Fig. 3. Knockdown of Beclin 1 blocks PP2-induced autophagy. (A) Ras-NIH 3T3 cells and Ras-NIH 3T3/Mdr cells were transiently transfected with Beclin 1-specific esiRNA (esiBecn1). Twentyfour hours post-transfection, cells were incubated for the indicated times at 37℃ with 5 μM PP2. Cell lysates were prepared, and Western blotting was performed with an antibody against Beclin 1 to analyze the extent of expression knockdown. The β-actin protein was used as a loading control. In (B) & (C), Ras-NIH 3T3 cells (B) and Ras-NIH 3T3/Mdr cells (C) were transiently transfected with GFP-LC3. The percentage of cells showing autophagy was quantified (mean ± SD) by counting the number of cells expressing the punctate pattern of GFP-LC3 among 200 GFP-positive cells. ^**p<0.01 as determined by Dunnett’s T-test compared with the mock-transfected group. The results presented are representative of at least three independent experiments.