American Society of Hematology

Figure 4

$Figure 4. RAD18 regulates chromatin loading of FANCD2 and FANCI. (A) RAD18-deficient and wild-type cells were treated with 500mM MMC for 24 hours and fractionated into chromatin and soluble fractions. Immunoblotting revealed that FANCD2 and FANCI are successfully loaded onto chromatin by 24 hours after treatment, but are undetectable in the chromatin fraction of untreated RAD18-deficient cells (lane 6). The same extracts run on a parallel gel were immunoblotted for core complex proteins FANCA and FANCE and show no difference in chromatin loading of these proteins in RAD18-deficient versus wild-type cells. (B) RAD18-deficient and wild-type cells were exposed to 500nM MMC for 24 hours and then fractionated into chromatin and soluble fractions. Western blotting shows no difference in chromatin loading of FANCL in RAD18-deficient versus wild-type cells or FANCL chromatin in RAD18-knockout cells. (C) HA-tagged RAD18 was expressed in HCT116 RAD18−/− cells using adenoviral vectors, treated with 500nM MMC for 24 hours, and then fractionated into chromatin and soluble fractions. Western blotting shows that FANCD2 and FANCI chromatin loading is restored in RAD18-deficient cells (lanes 5-6). (D) RAD18-deficient and wild-type cells were plated into 6 well plates and then trypsinized and counted every 24 hours for 5 days. Growth of each cell type was similar. (E) Cell-cycle analysis of RAD18-deficient and wild-type cells shows similar cell-cycle profiles.$

RAD18 regulates chromatin loading of FANCD2 and FANCI. (A) RAD18-deficient and wild-type cells were treated with 500mM MMC for 24 hours and fractionated into chromatin and soluble fractions. Immunoblotting revealed that FANCD2 and FANCI are successfully loaded onto chromatin by 24 hours after treatment, but are undetectable in the chromatin fraction of untreated RAD18-deficient cells (lane 6). The same extracts run on a parallel gel were immunoblotted for core complex proteins FANCA and FANCE and show no difference in chromatin loading of these proteins in RAD18-deficient versus wild-type cells. (B) RAD18-deficient and wild-type cells were exposed to 500nM MMC for 24 hours and then fractionated into chromatin and soluble fractions. Western blotting shows no difference in chromatin loading of FANCL in RAD18-deficient versus wild-type cells or FANCL chromatin in RAD18-knockout cells. (C) HA-tagged RAD18 was expressed in HCT116 RAD18^−/− cells using adenoviral vectors, treated with 500nM MMC for 24 hours, and then fractionated into chromatin and soluble fractions. Western blotting shows that FANCD2 and FANCI chromatin loading is restored in RAD18-deficient cells (lanes 5-6). (D) RAD18-deficient and wild-type cells were plated into 6 well plates and then trypsinized and counted every 24 hours for 5 days. Growth of each cell type was similar. (E) Cell-cycle analysis of RAD18-deficient and wild-type cells shows similar cell-cycle profiles.

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