Suppression of uPA and uPAR Attenuates Angiogenin Mediated Angiogenesis in Endothelial and Glioblastoma Cell Lines (original) (raw)
Figure 4
shRNA against uPA and uPAR inhibits secreted levels of ANG, Ang-1 and VEGF in cancer cells, endothelial cells and co-cultures.
(A–C) 1.5×105 U87 and U87 SPARC cells were transfected as described earlier. Conditioned medium was collected after 72 hrs and assayed for (A) ANG, (B) Ang-1, and (C) VEGF levels by ELISA. Data represented are the average of three independent experiments. *p<0.01, **p<0.001. (D–F) HMEC cells, U87 co-cultures and U87 SPARC co-cultures were transfected with jet Prime shRNA transfection reagent as mentioned in the Materials and Methods. The conditioned medium was collected and assayed for ANG, Ang-1 and VEGF by ELISA. Data represented are the average of three independent experiments. (G–I) HMEC cells transfected with shRNA constructs were lysed with RIPA lysis buffer and immunoblotted with primary antibodies for ANG, Ang-1 and VEGF and corresponding secondary antibodies. A representative blot of three independent experiments is shown. The blots were probed for GAPDH to show equal loading of lysates.