VEGF production and in vivo angiogenesis by parental, nonsense, and TfR1 knockdown MDA-MB-231 cells. (A) Levels of VEGF in tissue culture media of parental, nonsense (L-), and TfR1 knockdown (L3) cells treated with hypoxia or DFO. Bars represent the means + SD from four independent experiments (n = 4). (B) Matrigel plug xenografts (grown from MDA-MB-231 cells inoculated with Martigel™ into nude mice) and surrounding tissue were excised from the flanks after 72 h. Tissues were visually examined for the degree of vasculature recruitment to the xenograft site and arrows indicate neovascularization penetrating into the xenograft. Panel A: non-injected tissue; Panels B and C: parental cells; Panels D and E: nonsense shRNA-transfected cells (L-); Panels F and G: TfR1 shRNA stably-transfected cells (L3). * Significantly different from their respective controls (p < 0.05). +Significantly different from the parental cells (p < 0.05).