Fig. 3.

C/EBPδ augments FANCD2 monoubiquitination and cell survival in response to MMC. (A) C/EBPδ-KO MEFs were transfected with Flag-tagged WT mouse C/EBPδ or vector control for 16 h before treatment with 500 ng/mL MMC for 20 h. Whole-cell lysates were analyzed for expression of C/EBPδ and FANCD2 in comparison with WT MEFs. Tubulin was used as loading control. L/S, quantification of the FANCD2-L/S ratio in lysates of MMC-treated cells. (B) 293-C/EBPδ and 293P cells were seeded in 96-well plates and 16 h later were treated with tetracycline (Tet; 500 ng/mL) and/or MMC (1 μg/mL) for the indicated times before relative quantification of cell numbers. Data represent mean ± SEM of two experiments, each done in six wells per data point, shown relative to untreated controls (set at 1 for each time point). **P < 0.01 comparing MMC-treated cells with or without C/EBPδ induction by tetracycline. (C) PD20-D2 and PD20 cells were transfected with Flag-tagged WT C/EBPδ, the ΔD2ID mutant, or vector control for 8 h before being placed in 96-well dishes at 5,000 cells/well. The next day, MMC (100 ng/mL) was added, and cell viability was assessed 48 h later. Data are mean ± SEM of three experiments, each performed in triplicate, and relative to cells without MMC (set at 100%). **P < 0.01 relative to vector and ΔD2ID transfected cells. (D) Western blot analysis of FANCD2 and C/EBPδ in PD20-D2 and PD20 cells treated as in C. C/EBPδ was detected by anti-Flag antibodies. (E) PD20-D2 cells were transfected with shRNA constructs against C/EBPδ (+) or GFP (-) as control and 24 h later were treated with 500 ng/mL MMC for another 24 h. Whole-cell lysates were analyzed for FANCD2 and C/EBPδ expression.