Knockdown of Angptl4 suppresses tumor growth and angiogenesis. (A) The LN229vIII cells were transfected with a retrovirus vector encoding negative control shRNA (shNC) or Angptl4-targeted shRNA (shAngptl4). The knockdown efficiency of Angptl4 in the LN229vIII cells was examined by real-time PCR analysis. (B) Cell growth of LN229vIII transfected with shNC or shAngptl4 was measured using WST-8. (C) Balb/c nu/nu mice were implanted subcutaneously with LN229vIII shNC or shAngptl4 cells, and the tumor volumes were measured on day 21. The excised tumors were photographed. (D) Frozen sections were prepared from the extracted tumors and immunostained for CD31, an endothelial cell marker (a and b). The nucleus was stained with DAPI (c and d). Scale bars show 100 μm. (E) Quantitative analysis of the microvessel density in the tumors was performed. At least three fields were selected from each section. Vessel area/mm2 was measured as the average of the values in four sections/mouse. Data shown are the means ± SEM (n=5 mice). Significant differences are shown: * p<0.05, ** p<0.01.