Figure 4.

WZ35-induced H1975 apoptosis involves ROS-dependent ER stress. (A) EM evaluation of ER in H1975. H1975 cells were exposed to 5 μM WZ35 for 6 h, with or without pretreatment with 5 mM NAC for 2 h. The morphology of ER was examined with an electron microscope (×30,000) showing ER swelling (red arrows) in H1975 with WZ35 exposure. (B) H1975 cells were treated with 5 μM WZ35 for different time periods, and the protein levels of p-eIF2α, ATF4, and CHOP were determined by Western blotting. (C) H1975 cells were treated with WZ35 at different concentrations for 6 or 12 h. ER stress-associated proteins were then detected. (D) H1975 cells were pretreated with 5 mM NAC for 2 h before exposure to 5 μM WZ35 for 6 or 12 h. Same analysis as in panels B and C was performed. GAPDH was used as internal control. Data presented are representative of three independent experiments. (E) Immunostaining of H1975 for CHOP (red). Cells were pretreated with 5 mM NAC for 2 h before exposure to 5 μM WZ35 for 12 h. Cells were counterstained with DAPI (blue). (F) H1975 cells transfected with CHOP siRNA or negative control siRNA and then treated with 5 μM WZ35 for 12 h. CHOP expression in H1975 cells was determined by Western blotting. (G) Percentage of cell apoptosis following CHOP siRNA transfection and treatment of cells with WZ35 for 24 h. Apoptotic cells were measured by Annexin-V/PI staining.

Abbreviations: ROS, reactive oxygen species; EM, electron microscopy; ER, endoplasmic reticulum; NAC, N-acetylcysteine; DMSO, dimethyl sulfoxide; siRNA, small interfering RNA; PI, propidium iodide; FITC, fluorescein isothiocyanate; CHOP, C/EBP-homologous protein; ATF4, activating transcription factor 4; p-eIF2α, phosphorylated eukaryotic initiation factor α; DAPI, 4′,6-diamidino-2-phenylindole.