Fig 2. Genetically targeting NRP1 leads to enhanced GBM growth and progression in vivo.

(A); Immunoblot analyses of detergent-soluble lysates from six different human GBM cell lines reveals varying levels of Nrp1 and Nrp2 protein expression. (B); RNAi-mediated targeting of NRP1 in LN229 GBM cells with two different lentiviral-expressed shRNAs leads to reduced expression of Nrp1 protein, but not Nrp2 protein, as revealed by immunoblotting. (C-F); LN229 cells stably expressing control (non-targeting) shRNAs (C, D) or shRNAs targeting Nrp1 (E, F) were injected intracranially (n = 5 mice per cell type) and brain tumor xenografts were subsequently analyzed by bright field (C, E) and GFP fluorescence imaging (D, F). Note the obvious Nrp1-dependent differences in tumor size in the representative images. (G-L); Analysis of Nrp1-dependent angiogenesis and reactive gliosis in brain tumor xenografts by H&E staining (G, H), anti-CD31 (I, J) and anti-GFAP (K, L), respectively. Scale bars, 20 μm. (M, N); Quantitation of blood vessel densities based on CD31 expression (M) and tumor-associated astrocyte based on GFAP expression (N) in brain tumors generated from LN229 cells expressing control shRNAs or Nrp1 shRNAs, *p<0.05 for Nrp1 shRNA tumors in comparison to control shRNA tumors. All error bars represent standard deviation.