24 h doxycycline-induction of either BFP-NLS or NLS-BFP-Actin^R62D and optional treatment with CPT (100 nM, 1 h) or ETP (20 nM, 1 h). Total number of molecules analyzed per condition in brackets. e, f Bar graphs depict mean ± SD from three independent EM experiments (red and blue dots, respectively). Statistical analysis: ordinary one-way ANOVA. Source data are provided as a Source Data file.