Fig. 2: Reducing cytosolic calcium elevation suppresses the appearance of LC-3-positive autophagosomes caused by MPP+ treatment.

MN9D cells were treated with 50 μM MPP+ in the presence or absence of the indicated concentrations of BAPTA-AM for 30 h. a Cell lysates were subjected to immunoblot analyses using the anti-LC3 antibody. Anti-GAPDH antibody was used as a loading control. b–d Immunocytochemical analyses were performed using an anti-LC3 antibody (green) and nuclei counterstaining with Hoechst 33258 (blue). b Cells were then examined using a confocal microscope. Merged images are provided. The scale bar represents 10 μm. The number of LC3 puncta per cell (c) and average puncta area of LC3 (d) were quantified using ImageJ after examining 90 cells per condition. The bar represents the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; NS, not significant