VEGF-A competitively inhibits Nrp1-mediated canonical TGFβ receptor signaling in GBM cells.

<p><b>(A);</b> LN229 cells were treated with 1 ng/ml TGFβ1 for varying times in the presence or absence of 100 ng/ml VEGF-A. Detergent-soluble lysates were immunoblotted with anti-pSmad3 antibodies to access Nrp1-dependent canonical TGFβ signaling. Note that VEGF-A competitively inhibits TGFβ-mediated Smad3 phosphorylation in control cells, and these effects are enhanced in LN229 cells expressing Nrp1 shRNAs. <b>(B);</b> Quantitation of Nrp1-dependent Smad3 phosphorylation levels in response to TGFβ1 and VEGF-A treatment of LN229 cells. Error bars represent standard deviation, *p<0.05 for control shRNA/+TGFβ1 versus other treatments at 15 minutes and *p<0.05 for control shRNA/+TGFβ1 versus Nrp1 shRNA/+TGFβ1 and Nrp1 shRNA/+TGFβ1/VEGF-A treatments at 30 minutes. All error bars represent standard deviation. <b>(C);</b> Diagram showing the experimental strategy to generate drug-resistant U87 GBM cells by selecting for tumor growth in mice followed by bevacizumab treatment. <b>(D);</b> bevacizumab-sensitive (U87-bev^S, n = 4) and bevacizumab-resistant (U87-bev^R, n = 4) tumor lysates were immunoblotted with human-specific antibodies directed against Nrp1, Nrp2, or αv integrin proteins. Note that Nrp1 protein levels are diminished in U87-bev^R tumors, whereas αv integrin levels are increased and Nrp2 levels are unchanged.