Figure 4

Vesicles forming from mitochondria are autophagosomes. A. LCC9 cells were transfected with GFP-LC3, treated with 0.1% ethanol vehicle, 500 nM ICI, ERα shRNA, or 10 μM Imatinib for 24 hours and counterstained with DAPI. Confocal microscopy was used to determine LC3-positive puncta formation. B. LCC9 cells were treated with 0.1% ethanol vehicle, 100 nM ICI, ERα shRNA, or 10 μM Imatinib for 72 hours and protein isolated. Western blot hybridization was used to determine LC3-I/LC3-II and p62 levels. C. LCC9 cells were treated with vehicle, 500 nM ICI, serum starvation, 2 ng/mL tunicamycin, 10 μM Imatinib, or transfected with ATG7 siRNA, PARK2 (parkin) siRNA, or ERα shRNA for 24 hours. Autophagosome (modified monodansylcadaverine) and mitochondria (MitoTracker-GFP) fold change was determined by flow cytometry. D. LC3-immunogold EM of LCC9 cells. M indicates mitochondria; Av indicates an autophagic vesicle; * indicates mitochondria-autophagosome interaction; arrows indicate LC3-immunogold particles. E. Mitochondria were counted and represented as percent of mitochondria either labeled with LC3-immunogold or unlabeled. n = 3-4, *p < 0.05.