Characterization of resistance mechanisms to romidepsin. (A) DpP75 cells were transduced with lentiviral particles containing empty vector, a non-targeting shRNA, or an shRNA to BCL2L11 and selected with puromycin to generate stable cell lines. Cells were then incubated in 250 nM AZD6244 (AZD) or PD0325901 (PD) for 48 hours, after which whole-cell lysates were extracted and subjected to immunoblot analysis for Bim and cleaved PARP (c-PARP). GAPDH served as a loading control. (B) Cells from (A) were then treated with 25 ng romidepsin (Dp), 250 nM AZD6244 (AZD), or 250 nM PD0325901 (PD) for 48 hours, after which the cells were incubated with annexin V and PI, and percent live cells was determined. Results compiled from at least 5 independent experiments are shown. (C) HuT78 cells were transduced with lentiviral particles as outlined in (A), after which whole-cell lysates were extracted and subjected to immunoblot analysis for Bim and GAPDH. (D) Cells from (C) were then treated with 1 or 10 ng/mL romidepsin (Dp) for 48 hours after which the cells were incubated with annexin V and PI, and percent live cells was determined. Results compiled from at least 3 independent experiments are shown. (E) HuT78 and DpVp50 cells were incubated with 25 ng/mL romidepsin (Dp) or 1 or 2.5 µM ABT-737 for 48 hours, after which cells were stained with annexin and PI as in (A). Percent annexin-positive cells was determined; results from at least 3 experiments are shown. (F) RPMI-8226 cells were incubated with 25 ng/mL romidepsin alone (Dp) or with 250 nM AZD6244 (Dp AZD) or PD0325901 (Dp PD) for 6 hours, after which media was removed and cells were incubated with romidepsin-free medium alone or continuing with AZD6244 or PD0325901 for an additional 42 hours. As controls, cells were also incubated with 250 nM AZD6244 (AZD) or PD0325901 (PD) alone for 48 hours. Percent annexin positive cells was determined as in (A). Results from at least 4 independent experiments are shown. C, control.