Inhibition of autophagy by 3-MA at an early stage and by BafA1 at a later stage can be distinguished by individual AV analysis with OFACS.

Untransfected PC3 cells (A,B) or PC3 cells expressing mCherry-eGFP-LC3B (C) treated with 1 µM GDC-0941 or 5 µM GDC-0068 +/− 10 µM CQ for 24 hours with the addition of: nothing (upper panels), 0–10 mM 3-MA (middle panels) or 0–100 nM Bafilomycin A1 (BafA1; bottom panels). AVs were analyzed by OFACS after staining with AO (A,B) or unstained (C). Y axes represent the total “red” channel intensity of AO⁺ organelles normalized to cell number (A,B) or the percentage of mCherry⁺eGFP⁺ events of the total subcellular events (C). Note the different scales of the y-axes in (A) vs. (B). Error bars represent standard deviations (SD), n = 3.