Figure 5
8-Cl-Ado-induces autophagy. Immunoblot analysis of (A) phospho-ULK (Ser555) levels and (B) LC3B-I lipidation to form LC3B-II in MCF-7 and BT-474 cells treated with 10 μM of 8-Cl-Ado for the indicated times. (C) Fluorescent microscopy of MCF-7 and BT-474 cells transiently transfected with GFP-LC3B and treated with 10 μM of 8-Cl-Ado for 12-hours to assess aggregation of LC3B. (D) Immunoblot analysis of p62 levels in MCF-7 and BT-474 cells treated with 10 μM of 8-Cl-Ado for the indicated times. The normalized ratio of p62 to GAPDH relative to the untreated control is indicated beneath each lane. (E) Fluorescent microscopic imaging of autolysosomes. MCF-7 cells were untreated or treated for 2-days with 8-Cl-Ado or rapamycin. Cells were stained with MDC, blue, to visualize AVO and Syto 61 (DNA), red, for nuclei counterstaining. (F) Representative flow cytometery histograms of AVO stained with acridine orange in MCF-7 cells untreated or treated for 3 days with 50 nM rapamycin or 10 μM 8-Cl-Ado. Baf was added before 30 min prior to staining to neutralize AVO staining. (G) Quantification triplicate experiments of MCF-7 and BT-474 cells treated and analyzed as in E.