Figure 2.

Depletion of HSPA9 induces pexophagy in HeLa cells. (A) HeLa cells were transiently transfected with scrambled siRNA (Sc) or HSPA9-targeting siRNA (siHSPA9) for 5 d and then analyzed by western blotting with the indicated antibodies. (B and C) HSPA9-depleted HeLa cells were further incubated in the presence or absence of bafilomycin A₁ (Baf A1), and then the level of LC3 conversion and ABCD3 were analyzed by western blotting. The ratio of LC3-II to LC3-I and ABCD3 expression were determined using a densitometer (bottom). (D and E) HeLa cells stably expressing dKeima-PTS1 (HeLa/Pexo-Keima) were transfected with scrambled siRNA (Sc) or HSPA9-targeting siRNA (siHSPA9) for 4 d or treated with 1,10-phenantholoine (Phen, 50 μM) for 2 d. Thereafter, the cells were fixed and imaged with a confocal microscopy by different excitation wave length (D), and analyzed by flow cytometry (E). Green fluorescence of Pexo-Keima cells represents peroxisomes in the cytosol, whereas red fluorescence reflects peroxisomes in lysosomes. The data are presented as the mean ± SEM (n = 3, * p < 0.05). Scale bar: 5 µm