Fig. 6

NEO212 blocks autophagy flux. a A2780 cells expressing mRFP-GFP-LC3 were starved or treated with 1 nm Baf.A1 or 100 μM NEO212 for 48 h and imaged by confocal microscopy. Scale bars: 10 μm. Statistical analysis of the percentage of puncta mRFP signals that were positive for GFP using Fiji Image J. b Cells were treated with 100 μM TMZ, POH, TMZ + POH, NEO212 or DMSO respectively for 48 h in A2780 and SK-O-V3 cells. Western blot analysis demonstrated SQSTM1, EEA1, LAMP1, LAMP2, TFEB and ACTB expression. c A2780 cells were either starved or treated with NEO212, and stained with anti-LAMP1 and anti-LC3B antibodies and imaged by confocal microscopy. Scale bars: 10 μm. Statistical analysis of the colocalization coefficient of LAMP1 and LC3B using Fiji Image J. The results shown are means ± SD; ***p < 0.001, NS = no significance