Figure 3.

Autophagy inhibition reversed the expression of angiogenic protiens and improves cell death in PC-3 cells. (a) Inhibition of autophagy through 3-MA improves viability inhibited by Ery5 in PC-3 cells. PC-3 cells were plated in 96-well plates, and 3-MA (2 mM) was added 30 min before the addition of Ery5 for 6, 12 and 24 h. Cell viability was assessed through MTT assay as described in Materials and Methods. 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<0.001. (b) 3-MA reversed the expression of angiogenic proteins. PC-3 cells were pretreated with 3-MA (2 mM) before treatment with 10 μM of Ery5 for indicated time periods; cells were collected and lysed using RIPA buffer. Western blot analysis for indicated proteins was done as 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<0.001. (c) Autophagy inhibition through ATG7 siRNA reversed the cell death induced by Ery5 for different time period. PC-3 cells were seeded in six-well plates, ATG7 was silenced through siRNA as described in Materials and Methods. Cell viability was determined by MTT assay as described in materials and methods. 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<0.001. (d) ATG7 inhibition caused reversal of Ery5-mediated inhibition of angiogenic proteins. Cells were seeded in six-well plates; ATG7 was silenced through siRNA as described in Materials and Methods. ATG7 siRNA-transfected and non-transfected cells were treated with 10 μM of Ery5 for 6, 12 and 24 h. Cells were lysed in RIPA buffer and proteins were separated on SDS-PAGE and transferred to PVDF membrane. Membranes were probed with different angiogenic and autophagic proteins as 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<0.001. (e) LC3 silencing reversed the inhibitory effect of Ery5 on viability. LC3 was downregulated in PC-3 cells through siRNA as described in Materials and Methods. MTT assay was done to calculate cell viability as described in Materials and Methods. 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<0.001. (f) LC3 inhibition reversed the expression of angiogenic proteins. LC3 siRNA-transfected and non-transfected PC-3 cells were treated with 10 μM of Ery5 for indicated time periods. Cells were lysed and proteins were separated on SDS-PAGE, transferred to PVDF membranes and membranes were probed for different antibodies. β-actin was used as an internal control. (g) Cell death induced by Ery5 is autophagic. Combined silencing of ATG7 and LC3 improved the viability dramatically in PC-3 cells. siRNA-transfected and non-transfected cells were seeded in 96-well plates and treated with 10 μM of Ery5 for 6, 12 and 24 h. Viability was determined through MTT assay as described above. 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<0.001. (h) Combined knockdown of LC3 and ATG7 reduced the anti-angiogenic effect of Ery5. Cells were transfected with LC3 and ATG7 siRNA as described in Materials and Methods. Transfected and non-transfected cells were treated with 10 μM of Ery5 for indicated time periods. Western blot for indicated antibodies was done as described in Materials and Methods. Western blotting results revealed that combined silencing of LC3 and ATG7 reversed the expression of angiogenic proteins very significantly. β-actin was used as an internal control. (i) PC-3 cells (3 × 10⁶) were seeded in 90 mm dishes and incubated with Ery5 (10 μM) for different time points. Cells were collected and lysed in non-denaturing lysis buffer and immunoprecipitated with LC3 antibody as described in Materials and Methods section. After immunoprecipitation, western blot for indicated proteins was done with immunoprecipitated samples as described in Materials and Methods. (j) The figure represents western blot of the input from the IP experiment. Western blot was done as described in Materials and Methods