Fig. 3: PFN1 increases the stalling of stressed DNA replication forks. | Nature Communications

Fig. 3: PFN1 increases the stalling of stressed DNA replication forks.

From: Profilin-1 regulates DNA replication forks in a context-dependent fashion by interacting with SNF2H and BOD1L

Fig. 3

a, b Percentages of DNA fibers representing stalled (IdU only) and restarted forks (dual IdU-CIdU) in MCF-10A cells sequentially labeled with 25 μM IdU and 250 μM CIdU for 30 min with 2 h HU treatment in the middle. Data represent mean ± SEM of n = 2 independent experiments with around 2000 fibers per group. c–e Ratios of CIdU tract lengths in dual-labeled (IdU-CIdU) MCF-10A cells with and without HU in the middle. HU was used at 5 mM in c and 2 mM in d and e. CIdU labeling immediately followed IdU under the no-HU condition. Individual CIdU tract lengths of HU-treated fibers were normalized to mean CIdU lengths of untreated fibers. For c–e, at least 300 fibers were analyzed per condition. f Percentages of pSer4/8-RPA32-positive MCF-10A cells following HU exposure (4 mM, 4 h) and immunofluorescence staining. g Same cells as in (f) were labeled with 10 μM BrdU for 24 h, treated with 4 mM HU for 4 h, and immuno-stained for BrdU under native conditions. Percentages of BrdU-positive cells were analyzed by ImageJ. Data in f, g are mean ± SEM of n = 3 independent experiments with around 3000 cells per group. h Western blot analysis of whole cell extracts of MCF-10A cells after HU (5 mM) exposure. Equal amounts of proteins from control and PFN1 knockdown cells were loaded on separate gels but analyzed in parallel under identical conditions. Phospho-proteins were normalized to GAPDH. i Percentages of stalled and restarted forks in MCF-10A cells labeled with IdU and CIdU with HU treatment in the middle as in a, b. Data are mean ± SEM of n = 4 independent experiments with around 3000 fibers per group. j PLA between endogenous SNF2H and EdU in MCF-10A cells after 2 h HU treatment. PLA intensities of around 200 positive cells per condition were analyzed and shown as mean ± SEM. P-values in (a, b, f, g, i) were based on One-Way ANOVA and Dunnett’s multiple comparisons test. P-values in (c, d, e, j) were based on Kruskal–Wallis test with Dunnett’s multiple comparisons. 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.

Back to article page