Fig. 3.

Only CUGBP2 variant 1 inhibits cyclooxygenase-2 (COX-2) mRNA translation. A: HCT116 cells were transiently transfected with plasmid-encoding Flag-tagged CUGBP2 variants, whereas the control cells were transfected with the empty vector (Vec). RT-PCR analyses showed that all the 3 variants increase COX-2 mRNA expression. B: abundance of COX-2 mRNA following transfection of the CUGBP2 variants. COX-2 mRNA levels increased following expression of all 3 variants. *P < 0.05 from 3 independent experiments. PGE2, prostaglandin E₂. C: lysates from the CUGBP2 variant transfections were subjected to Western blot analysis for COX-2 protein. There was a marked decrease in COX-2 protein levels in the Flag-tagged CUGBP2 variant 1-transfected cells. D: abundance of COX-2 protein following expression of the CUGBP2 variants. COX-2 protein levels are significantly reduced following expression of variant 1. *P < 0.05 from 3 independent experiments. E: whole cell extracts (T) from Flag-tagged CUGBP2 variant 2-expressing cells were crosslinked and subjected to immunoprecipitation with anti-Flag antibody. RNA present in the immunoprecipitate (P) and supernatant (S) were isolated after reverse crosslink and subjected to RT-PCR for COX-2 mRNA. Data demonstrates the presence of COX-2 mRNA in the pellet. Actin was used as control to demonstrate specificity of CUGBP2 binding. F: schematic representation of control luciferase mRNA (Luc) and luciferase mRNA containing the full length mouse COX-2 3′untranslated region (3’UTR) (Luc-COX) that is encoded in the plasmid under the control of the SV40 promoter. G: relative expression of the firefly luciferase reporter activity in HCT116 cells following expression of the 3 CUGBP2 variants. Extracts were subjected to luciferase activity measurements. Data shows that over-expression of variant 1 resulted in significant reduction of luciferase expression. Data from 3 independent experiments. *P < 0.05.