Figure 3

EA induces autophagy in A498 cells. A498 cells were treated with 200 nM EA or 0.1% DMSO (control) for 46 h and with 500 nM rapamycin for 20 h. Autophagy was measured by staining autolysosomes and earlier autophagic compartments with the fluorescent probe Cyto-ID^® Green. Samples were then analyzed in the green (FL1) channel of the FACS Caliber flow cytometer (A). Cells were treated with 200 nM EA or 0.1% DMSO (control) for 45 h and then stained with Hoechst nuclear stain and Cyto-ID^® Green detection reagent followed by fixing with 4% formaldehyde. The stained cells were then analyzed by fluorescence microscopy. Panels a and c show cells treated with 0.1% DMSO and panels b and d show cells treated with EA. Nuclei are stained in blue. Autolysosomes and earlier autophagic compartments are stained in green (B). A498 cells were treated with 200 nM EA or with 0.1% DMSO (control) for 48 h and protein was extracted. Western blot analysis was performed using an anti-LC3B antibody. B-actin was probed as a control for protein loading (C).