Fig. 4

C-terminal region of RIF1 protects of reversed forks from degradation. a Schematic of full-length (FL) human RIF1 protein and deletion mutant constructs. Deleted region for each mutant is denoted by dotted line. b Western blot analysis for Rif1^−/− MEFs transfected with mutant construct of human RIF1. Lysates were probed with antibody against GFP. XPD was used as loading control. Expression of mutant protein is visualized as distinct bands in range of 198 kD to 310 kD, which is missing in mock-transfected samples. c DNA fiber assay to assess the rescue of Fork degradation in Rif1^−/− MEFs transfected with RIF1 mutant constructs (for 48 h) upon treatment with 4 mM HU for 3 h. d Percentage of fork reversal in Rif1^−/− MEFs transfected with different mutant constructs of human RIF1 and subsequent treatment with HU for 3 h (4 mM). Numbers of analyzed molecules are indicated in parentheses. e DNA fiber assay to determine the extent of fork degradation in WT and Rif1^−/− MEFs upon siRNA-mediated downregulation of PP1. f Percentage of reversed forks observed in WT and Rif1^−/− MEFs treated with 4 mM HU for 3 h with or without inhibiting PP1. Number of molecules analyzed are indicated within the parenthesis. At least 125 readings were taken for c and e and the mean values are represented by red bar. P-values were derived from Kruskal–Wallis ANOVA with Benjamini Hochberg post test for c and e and from unpaired t-test for d (ns, non-significant, ***P = 0.0009, **P = 0.0025) and f (ns, non-significant, ***P = 0.0003, **P = 0.0026). All the experiments were repeated for three times with similar outcomes (Supplementary Data 2 and Supplementary Fig. 8a, b). g DNA2 is hyper phosphorylated in Rif1^−/− MEFs during replication stress. Top panel: level of DNA2 in nuclear extracts from WT and Rif1^−/− MEFs before and after treatment with HU alone or in combination with PP1 inhibitor treatment (tautomycetin 225 nM for 2 h). Western blots were performed with antibody against DNA2 antibody. Histone H3 was used as loading control. Bottom panel: Immunoprecipitations were carried out with anti-DNA2 antibody or the corresponding IgG and were probed with p-(S/T) antibody