Fig. 2: Biochemical characterization of autophagy induced by 6-OHDA in MN9D cells.

a Cells were treated with or without 100 μM 6-OHDA for the indicated time periods. Immunoblot analyses were performed using anti-LC3 and anti-cleaved caspase-3 (c-cas-3) antibodies. Anti-GAPDH antibody was utilized as loading control. b The intensity of LC3-II signals at each timepoint was densitometrically measured using ImageJ, normalized by the intensity of GAPDH signal, and expressed as fold change relative to the untreated control value. Bars represent the mean ± standard deviation of three independent experiments (6 h, 2.7 ± 0.5; 12 h, 5.8 ± 0.6; 24 h, 9.7 ± 0.2). *P < 0.05; **P < 0.01; ***P < 0.001. c Cells treated with or without 100 μM 6-OHDA for the indicated time periods were subjected to immunocytochemical analyses using an anti-LC3 antibody (green) and nuclei counterstaining with Hoechst 33258 (blue). Cells were then examined under a confocal microscope. Merged images are provided in the right panels. Scale bars represent 10 μm. d, e The number (d) and area (e) of LC3 dots per cell were quantified using ImageJ in 100 μM 6-OHDA-treated cells for 12 h. Data are shown as the mean ± standard deviation of three independent experiments (number of LC3 dots, 2.0 ± 0.9 for control vs. 19.4 ± 0.7 for 6-OHDA-treated cell; area of LC3 dots, 0.2 ± 0.1 for control vs. 4.9 ± 0.1 for 6-OHDA-treated cell). ***P < 0.001