p130Cas/Cyclooxygenase-2 (Cox-2) modulates A17 in vivo growth. (A) Left panel: A17 cells expressing Ctr shRNA or p130Cas shRNAs were orthotopically injected in FVB-NeuN mice. Doxycycline has been supplied in drinking water at the time of injection. Tumor mean diameter at 21 days after injection is reported on the y-axis (**P <0.001). Right panel: cells were orthotopically injected in mice and allowed to grow for two weeks giving rise to tumors with a mean diameter of 1 mm. Doxycycline was then added to water of mice to induce the expression of Ctr or p130Cas shRNA and tumors diameter were measured every week for four weeks. Finally, at the beginning of the sixth week, doxycycline was removed and tumor growth was measured for other six weeks. n/n: number of measured tumors/injected mice for each group. (B) Representative images of hematoxylin and eosin staining and immunohystochemical analysis of Ctr or p130Cas silenced tumor sections. The antibodies against CD31 (c, d), pCNA (e, f) and cleaved caspase-3 (g, h) were used on paraffin-embedded sections (4X magnification). Quantification of immunohystochemical analysis was performed by counting CD31 positive vessels in eight different fields at 200X magnification, by expressing pCNA percentage of positive nuclei on the total nuclei, and by counting cleaved caspase-3-positive cells of 20 fields at 400X magnification. (C) Protein extracts from Ctr or p130Cas silenced tumors were blotted with antibodies to p130Cas, pSrc (pTyr416), c-Src, Cox-2, Cyclin D1, pJnk (Thr183/Tyr185), Snail, Slug, Twist, and Vinculin as loading control. (D) A17 cells expressing Ctr shRNA or Cox-2 shRNAs were orthotopically injected in FVB-NeuN mice. Doxycycline had been supplied in drinking water at the time of injection. Tumor mean diameter at four weeks after injection is reported on the y-axis (**P <0.001). n: number of measured tumors for each group.