Fig. 3

DNA2 drives reversed fork degradation in RIF1-deficient cells. a Western blot analysis for the downregulation of MRE11 and DNA2 in WT and Rif1^−/− MEFs. WT and Rif1^−/− MEfs were transfected with either siControl or siRNAs smart pool against MRE11 and DNA2. Lysates made were probed with antibody against MRE11 and DNA2. Tubulin is used as loading control. b Ratio of IdU versus CldU in WT and Rif1^−/− MEFs upon HU treatment after downregulating Mre11 or DNA2 (a). c Ratio of IdU versus CldU in WT and RIF1-KO HAP1 cells upon HU treatment after inhibiting Mre11 and DNA2 using mirin and DNA2 inhibitor. d Electron microscopic analysis of percentage of reversed forks observed in WT and Rif1^−/− MEFs subjected to HU (4 mM) for 3 h, with or without DNA2 inhibitor. Numbers of analyzed molecules are indicated in parentheses. At least 125 readings were taken for b and c and the mean ratio is represented by red bar. P-values were derived from Kruskal–Wallis ANOVA with Benjamini Hochberg post test except in d, where unpaired t-test (ns, non-significant, ****P < 0.0001, **P = 0.0024) was carried out. Similar observation was made from three independent experiments (Supplementary Data 2 and Supplementary Fig. 7g, i)