Figure 3.

Induction of VEGF by TGFβ in RIE and RIE-Ras(12V) cells. (A) Northern blots were prepared from RNA isolated from RIE and RIE-Ras(12V) cells treated with 2 ng/ml of TGFβ1 for the time intervals shown and probed for VEGF expression. Equivalent loading was confirmed by the signal intensity of IB15 (cyclophilin) a constitutively expressed gene. In (B), basal expression of VEGF is shown in RIE and RIE-Ras(12V) cells, confirming markedly increased basal expression in the Ras-transformed line. The bottom right shows densitometric values obtained from the Northern shown in the top panel with expression shown as a fold change overexpression at time 0. As shown in (C) VEGF protein was also differentially induced in RIE-Ras(12V) cells. Whole-cell protein lysates were obtained from RIE-1 and RIE-Ras(12V) cells treated with TGFβ for 24 hours. VEGF protein was quantified by ELISA, as described in the Materials and Methods section. (D) Effect of signaling pathway inhibitors on induction of VEGF by 2 ng/ml TGFβ1. Cells were treated with 10 µM SB203580 (p38 kinase inhibitor), 10 µM U0126 (a MEK inhibitor), 1 µM RafKI (BAY-439006, a Raf-1 kinase inhibitor), and 20 µM LY294002 (phosphoinositol 3-kinase inhibitor) for 24 hours and then treated with 2 ng/ml TGFβ1 for 1 hour before isolation of RNA according to procedures described in the Materials and Methods section. Equivalent loading was confirmed by hybridization with a cDNA probe complementary to cyclophilin. The results shown are representative of three separate experiments.