Figure 7

Atg5 deficiency aggravated EBSS-induced mitochondrial ROS generation of liver cells and subsequent cell necrosis. Chang liver cells were transfected with the indicated lentiviruses. (A) Atg5 expression of the transfected cells were determined by immunblotting. (B-H) The transfected cells were cultured with complete medium or EBSS for 24 h. (B) Cell viability was measured by CCK8. Data were shown as mean ± SEM (n = 6; *: p < 0.05). (C) The morphologies of cells were captured by a light microscope. (D) ROS generation was measured by flow cytometry using DCF-DA staining. ROS positive cells were counted and expressed as the mean ± SEM (n = 3). (E and F) The percentage of necrotic (E) and apoptotic (F) cells were assayed by Hoechst 33342/PI staining. Data were shown as mean ± SEM (n = 3; *: p < 0.05; **: p < 0.01; ns: no significance). (G) The cells were incubated with MitoSOX Red (red staining) and DAPI dye (blue staining). Representative images of cells were taken a magnification × 400 (left). Relative fluorescence intensity of MitoSOX Red per cell was quantitated and shown as mean ± SEM (n = 3; **: p < 0.01). (H) Oxidative damaged DNA was visualized by immunocytochemistry for 8-OHdG (green) and counter stained with DAPI (blue) at a magnification × 400.