Figure 2.

CUGBP1 inhibits p27 IRES activity and expression of endogenous p27. (A) MCF7 cells were cotransfected with the bicistronic reporter construct pTKLL472 and either empty vector (pCDNA3.1-HisB) or pCDNA3.1-HisB-CUGBP1 encoding Xpress-tagged CUGBP1. One day after transfection, the expression of CUGBP1 was tested by western blotting. (B) One day after transfection cell lysates were assayed for luciferase activity. Firefly luciferase activity represents p27 5′-UTR IRES-dependent expression and Renilla luciferase activity represents cap-dependent translation. The mean of two independent experiments is shown and error bars represent standard error. (C) An MCF7 cell line constitutively overexpressing CUGBP1 was generated as described in Methods. The levels of CUGBP1, p27 and β-actin in the parental MCF7 cell line and the CUGBP1 overexpressing cell line were compared by western blotting. (D) The bicistronic reporter construct pTKLL472 was transiently transfected into MCF7 cells or MCF7-CUGBP1 cells. Firefly and renilla Luciferase assays were performed as described in (A). (E) MCF7 cells were transfected with control (non-targeting) or CUGBP1-specific siRNAs. CUGBP1 siRNA transfected cells were harvest 24 hours or 48 hours after transfection. The levels of CUGBP1, p27 and β-actin were examined by western blotting. (F) MCF7 cells were transfected with control or CUGBP1-specific siRNAs. Three days later the cells were transfected with the pTKLL472 bicistronic reporter construct. After one additional day the cells were harvested for luciferase assays. Luciferase values were normalized to the levels observed in cells transfected with the control siRNA.