Figure 3.

Autophagy inhibitor PIKIII or silencing of LC3B blocked the protective effect of BBR in D407 cells. (a–d) D407 cells were pretreated with 5 μM PIKIII for 2 h and 6 μM BBR for 2 h and then incubated with or without H₂O₂ for a further 24 h. Cell viability was measured by MTT assay, and LC3B, P62, and β-actin were detected by western blotting with specific antibodies. D407 cells were pre-treated with 5 μM PIKIII for 2 h and 6 μM BBR for 2 h and then incubated with or without H₂O₂ for a further 24 h. Apoptotic cells were observed by flow cytometry of PI-Annexin-FITC (e, f) and TUNEL staining (scale bar = 100 μm) (g, h). (i) D407 cells transfected with si-CTRL and/or si-LC3B; the expression of LC3B was detected by western blotting with specific antibodies. (j) D407 cells transfected with si-CTRL and/or si-LC3B; then, cells treated with BBR (6 μM) for 2 h were exposed with or without H₂O₂ (100 μM) for 24 h in 96-well plate. Cell viability was measured by MTT assay. The assay was repeated for at least 3 times. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 were considered significantly different.