Figure 3: ET-1 stimulatory and ETAR inhibition effects on MCF-7 and MDA-MB-231 cells. Cells were serum deprived for 24 hours and then treated with ET-1 for the indicated times. Resulting cellular lysates were subjected to SDS-PAGE and Western blotting with the indicated antibodies (a). Cells (serum deprived for 24 hours) were treated with ET-1 for 15 minutes, then stained with p-Akt antibody and imaged by confocal microscopy with p-Akt staining (top) or phase contrast (bottom) (b). Silencing of ETAR by siRNA showed decreased ETAR protein by Western blot (c). Apoptosis in both cell lines was determined by flow cytometry using Annexin V and propidium iodide (PI) labeling (d). In the untreated control samples (left upper image for MSF-7 and left lower image for MDA-MB-231), the majority of cells were nonapoptotic (Annexin V−/PI− population). Silencing of ETAR decreased population of nonapoptotic cells and increased population of cells undergoing early apoptosis (Annexin V+/PI−) and late apoptosis (Annexin V+/PI+) as depicted in the images on the right.