Fig. 5: Blocking autophagy induces the amplification of ROS and VNR-induced apoptosis in the A549 and CL1-5 cells.

a A representative western blot analysis showing the expression of Akt, p-Akt, Keap1, ATG5 in the A549 cells with shLuc and shATG5. 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 A549 cells with shLuc and shATG5. For the flow cytometric analyses, the percentages are the means ± SDs of three individual experiments. *P < 0.5. c Nuclear PI staining and subsequent flow cytometric analysis determined cell apoptosis in the VNR-treated A549 cells with shLuc and shATG5. 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 A549 cells with shLuc and shATG5. β-actin was used as an internal control. The relative densities of the measured protein bands are also shown. f Nuclear PI staining and subsequent flow cytometric analysis determined cell apoptosis in the VNR-treated A549 and CL1-5 cells with or without autophagy inhibitor 3-MA. The percentages (%) of apoptotic cells are shown as the means ± SDs of three individual experiments. ***P < 0.001. CM-H2DCFDA staining, followed by a flow cytometric analysis, was used to determine the levels of ROS in the VNR-treated A549 and CL1-5 cells with or without autophagy inhibitor 3-MA. For the flow cytometric analyses, the percentages are the means ± SDs of three individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001. g A hypothetic model of this work. Targeting the regulators of autophagy modulates Keap1-mediated ARE activity, ROS generation, and VNR-induced apoptosis