Fig. 7

NEO212 blocks TFEB nuclear translocation. a-b A2780 and SK-O-V3 cells were treated with 100 μM NEO212 or not and stained with anti-TFEB antibody and imaged by confocal microscopy (a). Scale bars: 10 μm. Statistical analysis of the colocalization coefficient of LAMP1 and LC3B using Fiji Image J (b). c A2780 cells expressing Flag-TFEB were starved in present or absent with 100 μM NEO212 underwent cytoplasmic and nuclear extraction. Then the cytoplasmic and nuclear lysates were subjected to western blot analysis for demonstrating Flag-TFEB expression using anti Flag antibody. GAPDH acts as control for cytoplasmic protein, Lamin B1 acts as control for nuclear protein; (d) 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 p-AKT, AKT, p-ERK, and ACTB expression in above drug treated cells. The results shown are means ±SD, ***p < 0.001