Mutant PILSAP inhibits tumor growth and angiogenesis in vivo. (A) B16F10 cells were transiently transfected with a mock, Wt, or E343A expression plasmid. Thereafter, B16F10 cells were stimulated with 10% FCS for 12 hours and the in vitro–incorporated BrdU were measured by a chemiluminescent BrdU ELISA (n = 6). (B) A mock, Wt, or E343A expression plasmid was incorporated into the HVJ-envelope vector and mixed with Matrigel. Matrigel was subcutaneously injected into the abdomen of C57BL/6 mice. Twenty-four hours after Matrigel injection, B16F10 cells were inoculated subcutaneously on top of the Matrigel. Fourteen days after inoculation, tumors were excised and weighed (n = 4; *P < .01). (C) The number of microvessels in the tumor tissue was counted (n = 4; *P < .01). Error bars indicate SDs. (D) Tumor sections were stained with anti-VWF antibody (red) and nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Sections were observed using fluorescence microscopy (Olympus IX71 microscope and Color View Soft Image System). Objective lens: LCPlan Fl 20×; camera: CCD Color Camera System's ColorView II; software: analySIS Docu (Soft Imaging System, Münster, Germany).