Figure 4. Decreased COX2 inhibits the cell proliferation, colony formation, and in vivo tumorigenicity of Tsc2-deficient cells.

A and B. Tsc2−/− MEFs (A) or ELT3 cells (B) were infected with lentivirus harboring a vector encoding COX2 (LV-COX2) or the empty vector (LV). C. Two independent shRNAs targeting COX2 (shCOX2⁻¹ and shCOX2⁻²) or a control shRNA (shSc) were stably expressed in Tsc2+/+ MEFs. A-C. Cell lysates of the indicated cells were subjected to immunoblotting with the indicated antibodies. D. Proliferation of the indicated cells was examined using MTT assays. E. Representative images (upper panel) and quantifications (lower panel) of crystal violet-stained colonies formed by the indicated cells. Error bars indicate mean ± SD of triplicate samples. *P<0.05; **P<0.01; ***P<0.001. F and G. COX2-overexpressing Tsc2-null MEFs (F) or ELT3 cells (G) and the corresponding control cells were inoculated subcutaneously into nude mice, which were then monitored for tumor growth. H and I. Formalin-fixed, paraffin-embedded Tsc2-null MEF (H) and ELT3 cell (I) tumor sections were subjected to H&E and immunohistochemical staining, as indicated. Representative images were presented. Scale bar, 50 μm.