Figure 4.

SQSTM1 mediates pexophagy in HSPA9-depleted cells. (A-C) HeLa cells were transfected with scrambled siRNA (Sc) or HSPA9-targeting siRNA (siHSPA9) in the presence or absence of siRNAs against the receptor proteins SQSTM1 and NBR1 (siSQSTM1 and siNBR1). (A) After 5 d, nuclei were stained with DRAQ5 and peroxisomes were stained with anti-ABCD3 antibody. Then the cells were imaged by confocal microscopy (left). The efficient knockdown by the siRNA was confirmed by western blotting (right). (B) The number of peroxisomes per cell was calculated by assessing approximately 100 cells. (C) Cells were further analyzed by western blotting with the indicated antibodies. (D-F) and sqstm1 knockout MEFs were transfected with scrambled siRNA (Sc) or Hspa9-targeting siRNA (siHspa9). (D) After 5 d, the cells were stained with DRAQ5 (red) and an anti-ABCD3 antibody (green) and imaged by confocal microscopy. (E) The number of cells exhibiting pexophagy was counted and the cells were analyzed by western blotting with the indicated antibodies (F). Data are presented as the mean ± SEM (n = 3, * p < 0.05). Scale bar: 5 µm