The AO staining to show inhibition of rapamycin-induced autophagy in SK-N-BE2 and IMR-32 cells after LC3 shRNA plasmid transfection and GST treatment.

Here, cells treated with 200 nM rapamycin for 24 h were used as the treated control (CTL). Then, single and combination therapies were performed for another 24 h. Treatment groups: treated CTL, CTL shRNA plasmid, LC3 shRNA plasmid, GST, LC3 shRNA plasmid + GST, and untreated CTL. We used 50 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in SK-N-BE2 cells while 100 nM CTL or LC3 shRNA plasmid and 25 µM GST alone or in combination in IMR-32 cells. (a) Staining of cells with AO followed by fluorescence microscopy for detection of AVO in autophagic cells. (b) Flow cytometric analysis of the AO stained cells from all treatment groups for detection and determination of AVO in autophagic cells. (c) Presentation of amounts of autophagic cells in bar diagrams. Significant difference between treated CTL and another treatment or untreated CTL was indicated by *P<0.05 while significant difference between single treatment and double treatment was indicated by #P<0.05.