Figure 1.

Depletion of HSPA9 induces loss of peroxisomes in HeLa cells. (A and B) HeLa cells stably expressing turquoise2-Peroxi (HeLa/Peroxi) were transfected with scrambled siRNA (Sc) or HSPA9 -targeting siRNA (siHSPA9). (A) After 5 d, the cells were stained with DRAQ5 dye and imaged by confocal microscopy, and (B) the number of peroxisomes per cell was counted. 1,10-phenanthroline (Phen, 50 μM) was used as a control for induction of pexophagy. (C) HeLa cells were transfected with scrambled siRNA (Sc) or 2 different HSPA9-targeting siRNAs (siHSPA9 #1 and #2). After 5 d, the cells were harvested and analyzed by western blotting with the indicated antibodies. (D) HeLa cells stably expressing turquoise2-Peroxi, mitochondria-YFP, turquoise2-ER, or turquoise2-Golgi were transfected with Sc or siHSPA9 for 5 d, stained with DRAQ5, and fixed. Cellular organelles were imaged by confocal microscopy. (E) HeLa cells transfected with Sc and siHSPA9 were assessed by western blotting with antibodies for protein marker of subcellular organelles (ABCD3, peroxisome; TOMM20, mitochondria; P4HB, endoplasmic reticulum; FTCD, Golgi). Data are presented as the mean ± SEM (n = 3, * p < 0.05). Scale bar: 5 µm