Constitutively active 4EBP-1 stimulates p27 Kip1 mRNA 5'-UTR-mediated cap-independent translation. (A) MCF7 cells were transfected with the bicistronic expression vector pGL2CAT/Luc, pGL2CAT/Luc-p27UTR in which the full-length human p27Kip1 5'-UTR is inserted between the two cistrons in the forward direction, or pGL2CAT/Luc-p27UTR(rev) in which the 5'-UTR is inserted in the reverse orientation. After one day, the levels of both luciferase (left) and CAT (right) activities were determined. (B) The relative levels of cap-independent translation from the transfections in A were estimated from the luciferase/CAT ratio. (C) The construct pGL2CAT/Luc-p27UTR was cotransfected with an expression vector encoding 4EBP-1-5A or an empty vector as a control. One day after transfection luciferase and CAT activities were assayed and the luciferase/CAT ratio was calculated. All data represent the mean of a minimum of three replicates plus or minus the standard error.