Figure 3.

Pretreatment with ascorbic acid attenuates oxidative stress-induced apoptosis and autophagic flux blockage in B4G12 and ARPE-19 cells. B4G12 and ARPE-19 cells were cultivated in medium with or without 1 mM of ascorbic acid for two days, followed by the addition of paraquat (2 mM for ARPE-19 and 0.2 mM for B4G12) in the paraquat-treated groups (P only and C + P groups) for five days. (A) Paraquat-induced cellular accumulation of reactive oxygen species (ROS) and rescue by ascorbic acid was detected using ROS fluorescent dye (red color). ROS was induced by paraquat in both the B4G12 and ARPE-19 cells, and this was ameliorated by treatment with ascorbic acid. (B) Paraquat-induced apoptosis and rescue by ascorbic acid was examined using the TUNEL assay (green). (C) Paraquat-induced autophagosome formation and rescue by ascorbic acid was examined using immunofluorescence staining for LC3-II (autophagosome formation biomarker; green). (D) Effects of the paraquat and ascorbic on the protein expression of anti-apoptosis (Bcl-2), pro-apoptosis (lamin A, including cleaved forms), and autophagic flux (LC3 I/II and p62) biomarkers in B4G12 and ARPE-19 cells were probed using Western blotting. Paraquat induced altered protein expression in both B4G12 and ARPE-19 cells, and this could be reversed by ascorbic acid. The scale bars represent 100 µm (A) and 50 µm (B–C). Nuclei were counterstained with Hoechst 33342 (blue; B–D). (n = 3, * p < 0.05, ** p < 0.01).