Figure 3.

Hypoxia-induced cell death is blocked by knock-down of Beclin-1 and ATG5. HEK293 cells were untransfected (control) or transiently transfected with siRNA against beclin-1 or Atg5, or a non-targetting control siRNA. (A) Western blot analysis with β-actin as a loading control at 72 hours post-transfection. Numerical values indicate protein quantification by densitometry, normalized to β-actin. (B) 72 hours post-transfection, cells were incubated in normoxia or hypoxia for a further 48 hours. Autophagy was assessed by detection of AVOs as described. (C) (Top) Total cell death was determined by trypan blue membrane permeability assay using flow cytometry as described in Materials and Methods. Red fluorescence yields two observable peaks, representing viable cells (first peak – weak fluorescence) and dead cells (second peak, unable to exclude the dye and therefore strongly fluorescent). (Bottom) Quantification of trypan blue assay using CellQuest software, representing four independent experiments. Error bars indicate standard deviation; statistical analysis was by unpaired student’s t-test: *p<.05, **p<.01, ***p<.001.