Fig. 5.

A: Prolif as well as day 10 or 12 Sp Caco-2 cells were transfected with a luciferase reporter construct driven by the 808-bp human Mybl2 promoter [containing all known cis-acting elements that repress Mybl2 transcription, including the major repressive E2F binding site (7, 35, 36)] or the T-cell factor-responsive TOP flash promoter. After 48 h, luciferase reporter activity was normalized to co-transfected pRL-TK renilla reporter activity and expressed relative to that in Prolif. B: primers flanking the indicated intron/exon boundaries of Mybl2, cyclin D2, and c-myc genes were used to amplify pre-mRNA (a measure of ongoing transcription) by quantitative RT-PCR of cDNA from Prolif or day 14 Sp Caco-2 cells. pre-mRNA levels are normalized to GAPDH mRNA and expressed as a percentage of those in Prolif. Each bar represents the mean + SE of 3 independent experiments, each performed in triplicate (A) or duplicate (B). Unpaired, two-tailed Student's t-test (relative to Prolif): *P < 0.01, **P < 0.05.