Figure 2

BA145 triggers autophagy and suppresses VEGFR signaling in cancer cells. (A) Western blot analysis of the indicated proteins in VEGF activated PC-3 cells with or without BA145 treatment for 24 h. (B) Western blot analysis of the expression of the autophagy marker proteins LC3 and p62 in BA145 treated PC-3 cells and HUVECs after 24 h. (C) Detection of acidic autophagic vesicles in PC-3 cells and HUVECs. After 24 h treatment with BA145, cells were stained with acridine orange (1 μg/ml) in serum free media for 15 min and fluorescent micrographs were obtained by microscopy. Autophagy is indicated by the red fluorescence while the untreated control cells are green. (D) Detection of LC3-II protein in PC-3 cells by immunofluorescent microscopy. (E) Quantification of acridine orange positive cells by flow cytometry. PC-3 cells were treated with BA145 at the indicated concentrations for 24 h, stained with acridine orange (1 μg/ml) for 15 min, and analyzed with the FL3-H (red color intensity) and FL1-H (green color intensity) channels of the flow cytometer. (F) Time dependent accumulation of LC3-II and loss of p62 in BA145 treated PC-3 cells and HUVECs.