(B) Depolarized mitochondria are selectively targeted for autophagy after a multi‐hour time lag. APs are labeled with LC3:GFP, which translocates from the cytosol to the AP's isolation membrane. MTR, a membrane potential dye that stains mitochondria irreversibly and is retained during depolarization, is used to pulse label mitochondria in INS1 cells (14 min, 50 nM) at different time points before detection of APs content. At the time set for detection, cells were treated with pepstatin A (10 μM) and E64d (10 μM) for 30 min to arrest digestion inside the APs, and then subjected to confocal microscopy (see schematic illustration of the Materials and methods). MTR pulse is used here to report on Δψm during the staining period. While mitochondria outside APs show bright MTR fluorescence, those localized in APs varied in fluorescence based on the time at which they were pulsed with MTR. Note that mitochondria inside APs have dim MTR FI if they were pulsed with MTR 1 h before autophagy was detected (circle, top panel), but bright MTR FI if pulsed 24 h before detection of autophagy (circle, bottom panel). Scale bar, 10 μm. 