FIG 1.

IR/IGF-1R contributes to IRS2-mediated tumor cell invasion. PyMT:Irs1/2^−/− cells expressing EV or IRS2 were treated with dimethyl sulfoxide (DMSO) or BMS754807 at the concentrations indicated for 4 h. (A) Matrigel Transwell invasion assays were performed for 5 h. The data shown represent the means ± standard errors of the mean (SEM) from three independent experiments. ns, no significant difference; **, P < 0.01 relative to EV-DMSO; ##, P < 0.01 relative to IRS2-DMSO. (B) Cells were stimulated with IGF-1 (50 ng/ml) for 5 h in the presence or absence of BMS754807. Cell extracts containing equivalent amounts of protein were immunoblotted with antibodies specific for IRS2, p-IGF-1R (Y1135/1136)/pIR (Y1150/1151), IGF-1R, pAKT (S473), AKT, or tubulin. (C) Schematic of Irs2 and Irs2-Y5F proteins. (D) Cell extracts from PyMT:Irs1/2^−/− cells expressing EV, Irs2, or Irs2-Y5F were immunoblotted with antibodies specific for IRS2 and tubulin. (E and F) PyMT:Irs1/2^−/− cells expressing EV, Irs2, or Irs2-Y5F were assayed for invasion. (E) Matrigel Transwell invasion assay. The data shown represent the means and SEM of the results of three independent experiments. (F) Matrigel-collagen I 3D invasion assay. The data shown represent the means and SEM of the results of a representative experiment performed three times independently. Representative images of colonies are shown on the right (magnification, ×10). **, P < 0.01 relative to EV; #, P < 0.05 relative to IRS2; ##, P < 0.01 relative to IRS2. Molecular weight markers (in kilodaltons) are indicated to the left of the immunoblots.