Figure 5.

Depletion of HSPA9 increases the level of peroxisomal ROS. (A and B) HeLa cells were transfected with scrambled siRNA (Sc) or HSPA9 siRNA (siHSPA9) for 4 d, treated with or without NAC (1 mM). Subsequently the cells were incubated with DCFH-DA to obtain fluorescence images (A) and the fluorescence intensity was measured using a microplate reader (B). (C and D) HeLa cells stably expressing HyPer-PTS1 (HeLa/HyPer-PTS1) were transfected with Sc or siHSPA9. After 3 d, the level of peroxisomal ROS was monitored under a fluorescence microscope in the presence or absence of NAC. Scale bar 20 µm. (E and F) HeLa cells were transfected with Sc or siHSPA9 for 4 d, treated with or without NAC (1 mM), stained with DRAQ5 (red) and anti-ABCD3 antibody (green), and imaged with a confocal microscope (E). (F) The number of peroxisomes per cell was calculated by assessing approximately 100 cells. Data are presented as the mean ± SEM (n = 3, * p < 0.05). Scale bar: 5 µm