Figure 1.

HIF-1α and NICD3 are both required for the expression of CA9. (A) Schematic representation of the CA9 luciferase and CA9 mutant reporter. The HRE was mutated to create a CA9 promoter defective in HIF-1 binding (CA9M-Luc). (B) CA9 reporter assay: MCF10A cells were transfected with CA9-Luc or CA9M-Luc and HIF-1α and/or NICD3 and exposed to hypoxia. (C) CA9 expression by real-time RT-PCR: MCF10A cells were transfected with HIF-1α and NICD3 shRNA against HIF-1α or Notch3 or both HIF-1α and Notch3 and the relative CA9 mRNA expression as compared to the β-actin expression was analyzed. In addition, the cells were transfected with HIF-1α or Notch3 with Notch3 shRNA or HIF-1α shRNA, respectively, and CA9 mRNA expression was analyzed by real-time RT-PCR. (D) Western blot analysis showing the expression of CA9: MCF10A cells were transfected with vector alone or HIF-1α or Notch3 shRNA or both, and protein lysates were analyzed for the expression of CAIX, HIF-1α, NICD3, and β-actin (loading control). #Significant difference between normoxia- versus hypoxia-induced CA9 reporter activity and loss of CA9M activity during hypoxia at P ≤ 0.0005. *Significant lack of CA9M-Luc induction with overexpression of HIF-1α and NICD3 as compared to the wild-type CA9 promoter with overexpression of HIF-1α and NICD3 at P ≤ 0.05. &Significance between normoxia- versus hypoxia-induced CA9 mRNA expression at P ≤ 0.005. $Significant reduction in CA9 mRNA expression with HIF-1α shRNA or Notch3 shRNA or both HIF-1α and NICD3 shRNA at P ≤ 0.001. *Significant loss of CA9 mRNA expression with overexpression of HIF-1α and NICD3 with Notch3 shRNA and HIF-1 shRNA, respectively, as compared to the overexpression of HIF-1α and NICD3, respectively, at P ≤ 0.01. Each experiment has been repeated at least 3 times. H = hypoxia; N = normoxia.