Analysis of the M3 promoter responsivness to RTA by using reporter assays in BHK-21, 293T, and NIH 3T3 cells. Two regions of the M3 promoter were cloned into a pGL3-Basic vector to drive the expression of firefly luciferase as a reporter. (A) Luciferase activities of reporters cotransfected with pCMV-FLAG (−) or pCMV-FLAG/RTA (+). The M3 promoter constructs were cotransfected into BHK-21, 293T, and NIH 3T3 cells with pCMV-FLAG or pCMV-FLAG/RTA in the presence of a control vector, pRLSV40, that constitutively expresses Renilla luciferase driven by the SV40 promoter. At 24 h posttransfection, cells were harvested and dual luciferase assays were performed. Firefly luciferase activity in reporter constructs was normalized to the corresponding Renilla luciferase activity. (B) Activation of the M3 promoters by RTA in BHK-21, 293T, and NIH 3T3 cells. Fold activation of the reporter by RTA was obtained by comparing the normalized firefly luciferase activity of pCMV-FLAG/RTA-transfected cells to that of pCMV-FLAG-transfected cells. The data are an average of two separate experiments. Bars: □, BHK; ░⃞, 293T; ▪, 3T3.