Figure 4
Autophagy inhibition potentiates the anti-angiogenic effects of BA145. (A) PC-3 cells were treated with BA145 (30 μM) along with autophagic inhibitors for 16 h. Whole cell protein lysates were prepared for western blotting, and the detection of the indicated proteins. (B) LC3 specific siRNA were used to silence the expression of LC3 in PC-3 cells and HUVECs that were further treated with BA145 for 12 h. Protein lysates were analyzed by western blotting for the indicated proteins. In the case of HUVECs, prior treatment of VEGF (20 ng/ml) for about 40 min was given before the addition of BA145 (7 μM). For HIF-1α expression in PC-3 cells, VEGF was added 30 min before the BA145 treatment as PC-3 cells have less endogenous HIF-1α expression. (C) Effect of BA145 on VEGF production in PC-3 cells. Cells were seeded in 6 well plates, grown to 90% confluency, and then exchanged with fresh media containing BA145 for 16 h. Secreted VEGF in the culture media as well as intracellular VEGF were measured by ELISA and western blot, respectively. Columns, mean; bars, SD; with ***p < 0.001, **p < 0.01, *p < 0.05 versus control. (D) Effect of autophagic inhibitors on VEGF production in BA145 treated PC-3 cells under hypoxic and normoxic conditions. Hypoxia was created by treating cells with 100 μM cobalt chloride (CoCl2) for 24 h. After incubation, the media was replaced and cells were treated with BA145 (30 μM) and autophagic inhibitors for the next 16 h. Extracellular VEGF in the media was measured by ELISA. Columns, mean; bars, SD; with **p < 0.01 versus BA145 alone. (E) Western blot analysis of the indicated proteins following treatment of PC-3 cells with BA145 and autophagy inhibitors under normoxic and hypoxic conditions.