(A) Left, IBs of Sema3A in 387 GBM cells transduced with either NT control or Sema3A shRNA-expressing lentivirus. β-Actin was used as a loading control. Right, limiting dilution assay (LDA) analysis to determine clonogenic growth of GBM cells with NT or Sema3A shRNAs. *P < 0.01 by pairwise t test. (B and C) IBs of NRP1 expression and proliferation index in 131 GBM cells transduced with either NT control or NRP1 shRNA-expressing lentivirus. n = 4. *P < 0.01 by 1-way ANOVA with Tukey’s multiple-comparison test. (D) Left, representative H&E brain sections of the mice that were injected with either NT or Sema3A KD cells. Right, quantitation of tumor volumes in the brain sections. n = 4. *P < 0.01 by unpaired, 1-tailed Student’s t test. (E) Top, representative H&E brain sections of the mice that were injected with either NT or NRP1-KD 131 cells. Bottom, Kaplan-Meier survival curves of mice orthotopically implanted with 131 cells transduced with either NT shRNA– (n = 8) or NRP1 shRNA-expressing lentivirus (n = 9). *P < 0.001 by log-rank test. (F) Scheme of in vivo tumor growth competition assay. Equal numbers of NT shRNA–expressing GBM cells (GFP-labeled) and NRP1 shRNA–expressing GBM cells (RFP-labeled) were mixed and injected into the brains of mice. The resultant tumors were dissociated into single cells and processed for FACS analysis. (G) Representative images of 559 xenograft tumor. Inset shows a high-power image. (H) Quantitation of GBM tumors derived from the mixture of NT shRNA– and NRP1 shRNA–expressing cells. Tumors were harvested when the animals showed neurological signs, and tumor latency per each tumor is indicated in x axis. Data represent mean ± SD. Scale bars: 2 mm (D and E), 50 μm (G).