Fig. 3 | Nature Communications

Fig. 3

From: The concerted roles of FANCM and Rad52 in the protection of common fragile sites

Fig. 3

FANCM translocase activity is important for CFS protection. a, b Plasmid stability assay was performed in cells carrying pCEP4-Flex1 or pCEP4-Luc plasmids after culturing cells without hygromycin for 1 week. U2OS cells expressing indicated shRNAs or control vector (a), and HCT116 WT or FANCM KO cells reconstituted with Flag-FANCM (WT or K117R) (b) were used. c Metaphase spread of HCT116 cells was performed with or without expressing FANCM shRNA or vector before and after APH treatment (0.4 μM, 24 h). Representative images of metaphase spread are shown on the left, and overall chromosome gaps and breaks per cell are shown on the right. d FISH images of HCT116 cells after DAPI staining using probes against FRA3B or FRA16D are shown (left). Purple and green arrows indicate broken and normal chromosomes, respectively. Frequency of CFS expression at FRA3B or FRA16D in HCT116 cells expressing FANCM shRNA or control vector before and after APH treatment was determined (right). FANCM expression is shown by western blots. e Anti-γH2AX ChIP analysis at FRA3B locus using primer sets 3B-P1 and 3B-P2 was performed in U2OS cells expressing FANCM shRNA or vector before and after APH treatment (0.4 μM, 24 h). ChIP value in Ctrl is set up as 1 for normalization. f Frequency of CFS expression at FRA3B or FRA16D in HCT116 cells expressing Flag-FANCM (WT or K117R) with endogenous FANCM silenced before and after APH treatment (0.4 μM, 24 h) was determined. Expression of indicated Flag-FANCM alleles is shown by western blots. Bars, 5 μm