Figure 5

Inhibition of autophagy accelerated nutrient deprivation-induced ROS accumulation and subsequent cell necrosis. (A) Chang liver cells were cultured with complete medium or EBSS in the absence or presence of CQ or 3-MA for 12 h. At the end of treatments, cells were stained with DCF-DA and observed under a fluorescent microscope at magnification × 400 (left panel). The fluorescent intensity of each group was also quantified (right panel) and expressed as mean ± SEM (n = 3; *: p < 0.05; **: p < 0.01). (B) Chang liver cells were cultured with complete medium or EBSS in the absence or presence of CQ or 3-MA for 24 h. Intracellular ROS generation was measured by flow cytometry using DCF-DA staining. ROS positive cells were counted and expressed as the mean ± SEM (n = 3). (C, D and E) Chang liver cells were cultured with complete medium or EBSS in the absence or presence of CQ or 3-MA and/or NAC for 24 h. Cell viability was detected by CCK8, and necrosis and apoptosis of Chang liver cells were assayed by Hoechst 33342/PI staining. Cell viability (C), the percentage of necrosis (D) and apoptosis (E) were shown. Data were shown as mean ± SEM (n = 3; *: p < 0.05; ns: no significance).