Figure 6.

Depletion of HSPA9 induces pexophagy in neuroblastoma cells. (A-C) SH-SY5Y cells were transfected with scrambled siRNA (Sc) or HSPA9-targeting siRNA (siHSPA9) in the presence or absence of ATG5- or SQSTM1-targeting siRNA (siATG5 and siSQSTM1, respectively). After 5 d, the cells were stained with DRAQ5 and anti-ABCD3 antibody to get images with confocal microscopy (A). The number of peroxisomes per cell was calculated (B). The cells were further analyzed by western blotting with indicated antibodies (C). (D) SH-SY5Y cells stably expressing dKeima-PTS1 (SY5Y/Pexo-Keima) were transfected with Sc or siHSPA9 for 4 d. The cells were analyzed by flow cytometry. (E and F) SH-SY5Y cells stably expressing HyPer-PTS1 (SY5Y/HyPer-PTS1) were transfected with Sc or siHSPA9 and treated with or without NAC (1 mM). After 3 d, peroxisomal ROS were imaged by fluorescence microscopy (E) and the fluorescence intensity was measured (F). Data are presented as the mean ± SEM (n = 3, * p < 0.05). Scale bar: 5 µm