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Fig. 3. Rotenone and TTFA induce autophagic cell death in HEK 293 and U87 cells. Cells were treated with 50.0 µM rotenone (R); 0.5 mM TTFA (T); 3-MA, 2.0 mM (autophagy inhibitor); and/or zVAD, 0.1 mM (caspase inhibitor). (A) Effect of autophagy and apoptosis inhibitors on rotenone- or TTFA-induced cell death after treatment for 48 hours was determined. HEK 293 or U87 cells were treated with TTFA or rotenone alone or with each in combination with 3-MA, zVAD or both. Etoposide (100 µM) was used as an apoptotic stimuli. The amount of cell death was determined by membrane permeabilization, as described in the Materials and Methods section. (B) The amount of apoptotic cell death was determined by sub-G1 peak or TUNEL analysis. HEK 293 or U87 cells were treated with rotenone, TTFA or etoposide and the percentage of apoptotic cells was determined. (C) Effect of siRNAs against the autophagic genes beclin 1 and ATG5 on rotenone- or TTFA-induced autophagy, cell death and apoptosis in U87 cells after a treatment of 48 hours was determined. U87 cells were not transfected (non siRNA) or were transfected with scrambled siRNA or siRNAs against beclin 1 or ATG5. (i) Cells were lysed and western blotted for beclin 1 and ATG5. Blots were stripped and re-probed with anti-actin antibody for equal loading. (Cii,Ciii,Di,Dii) The effects of siRNA against beclin 1 and ATG5, and of scrambled siRNA, on AVO formation (Cii), GFP-LC3-labeled vacuoles (Ciii), cell death (Di) and apoptosis (formation of sub-G1 peaks) (Dii) following rotenone or TTFA treatment were determined. Error bars represent s.e. from three independent experiments. * Represents significant difference from control conditions (P<0.05). # (A) Represents a lack of significant difference from control conditions (P>0.05).
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