Fig. 7.

Mitophagy suppression causes alterations in mitochondrial functions. (A) Mitochondria membrane potential in ART-treated HeLa cells after PINK1 knockdown. The cells were transiently transfected with the PINK1-specific siRNA and then treated with ART (10 µM, 12 h). Cells were incubated with JC-1 (10 μg/ml) to assess mitochondrial membrane potential as described. Fluorescence of J-aggregates (red) and J-monomers (green) was detected under confocal microscope. Scale bar: 10 µm. (B) Glycolysis level of ART-treated HeLa cells after PINK1 knockdown. The cells were treated as indicated in (A). Cellular lactate levels were measured in cells as reported. The numeric data was analyzed using Student's t-test. * P < 0.05, ** P < 0.01. (C) Mitochondrial ROS level in ART-treated HeLa cells after PINK1 knockdown. The cells were treated as indicated in (A). Mitochondrial ROS were measured in the cells as reported. The numeric data was analyzed using Student's t-test. * P < 0.05, ** P < 0.01. (D) Effects of PINK1 knockdown on ART-induced cell death. HeLa cells were transiently transfected with a nonspecific siRNA or the PINK1-specific siRNA. After 72 h, cells were treated with ART (20 μM) for 24 h and morphological changes of HeLa cells with respective treatments were examined and photographed with an inverted microscope. Scale bar: 500 µm. (E) Cellular apoptosis of ART-treated HeLa cells after PINK1 knockdown. The cells reflected in (D) were harvested and resuspended in 1x annexin-binding buffer. Cells were then labeled with Pacific Blue™ Annexin V (5 µL) and cell fluorescence emission was measured. The numeric data was analyzed using Student's t-test. * P < 0.05, ** P < 0.01.