Figure 4.

Angiogenesis induction reverses Ery5-induced autophagic cell death in PC-3 cells. (a and b)VEGF and Cocl2 improve viability in PC-3 cells. PC-3 cells (6 × 10³ per well) were plated in 96-well plates. Cells were allowed to attach for 24 h, VEGF (20 ng/ml) and Cocl2 (100 μM/ml) were treated 30 min before treatment of Ery5 for 6, 12 and 24 h. MTT dye was added 3 h before termination of the experiment and MTT crystal were dissolved in 150 μl of DMSO. OD was measured at 570 nm. Percent viability was calculated as described in Materials and Methods section. Data are mean±S.D. of three similar experiments; statistical analysis was done by using the Bonferroni method and P-value<0.05 was considered to be significant with *P<0.05 and ***P<0.001. (c and d) Angiogenesis induction through VEGF and Colc2 inhibited autophagy in PC-3 cells. PC-3 cells (1 × 10⁶) were seeded in 60 mm dishes, cells were allowed to attach followed by treatment with VEGF (20 ng/ml) and Cocl2 (100 μM/ml) 30 min before treatment of Ery5 for indicated time periods. After treatment for various indicated time periods, cells were detached and lysed in RIPA buffer; proteins were separated on SDS-PAGE and transferred to PVDF membranes. Membranes were probed with different antibodies. The detailed procedure is described in Materials and Methods. Quantification for LC3-II was done using image J software. Data are mean±S.D. of three similar experiments; statistical analysis was done by using the Bonferroni method and P-value<0.05 was considered to be significant with **P<0.01 and ***P<0.001