Inhibition of mitochondrial Hsp90s sensitizes HeLa cells toward thapsigargin. (A) Cytoplasmic calcium and mitochondrial membrane potential by suboptimal dose of gamitrinib. Fluo-4 or TMRM/MitoTracker-labeled HeLa cells were incubated with 5 μM gamitrinib for 24 hours and analyzed by confocal microscope. Bar, 20 μm. (B) Combination effect in HeLa. HeLa cells were treated with various concentrations of Thap in the presence of 5 μM of either 17AAG or gamitrinib, and analyzed by MTT assay (left). Alternatively, HeLa cells were treated with 5 μM gamitrinib and/or 0.06 μM Thap for 24 hours and analyzed by the MTT assay. ***, p < 0.0001. (C) Combination effect in 22Rv1. 22Rv1 cells were treated with various concentrations of thapsigargin in the presence of 2.5 μM of either 17AAG or gamitrinib for 24 hours, and analyzed by the MTT assay (left). Alternatively, 22Rv1 cells were treated with 2.5 μM gamitrinib (Gami) and 0.06 μM Thap as indicated for 24 hours and analyzed by the MTT assay. **, p = 0.0006. (D) Combination treatment induces apoptosis. HeLa cells were treated with 10 μM gamitrinib and 0.5 μM Thap as indicated, labeled with FITC-DEVD-fmk and propidium iodide, and analyzed by flow cytometry. (B) and (C) represent mean ± SEM from three independent experiments.