Knockdown of Beclin1 rescued Bcl-2 knockdown-induced cell death in response to starvation.

<p>A and B: Western blot analysis of Beclin1 expression in SH-SY5Y cells 72 hr after transfection and quantitative analysis of optical densities of Beclin1 protein bands with Sigma Scan Pro 5 and normalized to the loading control (B). Bars represent Mean ± SD (n = 3). Statistical analysis was carried out with ANOVA followed by Dunnett t-test (*p<0.05). C and D: After Beclin1 was knocked down, the cells were subjected to serum starvation for another 12 hrs, and then Beclin1 and LC3 were detected (C) and quantitative analysis of optical densities of the LC3-II/LC3-I protein bands with Sigma Scan Pro 5 (D, **p<0.01). Bars represent Mean ± SD (n = 3). C: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were co-transfected with Bcl-2, Beclin1 or Bcl-2/Beclin1 siRNA for 72 hrs, and the cells were subjected to serum starvation for 12 hrs or 24 hrs in the presence or absence of 50 µM Z-VAD. ***P<0.001 represent the indicated groups vs Bcl-2 RNAi #6 starvation 12 hrs group; ###p<0.001 represent the indicated groups vs Bcl-2 RNAi #6 starvation 24 hrs group. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. Data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.