Figure 7. NOR-1 mediates the regulation of cIAP2 by hypoxia and CoCl₂.

(a) cIAP2 mRNA levels analyzed by real-time PCR in VSMC treated with CoCl₂ (black bars) for 4 hours in the presence or absence of actinomycin D (ActD, 4 μM). Results are expressed relative to mRNA levels from VSMC maintained in normoxia and non-treated with ActD (controls) (n = 6). *P < 0.0001 vs. controls; ^#P < 0.0001 vs. CoCl₂ without ActD. 18S rRNA was used as endogenous control. (b) cIAP2 promoter activity in VSMC transfected with the constructs pcIAP2-1808, pcIAP2-349 or pcIAP2-1808-mut and maintained in normoxia (white bars), or exposed to hypoxia (grey bars) or to CoCl₂ for 6 hours (black bars). Luciferase activity (normalized by Renilla) was measured and results are expressed relative to transcriptional activity in control VSMC (cells under normoxia conditions; white bars) (n = at least 8). *P < 0.0001 vs. cells maintained in normoxia; ^†P < 0.001 vs. cells transfected with pcIAP2-1808 and exposed to hypoxia; ^#P < 0.001 vs. cells transfected with pcIAP2-1808 and exposed to CoCl₂. (c) Representative autoradiograms of EMSA performed with a cIAP2 probe containing the NBRE(−358/−351) site (NBRE) and nuclear protein extracts from VSMC maintained under normoxia, or exposed to hypoxia or CoCl₂ for 4 hours. The position of the complex up-regulated by hypoxia and CoCl₂ (arrowhead) is indicated. Competition assays with a molar excess of unlabelled probe (100-fold; Competitor) were performed. EMSA carried out with a mutated NBRE probe (m-NBRE) is also shown.