Fig. 2.

MET suppresses VEGFR2 via PI3K-induced intracrine VEGF action.

(A) Effect of HGF (100 ng/ml), METi (GDC-0712, 1 μM) or combinations thereof on VEGF secretion by PC3 and H441 cells after 24 h treatment.

(B) Effect of MET or VEGFR2 knockdown on VEGF production. H441 cells were subjected to knockdown of MET (shMET) or VEGFR2 (siVEGFR2) or both for 2 days and VEGF protein levels in conditioned media or washed cell monolayers were measured by ELISA.

(C) Effect of MET disruption on mRNA levels of MET and VEGF. H441 cells were subjected to MET inhibition (METi) or knockdown (shMET) and mRNA levels were determined by qPCR.

(D and E) Effect of 1 μM of MET, PI3K, MEK, or JAK SMI on modulation of VEGF (D) and VEGFR2 (E) by HGF. PC3 cells were treated with PBS or HGF (100 ng/ml) for 24 h and VEGF levels in washed cell layers were determined as above (D), while levels of VEGFR2 or other indicated markers were determined by immunoblot (E).

(F and G) Effect of MET SMI (F) or PI3K SMI (G) on secreted levels of VEGF (bar graphs) or VEGFR2 (immunoblots) in H441 cells. 1 × is 1 μM for METi and 10 μM for PI3Ki.

(H) Effect of VEGF transfection on VEGFR2 modulation by MET shRNA knockdown (shMET) or kinase inhibition (METi, 1 μM) in H441 cells.

(I) Effect of MET knockdown (shMET) in H441 cells cultured under normoxia or hypoxia on VEGF levels in washed cell layers.

(J) Effect of MET knockdown (shMET) or inhibition (METi, 1 μM) on VEGFR2 levels in H441 cells cultured under normoxia or hypoxia with or without siRNA knockdown of VEGF.

(K) Effect of extracellular VEGF neutralization (anti-VEGF, 10 μg/ml) or VEGF siRNA knockdown (siVEGF) on VEGFR2 modulation by MET knockdown (shMET) or inhibition (METi, 1 μM) in H441 cells. Scrambled siRNA (siNTC) was used as control.

Error bars indicate S.E.M for a minimum of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.005 for indicated comparisons.