Fig. 3. mTOR knockdown induces increased autophagy activity followed by diminished ROS and VNR-induced apoptosis in the AS2 cells.

a A representative western blot analysis showing the expression of Akt, p-Akt, Keap1, mTOR and p-S6K in the AS2 cells with shLuc and shmTOR. β-actin was used as an internal control. The relative densities of the measured protein bands are also shown. b CM-H₂DCFDA staining, followed by a flow cytometric analysis, was used to determine the levels of ROS in the AS2 cells with shLuc and shmTOR. For the flow cytometric analyses, the percentages are the means ± SDs of three individual experiments. ***P < 0.001. c Nuclear PI staining and a subsequent flow cytometric analysis determined cell apoptosis in VNR-treated AS2 cells with shLuc and shmTOR. The percentages (%) of apoptotic cells are shown as the means ± SDs of three individual experiments. ***P < 0.001. A representative western blot analysis showing the expression of p62 (d) and LC3 I and II (e) in the AS2 cells with shLuc and shmTOR. β-actin was used as an internal control. The relative densities of the measured protein bands are also shown