RAD18 regulates FANCD2 and FANCI monoubiquitylation. (A) H1299 cells were depleted of RAD18 by siRNA transfection and then treated with 500mM MMC over several time points for 24 hours. Immunoblotting for FANCD2 shows a 6- to 8-hour delay in the monoubiquitylation of FANCD2 compared with control knockdown. (B) Rad18-deficient and wild-type cells were treated with 500mM MMC over several time points for 24 hours and assessed for FANCD2 and FANCI monoubiquitylation by Western blot. FANCD2 and FANCI monoubiquitylation is delayed by 6-8 hours in RAD18-deficient cells compared with wild-type cells. (C) RAD18-deficient and wild-type cells were treated with 75uM cisplatin over several time points for 24 hours. Immunoblotting reveals that FANCD2 and FANCI monoubiquitylation was again delayed by nearly 6-8 hours in RAD18-deficient cells. (D) HA-tagged RAD18 was expressed in Rad18-deficient and wild-type cells, which were then treated with 500mM MMC for 24 hours and assessed for FANCD2 and FANCI monoubiquitylation by Western blot. FANCD2 and FANCI monoubiquitylation was restored in untreated RAD18-deficient cells compared with the expression of green fluorescent protein as a control. Topo II, tubulin, and Ku86 western blots represent loading controls. The ratio of FANCD2-L:FANCD2-S was calculated from densitometry of each Western blot using ImageJ software Version 1.44. (E) Endogenous PCNA was depleted from MRC-5 cells expressing a ubiquitylation-resistant form of PCNA, followed by treatment of the cells with 500mM MMC over several time points for 24 hours. Immunoblotting shows that FANCD2 monoubiquitylation is unaffected by PCNA status, indicating that modification of PCNA does not regulate monoubiquitylation of FANCD2. (F) HeLa cells were depleted of PCNA by siRNA transfection and then treated with 500mM MMC over several time points for 24 hours. Immunoblotting for FANCD2 shows no delay in the monoubiquitylation of FANCD2 compared with control knockdown.