Figure 3abcd.

Beclin-1-dependent autophagy stimulated by ABT737. (A, B) Detection of autophagic vacuoles by LC3-GFP and their modulation by ABT737 and by Beclin-1-specific siRNAs. HeLa cells were transfected with control or Beclin-1-specific siRNAs, 24 h later re-transfected with LC3-GFP, cultured in complete medium (CM) for 24 h, and finally kept 12 h either in CM or in nutrient-free (NF) conditions, in the presence or absence of 1 μM ABT737. Representative microphotographs of cells cultured in NF medium are shown in (A) and the percentage (means±s.d., n=3 separate experiments) of LC3-GFP-transfected cells bearing LC3-GFP aggregates in the cytoplasm (LC3-GFPvac) are quantified in (B). The insert in (B) demonstrates the efficiency of the Beclin-1-specific siRNAs, as quantified by immunoblot. (C, D) Detection of cytoplasmic vacuoles using chloromethylfluorescein diacetate (CMFDA). Cells were transfected with control or Beclin-1-specific siRNAs, cultured for 48 h in CM, washed, cultured in CM (D) or NF (C, D) for 12 h, stained with CMFDA, and either photographed (C) or subjected to the quantification of the cells that bear at least one discernible cytoplasmic vacuole (arrow head) (means±s.d., n=3 separate experiments).