3-MA-induced cell death occurred independently of the inhibition of autophagy.

<p>(A) Immunoblot showing the efficiency of beclin1 silencing in HeLa cells. Cells were transiently transfected with negative control (NC) siRNAs or siRNAs specific for beclin1 mRNAs. The cells were collected and subjected to immunobloting 48 hours post-transfection. The blot shown is representative of three independent experiments. (B) Beclin1 KD did not cause cell death in HeLa cells. Cells were transiently transfected with negative control (NC) siRNAs or siRNAs specific for beclin1 mRNAs. Cell viability was determined using a trypan blue exclusion assay at 24, 48 and 72 hours post transfection. (C) Beclin1 KD had no effects on 3-MA-induced cell death. Twenty four hours post transfection with NC siRNAs or siRNAs against beclin1, HeLa cells were treated with 0, 5 or 10 mM 3-MA. Cell viability was determined 48 hours post treatment using trypan blue exclusion assay. (D) Immunoblot showing atg5 and LC3 levels in atg5+/+ and atg5−/− MEFs. (E) Atg5+/+ and atg5−/− MEFs were treated with 0, 5 or 10 mM 3-MA. Then cell viability were determined 48 h post treatment using trypan blue exclusion assay. At least 300 cells were counted for each treatment to determine cell viability and bars represent the standard deviations from three independent experiments.