Neuropilin-2 Expression Promotes TGF-β1-Mediated Epithelial to Mesenchymal Transition in Colorectal Cancer Cells
Figure 3
NRP2 enhances colorectal cancer xenograft formation.
A, 1.106 HT29ctrl and HT29NRP2 cells were injected subcutaneously in nude mice (15 mice in each group included in four independent experiments). NRP2 significantly enhanced tumor growth of colon cancer cells in xenograft experiments. Tumor volume was calculated by the formula V = 1/2 a × b2, where a is the longest tumor axis, and b is the shortest tumor axis. Data reported are the mean average tumor diameter ± SEM. A representative experiment out of 3 is shown. (**, P<0.01). B, 1.106 Colo320siRNA-NRP2 or Colo320siRNA-ctrl were injected s.c in nude mice (15 mice in each group included in four independent experiments). Mice receiving Colo320siRNA-NRP2 did not develop tumor, even after 2 months. Data represent results of a representative experiment out of 3. C, 5000 Colo320 cells treated with control siRNA or NRP2 siRNA were cultured in soft Agar containing medium during 10 days. Colonies were then photographed (magnitude*10) and counted. SiRNA-NRP2 prevents colony formation in soft agar assays, in comparison to siRNA-ctrl in Colo320 cancer cells. (**, P<0.01). D, Invasion assays were performed using HT29ctrl and HT29NRP2. HT29NRP2 were resuspended in serum-free medium and placed in the top compartment of a standard 8 µM pore Boyden chamber. Following 16 hours of culture (37°C, 5% CO2), invasive cells were incubated with cell detachment buffer, lysed and marked with CyQuant GR dye. Fluorescence was then evaluated with a fluorescence plate reader using a 480-520 nm filter set.