Figure 4: mTORC1 signaling inhibition is not sufficient to enhance apoptosis induced by GTN/H89. SW480 cells (3 × 105/mL) were pretreated for 1 h 30 with rapamycin (50 nM) before exposure to GTN or/and H89 (10 μM each) (A) for 24 hours to analyze by western blot p70 and pp70 expression or (B) for 48 h to count apoptotic cells after Hoechst 33342 staining. (C) SW480 cells were transfected with a p70-specific siRNA. Total protein was isolated and subjected to western blot analysis for p70 expression. HSC70 was used as a control for equal protein loading (1 representative of 3 independent experiments). (D) p70-specific siRNA-transfected SW480 cells were treated with GTN or/and H89 (10 μM each) for 48 h at 37°C. Then, apoptotic cells were counted after Hoechst 33342 staining (Results are the mean SD of 3 independent experiments).