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. Author manuscript; available in PMC: 2011 Mar 23.
Published in final edited form as: Oncogene. 2010 Jul 26;29(38):5241–5253. doi: 10.1038/onc.2010.264

Fig. 5. Sprouty-2 up-regulation increases hepatocyte growth factor (HGF)-induced signaling, cell migration and invasion.

Fig. 5

(A) Upregulation of c-Met receptor expression in sprouty-2 transfectants. Cell lysates from EV or sprouty-2 transfectants were probed for c-Met expression by Western blotting expressed as fold EV cells. (B) Total RNA was isolated from EV and sprouty-2 transfectants and c-Met mRNA levels quantified and expressed as fold of EV (Mean ± SD) as described in “Materials and Methods”. *p< 0.05, compared with EV. (C) HGF increases c-Met tyrosine phosphorylation in sprouty-2 transfectants. Cells were treated with HGF (50 ng/ml) for 5 min, c-Met was immunoprecipitated and Western blotted with anti-phosphotyrosine antibody as described in “Materials and Methods”. (D) Increased phospho-active Akt (p-serine 473) and (E) phospho-active ERK-1,2 (p-ERK 42, 44) induced by HGF in sprouty-2 transfectants. EV or sprouty-2 transfectants were treated with HGF (50 ng/ml) for the indicated times and cell lysates probed for phospho-Akt (serine-473) and phospho-ERK-1,2 expression by Western blotting. Quantitative densitometries were expressed as fold increases compared to untreated controls. (F) HGF increases cell migration in sprouty-2 transfectants. EV or sprouty-2 cells (30×103) were plated on upper chambers of transwells (permeable support, Corning, Inc). The lower chamber contained media with 2% FBS and HGF (5 ng/ml). Sixteen hours later migrated cells were counted as described in “Materials and Methods” (p<0.05 compared to respectively treated EV cells). (G) HGF increases cell invasion in sprouty-2 transfectants. EV or sprouty-2 cells (50×103) cells were plated into upper chambers of transwells coated with matrigel (BD matrigel invasion chamber). The lower chamber contained media with 2% FBS and HGF (1 ng/ml). Forty-eight hrs later migrated (invading) cells were counted as described in “Materials and Methods”. Data was expressed as relative increase in cell migration and invasion compared to EV transfected untreated cells. *p< 0.05, compared to respectively treated EV cells.