Analysis of TOM20 ubiquitination, as well as levels of TOM20, TIM23, and TOM40 in the presence or absence of A/O treatment in Hela cells expressing Parkin or Parkin/Myc-tagged USP30. Shown is one representative western blot from three independent experiments, all providing similar results. (b) Chemical structure of ST-539. (c) Representative immunoblotting for TOM20 TOM40 and TIM23 in Hela cells expressing Parkin or Parkin/Myc-tagged USP30. Prior to A/O treatment, cultures were treated with ST-539 at indicated concentrations. (d) Representative FACS analysis of AO-induced mitophagy using mt-Keima fluorescence. Cells were treated with A/O for 2h and analyzed by FACS for lysosomal positive mt-Keima (pH4). Prior to A/O treatment, cultures were treated with DMSO or ST-539(3μg/ml).