Fig. 4: Replication stress-induced RIF1-BRCA1 proximity occurs in S phase and depends on ATR signalling. | Nature Communications

Fig. 4: Replication stress-induced RIF1-BRCA1 proximity occurs in S phase and depends on ATR signalling.

From: The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

Fig. 4

A Western blot analysis of 53BP1 depletion by siRNA in RPE-1 cells. B RIF1-BRCA1 PLA analysis in Control or 53BP1-depleted RPE-1 cells with indicated treatments. HU: 4 mM 24 h. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. C Experiment procedure to label S phase cells with EdU, followed by RIF1-BRCA1 PLA analysis. An asynchronous RPE-1 cell culture was pulse labelled with EdU for 15 min, and then treated with no or 4 mM HU for 4 h. Cells were fixed at the end of HU treatment and subjected to RIF1-BRCA1 PLA procedures. D Left: representative images acquired from the experiment described in (C). unt: not treated with HU; HU: 4 mM 4 h. Scalebar: 10 µm. Right: quantification of RIF1-BRCA1 PLA foci per nucleus. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. E RIF1-BRCA1 PLA analysis in RPE-1 cells collected at indicated timepoints after removal of HU. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. F RIF1-BRCA1 PLA analysis in RPE-1 cells following indicated kinase inhibitor and HU treatments. HU: 4 mM 24 h; ATRi (VE-821): 1 µM 24 h; Chk1i (PF-477736): 1 µM 24 h. p values calculated by Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups.

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