Figure 6

8-Cl-Ado-induces autophagic cell killing. (A) Western blot analysis of beclin1 and ATG7 levels in MCF-7 cells transfected with either a pool of control siRNA (siCONT), siRNA targeting the expression of the beclin1 gene (siBECN1), or targeting the expression of the ATG7 gene (siATG7). Immunoblot analysis of LC3B lipidation and PARP cleavage were assessed as markers of autophagosome formation and apoptosis, respectively. GAPDH was used as loading control. Flow cytometric analysis of cells transfected with siCONT, solid bars, siBECN1, hatched bars, or siATG7, checkered bars, treated with 10 μM 8-Cl-Ado and stained with (B) annexin V and PI, as well as (C) acridine orange. Effect of autophagy on 8-Cl-Ado-inhibiton of clonogenic survival. Cells transfected with (D) siCONT, ○, or siBECN1, ●, and with (E) siCONT, ○, or siATG7, ●, were treated with the indicated doses of 8-Cl-Ado for 3 days, washed with PBS, and cultured in fresh medium for 10 days. Colonies of >50 cells were counted under a dissecting microscope.