c-Myc cooperates with ZO-2 in down-regulating the cyclin D1 promoter. (A) Western blot done with c-Myc antibody reveals how c-Myc is overexpressed in MDCK cells transfected with the c-Myc-LTR/pBR322 or the empty vector LTR/pBR322 (indicated as pBR322). HeLa cell extract was included as a positive control. (B) c-Myc overexpression represses cyclin D1 promoter activity in a dose-dependent manner and displays specificity for the E box element. This assay was done with sparse MDCK cells transfected with 1 μg of the intact cyclin D1 promoter construct (pXP2-CD1) or with a plasmid lacking E box/E2F element (−492ΔCycD1) and indicated amounts of c-Myc-LTR/pBR322 plasmid or the empty vector LTR/pBR322. (C) Simultaneous overexpression of c-Myc and ZO-2 enhanced the repression of cyclin D1 promoter activity. MDCK cells were cotransfected as indicated with different combinations of the following plasmids: pXP2-CD1, ZO-2, pBR322, and c-Myc. (D) Addition of ZO-2 siRNA modifies c-Myc repression abilities. Over a constant c-Myc expression, ZO-2 siRNA but not NC interferes with repression. Recombinant ZO-2 expression abrogates the ZO-2 siRNA effect. Relative luciferase activities are expressed as a percentage of the control activity (pXP2-CD1/pBR322 or in combination with NC siRNA). Data are mean values ± SE of at least three independent experiments (*p < 0.05, **p < 0.001, Student's t test).