Fig. 2.

ADAM12 is a direct HIF target gene. (A) Two matches to the HIF consensus binding site (5′-RCGTG-3′) were identified in the human ADAM12 gene at +0.1 kb (site 1) and +37 kb (site 2), relative to the transcription start site. (B) SUM159 cells were incubated at 20% or 1% O₂ for 16 h, and ChIP assays were performed using antibodies against HIF-1α, HIF-2α, or HIF-1β. Primers flanking site 1 or site 2 were used for qPCR, and results were normalized to IgG at 20% O₂ (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus 20% O₂ (Student’s t test). (C) The following reporter plasmids were generated: pA12-HRE, containing a 55-bp candidate hypoxia response element (HRE1 or HRE2, encompassing site 1 or 2, respectively), which was either WT or mutant (MUT), downstream of an SV40 promoter and firefly luciferase coding sequences (Upper); and pSV-Renilla, a control plasmid containing Renilla luciferase coding sequences downstream of the SV40 promoter (Lower). (D) SUM159 cells were cotransfected with pSV-Renilla and firefly luciferase reporter pA12-HRE1 or pA12-HRE2 (WT or MUT), and exposed to 20% or 1% O₂ for 24 h. Fluc:Rluc ratio was determined and normalized to WT at 20% O₂ (mean ± SEM; n = 3). **P < 0.01 versus WT at 20% O₂; ^##P < 0.01, versus WT at 1% O₂ (two-way ANOVA with Tukey’s posttest).