Inhibition of mitochondrial Hsp90s depletes stored calcium in both mitochondria and the ER. (A) Mitochondrial calcium depletion. After 30 μM gamitrinib and 10 μM FCCP treatment, confocal FRET images of mtCameleon-expressing HeLa cells were reconstructed from their emission fluorescence ratios at 535/480 nm with excitation at 440 nm (left). FRET ratios at the indicated time intervals were averaged and plotted (right). (B) ER calcium depletion. FRET images of HeLa cells transiently expressing D1ER were acquired at the indicated time points after gamitrinib treatment (left) and analyzed to plot the FRET ratio (right). Selected ROIs are indicated as white circles. Bar, 10 μm. Data in (A) and (B) are mean ± SEM collected from 30 ROIs. R.U., relative units. (C) CHOP induction and eIF2α phosphorylation. HeLa cells were treated with 30 μM gamitrinib, 5 μM CsA, and 10 μM BAPTA as indicated and analyzed by western blotting. #, not significant; *, p < 0.001; **, p < 0.0001.