Figure 5

Gamitrinib and Thap combination treatment elevates CHOP expression. (A) Synergistic increase in CHOP induction. 22Rv1 and H460 cells were treated with 5 μM gamitrinib and 0.06 μM Thap as indicated and analyzed by western blotting. (B) CHOP reporter assay. PC3 cells stably transfected with a CHOP::GFP reporter plasmid [50] were incubated with 2.5 μM gamitrinib and/or 0.02 μM Thap as indicated and analyzed as described in Materials and Methods (left). Cells with more than twice the background fluorescence intensity were considered as positive cells (right). Bar, 100 μm. Mean ± SEM. **, p = 0.0036. (C) Silencing CHOP expression. Control or CHOP siRNA-transfected HeLa cells were treated with 0.06 μM Thap and 5 μM gamitrinib for 24 hours, and analyzed by MTT assay. Knockdown efficiency analyzed by western blotting (bottom right). Mean ± SEM. *, p < 0.05. (D) Silencing RyR expression. Control or RyR siRNA-treated HeLa cells were incubated with 0.06 μM Thap and 5 μM gamitrinib for 24 hours, and analyzed by MTT assay. Knockdown efficiency analyzed by western blotting (bottom right). Mean ± SEM. *, p < 0.05. (E) Knockdown of RyR2 by siRNA. Control or RyR2 siRNA-transfected 22Rv1 cells were incubated with 2.5 μM gamitrinib and/or 0.06 μM Thap as indicated and analyzed by western blotting.