TGF beta1 increases pSTAT3, HIF1 alpha, and VEGF-A expression in and invasiveness of GSCs. (A) GSC272 cells were seeded in 6-well plates for 24 h and treated with TGF beta1 (in medium without EGF or bFGF) for the indicated times. Secretion of POSTN and expression of SMAD3 in GSC272 cells were assessed by ELISA and Western blot analysis, respectively. *P < 0.05, **P < 0.01 versus control. (B) GSC272 cells infected with vector shRNA or POSTN shRNA were treated with TGF beta1 for 24 h; expression of VEGF-A was determined by ELISA and expression of p-STAT3, STAT3, and HIF1 alpha was determined by Western blot. *P < 0.05, ***P < 0.001 versus control; ##P < 0.01 versus TGF beta1. (C) GSC272 cells infected with a vector or POSTN shRNA were added to the upper chambers of a transwell insert (2 × 104 cells/well), and medium containing recombinant TGF beta1 was added to the lower chambers; the plate was incubated for 18 h at 37°C. *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus TGF beta1.