Figure 3.

ATG depletion enhances apoptosis in ECM-detached cells. (A) MCF-10A cells transfected with pooled nontargeting control (siControl) or siRNA oligonucleotides against ATG5, ATG6 (Beclin 1), or ATG7 were lysed and immunoblotted with α-ATG12 to detect the ATG5:12 complex, α-Beclin, α-ATG7, and α-tubulin. (B) Cells transfected with the indicated siRNAs were grown attached (A) or suspended (S) for 24 h, lysed, and subjected to α-LC3 and α-tubulin immunoblotting. LC3-I detection was minimal in this experiment. (C) Confocal images of MCF-10A cells transfected with the indicated siRNAs, suspended for 24 h, fixed, and immunostained with α-cleaved caspase-3 antibody (green). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Bars, 20 μm. (D) Percentage of cleaved caspase-3–positive cells in MCF-10A cells transfected with the indicated siRNAs and grown attached (white) or suspended (black) for 24 h. Results are the mean ± SEM of five or more independent experiments; in each experiment, 200–300 cells were analyzed. (E) Clonogenic replating efficiency of cells transfected with the indicated siRNAs after 48-h attachment (white) or suspension (black). Replating efficiency was calculated as the number of colonies formed divided by the number of cells originally replated. Results are the mean ± SEM from three to five independent experiments.