Figure 3

9-cis-RA activates cIAP2 transcription through NF-κB response elements. (A) Illustration of the reporter constructs containing 5'-deletion fragments of the cIAP2 upstream regulatory region. Luciferase activity in SK-BR-3 cells transiently transfected with the indicated cIAP2 reporter gene in the presence (black bars) and absence (white bars) of 9-cis-RA. The data shown represent the mean ± SD of three independent experiments performed in duplicate. The luciferase levels were normalized with those of β-galactosidase and expressed as the induction over the controls. (B) Transfection experiments as described in (A) but using site-specific mutants derived from -247-cIAP2 promoter as depicted (Black triangle), the wild-type promoter (Black square) and the backbone vector pGL3 (Black diamond). After transfection, SK-BR-3 cells were treated with different doses of 9-cis-RA, as indicated. The data shown represent the mean ± SD of three independent experiments performed in duplicate. The luciferase levels were normalized with those of β-galactosidase and expressed as the induction over the controls. Asterisks denote the existence of statistically significant differences between the wild-type and mutant promoter constructs. (C) SK-BR-3 cells were transiently co-transfected with the -247- cIAP2 reporter gene and pSG5, pSG5-IκBαSR(S32A/S36A) or pcDNA-TAM54 (a dominant-negative version of c-JUN) and either untreated or treated with 9-cis-RA, as described in (B). The luciferase levels were normalized with those of β-galactosidase and expressed as the induction over the controls. The values represent the mean ± SD of three experiments performed in duplicate. Asterisks denote statistically significant differences against cells transfected with an empty vector.