Fig 6. BLM and FANCM play distinct roles in protecting Flex1 stability.

A. Spontaneous recombination was assayed in U2OS (HR-Flex) cells 6 days after infecting with lentiviruses encoding vector, BLM shRNA or FANCM shRNA alone or both. The expression of BLM and FANCM was examined by Western blot with β-actin as loading control. B. Spontaneous recombination was assayed in U2OS (HR-Flex) cells expression FANCM WT or MM2 mutant with endogenous FANCM silenced by shRNA. The expression of FLAG-FANCM WT or MM2 mutant was examined by Western blot analysis with β-actin as loading control. C. U2OS cells containing pCEP4-Flex1 or pCEP4-Luc plasmids were infected with lentiviruses expressing BLM shRNA or FANCM shRNA or both. Two days after shRNA lentiviral infection, cells were treated with HU (2 mM, 6 hr) and ChIP analysis of γH2AX at Flex1 or Luc on pCEP4-Flex1 or pCEP4-Luc plasmids was performed with PCR products resolved on agarose gel (left). The expression of BLM and FANCM was examined by Western blot with β-actin as loading control (right).