Figure 3. Drug interactions promoting eIF2α-dependent autophagosome formation and autophagic flux. (A) HCT116 ATG16L1 T300 and GBM6 cells were transfected with a plasmid to express LC3-GFP-RFP and in parallel with a scrambled siRNA or with siRNA molecules to knock down the expression of eIF2α or AMPKα1. After 24h, cells were treated with vehicle control, AR12 (2 μM), neratinib (50 nM), MMF (5 μM), fingolimod (FTY, 100 nM) or the drugs in combination as indicated in the graphs. Randomly cells (> 50 per data point) were examined 4h and 8h after drug exposure and the mean number of GFP+ and RFP+ intense staining punctae determined under each condition (n = 3 +/-SD) * p < 0.05 less than corresponding value after 4h; # p < 0.05 greater than corresponding value after 4h; ¶ p < 0.05 less than corresponding value in siSCR cells. (B) HCT116 ATG16L1 T300 cells were transfected with a plasmid to express wild type Tau-GFP. After 24h, cells were treated with vehicle control, AR12 (2 μM) or neratinib (50 nM). Cells were fixed after 1h. Cells were fixed in place and the detection of Tau levels determined using GFP tag fluorescence (10X).