Fig. 6.
VEGFR2 upregulation upon MET disruption facilitates compensatory cell growth.
(A) Effect of MET or VEGF knockdown on cell growth. H441 cells were subjected to knockdown of MET (shMET) or VEGF (siVEGF) for 48 h and analyzed at 72 h for growth under the indicated treatment.
(B) VEGF concentration in whole cell lysates from (A).
(C) H441 cells were subjected to knockdown of MET (shMET) or VEGFR2 (siVEGFR2) for 48 h and incubated with activated sodium ortho-vanadate (Na3VO4) for 24 h. Total and phospho-VEGFR2 were then analyzed by IP and immunoblot.
(D) H441 cells were subjected to knockdown of MET (shMET) or VEGFR2 (siVEGFR2) or both and analyzed for growth as in A. Error bars indicate S.E.M., n = 3; **p < 0.01 for indicated comparison.
(E) Effect of MET disruption on VEGFR2 levels and on downstream signaling. H441 cells were treated with or without shMET or METi (GDC-0712, 1 μM) for 24 h and analyzed for indicated markers by immunoblot.
(F) Effect of VEGFR2 kinase inhibitor (su4312, 4 μM) and PI3K inhibitor (GDC-0941, 1 μM) on phosphorylation of downstream markers upon MET knockdown or inhibition in H441 cells. Densitometric analyses were normalized to the no MET inhibition.
(G) Effect of MET shRNA knockdown (shMET) or inhibition (METi, 1 μM) on VEGFR2 and pVEGFR2 levels in H441 cells with or without addition of exogenous VEGF (100 ng/ml) for 10 min.
(H) H441 cells were treated with or without VEGF (100 ng/ml) for 5 days in the presence of vehicle or Dox (shMET). Cells were treated with PBS, PI3Ki (GDC-0941, 1 μM) or MEKi (cobimetinib, 1 μM). Growth % was then analyzed by Incucyte. Error bars indicate S.E.M., n = 4; *p < 0.05; **p < 0.01 for indicated comparison.
(I) Effect of MET inhibitor (GDC-0712, 1 μM) and MEK inhibitor (cobimetinib, 1 μM) on phosphorylation of downstream markers in H441 cells.
(J) H441 cells were subjected to SMI blockade of MET (GDC-0712, 1 μM) or MEK (cobimetinib, 1 μM) or both and analyzed for growth as in A. Error bars indicate S.E.M., n = 5; ***p < 0.005 for indicated comparison.