FIGURE 5.

Depletion of angiopoietin‐like 4 (ANGPTL4) inhibits epidermal growth factor (EGF)‐ and prostaglandin E₂ (PGE₂)‐induced invasion of tumor cells and interaction with endothelial cells. A, FaDu cells were transfected with 20 nmol/L ANGPTL4, COX‐2, or MMP1 siRNA oligonucleotides (siANGPTL4, siCOX‐2, and siMMP1, respectively) and scrambled control oligonucleotides (SC) by lipofection. Endothelial cells were grown to form a monolayer on the bottom of a thick layer of ECM proteins to mimic intravasation in transendothelial invasion assays. After 50 ng/mL EGF (i) and 20 μmol/L PGE₂ (ii) treatment for 6 h and stained with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine perchlorate, cells were loaded in the upper chamber of filter inserts. After incubation for 48 h, noninvasive cells were removed. Invasive images were examined using a microscope (left panel). Original magnification, ×200. Invasive cells were counted (right panel). Values represent mean ± SEM of 3 determinations. B, Tumor cells were further cultured with endothelial cells for 3 h. Attachment of tumor cells was examined using a microscope (left panel). Number of attached cells was counted using 3 randomly chosen fields under the microscope from 3 independent experiments (right panel). Values represent mean ± SEM of 3 replicate analyses