Figure 3

Ryanodine receptor (RyR)-mediated cytosolic calcium elevation. (A) IP₃R silencing. After IP₃R siRNA treatment, Fura-2 labeled HeLa cells were treated with 30 μM gamitrinib for 1 hour. The fluorescence ratio (340/380) was plotted. The data are mean ± SEM collected from 30 ROIs in two independent experiments. (B) IP₃R knockdown effect on CHOP expression. Control or IP₃R1 siRNA-transfected HeLa cells were incubated with or without 30 μM gamitrinib for 2 hours. Cell extracts were analyzed by western blotting. (C) RyR inhibitors compromise cytosolic calcium increase. Fura-2 labeled HeLa cells were treated with 30 μM gamitrinib for an hour in the presence or absence of 300 μM tetracaine, 100 μM ryanodine, and 5 μM CsA, and emission fluorescence intensity ratios (340/380 nm excitation) were measured. Data are mean ± SEM calculated from 40 ROIs in two independent experiments. (D) Fura-2 imaging and RyR2 silencing. Control or RyR2-#2 siRNA-treated HeLa cells were labeled with Fura-2 and imaged after 30 μM gamitrinib treatment for an hour (left). The fluorescence ratio (340/380) was plotted (middle). Knockdown efficiency of RyR2-#2 siRNA by western blotting (right). The data are mean ± SEM collected from 30 ROIs in two independent experiments. Bar, 50 μm. (E) Inhibition of CHOP induction by RyR inactivation. HeLa cells were treated with 30 μM gamitrinib in the presence or absence of 100 μM ryanodine. Cell extracts were analyzed by western blotting. (F) RyR2 silencing and CHOP expression. HeLa cells were treated with two different RyR2 siRNAs, incubated with 30 μM gamitrinib, for 2 hours and analyzed by western blotting. #, not significant; *, p < 0.0001.