Fig. S1.

(A) HeLa cell loaded with 30 nM TMRE displays high fluorescence intensity before addition of 20 μM CCCP. Within 2 min, the majority of mitochondria have been depolarized, resulting in decreased TMRE fluorescence. By 6 min, all mitochondria have expelled the TMRE dye. (B) Confocal time series showing individual mitochondria labeled with TMRE and Mito-SNAP, a genetically encoded mitochondrial marker that is insensitive to membrane potential. Before CCCP treatment, mitochondria are double-labeled with Mito-SNAP and TMRE, and YFP-Parkin is cytosolic. Within 1 min of CCCP treatment, the mitochondria can still be visualized by Mito-SNAP, but TMRE is expelled from the mitochondria (white arrow). Forty min later, Parkin is recruited to the depolarized mitochondria (yellow arrows). (C) Normalized intensity of Mito-SNAP and TMRE after 20 μM CCCP addition (dashed line). (D) CCCP treatment for 180 min fails to induce mitophagy in HeLa cells due to low levels of endogenous Parkin. [Scale bars: A and D (full size), 10 μm; B and D (zoom), 2.5 μm.]