Fig. 2
From: The concerted roles of FANCM and Rad52 in the protection of common fragile sites
FAAP24 and MHF are important for recruiting FANCM to Flex1 to prevent DSB formation and mitotic recombination. a DSB formation at Flex1 or Luc surrounding regions was analyzed by ChIP analysis of pCEP4-Flex1 or pCEP4-Luc10 using H2AX-S139p (γH2AX) antibody. Anti-γH2AX ChIP was performed in U2OS cells expressing FANCM shRNA (left), expressing Flag-FANCM-WT or K117R with endogenous FANCM silenced by shRNA (middle), or expressing FAAP24 shRNA with or without HU treatment (2 mM, 6 h) (right). Western blots for the expression of FANCM, Flag-FANCM WT and K117R, and FAAP24 are shown in Supplementary Fig. 5b. b Flag-FANCM or empty vector (Ctrl) was expressed in U2OS (HR-Flex) reporter cell line with Flex1 stably integrated into the genome. Anti-Flag ChIP was performed using indicated primer sets (P1 or P2), or primers corresponding to GAPDH (left); or using P1 before or after APH treatment (0.4 μM, 24 h) (right). ChIP value in Ctrl is set up as 1 for normalization. c Anti-Flag ChIP at Flex1 was performed in U2OS (HR-Flex) cells expressing Flag-FANCM and shRNAs for FAAP24 (left) or MHF1 (right) with vector as a control. Western blots for the expression of FAAP24 and MHF1 are shown in Supplementary Fig. 5d. d Spontaneous recombination was assayed in U2OS (HR-Flex) cells expressing shRNA for FAAP24 (left) or MHF1 (right) with a vector control. e Anti-Flag ChIP at Flex1 was performed in U2OS (HR-Flex) cells expressing Flag-FANCM-WT or indicated mutants with the vector as a control. Western blots showing FANCM expression are in Supplementary Fig. 5e. f Anti-Flag ChIP at Flex1 was performed in U2OS (HR-Flex) cells expressing Flag-FAAP24 WT or mutants (left), or Flag-FANCM and HA-FAAP24 WT or indicated FAAP24 mutants with endogenous FAAP24 silenced by shRNA (right). ChIP in the vector expressing cells serves as the control. DB DNA binding mutant: K171A/K173A; MB FANCM binding mutant: V198A/V199A/G200A. Western blots showing relevant protein expression are in Supplementary Fig. 5f, g. g Spontaneous recombination was assayed in U2OS (HR-Flex) cells expressing Flag-FAAP24 WT and mutant alleles with endogenous FAAP24 silenced by shRNA
