Fig. 2.

Hypoxic activation of CDCP1 is HIF-2α–dependent. (A) Stable knockdown of HIF-1α, HIF-2α, and ARNT in MCF10A cells. (B) Stable cell lines exposed to 21% O₂ (N) or 1% O₂ (H) for 48 h. Lysates were immunoblotted for CDCP1, P-734 CDCP1, P-416 SFK, SFK, and α-tubulin for loading control. (C) Quantitative real-time PCR for Cdcp1 on mRNA isolated from MCF10A stable cell lines cultured in N or H for 24 h. Data are represented as the means ± SEM (n = 3). (D) MCF10A cells were exposed to N or H for 24 h and subsequently ChIP was carried out on DNA–protein complexes with anti–HIF-1α, anti–HIF-2α, anti-ARNT, or control IgG antibodies followed by qRT-PCR. Antibodies used are indicated on the x axis. Data are represented as the means ± SEM (n = 3). ***P < 0.0001, two-tailed Student’s t test. (E) Alignment of Cdcp1 (chromosome 3) with the predicted HRE/ARNT binding site using MAPPER2 (http://genome.ufl.edu/mapper). Red indicates the predicted HRE/ARNT binding site. (F) HT1080 cells were transfected with the R01-scrambled control vector or with the Cdcp1-promoter vector. At 24 h after transfection, cells were exposed to 21%O₂ (N) ±100 μM DFO or 1 mM DMOG. RenSP luciferase was measured and the data are reported as fold induction over untreated normoxic R01 control vector cells. The data presented are the result of triplicate analyses and the error bars represent SEM. *P = 0.02, ***P = 0.0002. (G) Stable MCF10A cell lines were serum-starved and exposed to 1% O₂ for 24 h; subsequently, the cells were seeded in transwells and returned to 1% O₂ for 24 h. CUB1 mAb was added at the time of seeding to the top and bottom chambers. Cells were fixed and stained with crystal violet (0.1%; Lower). The number of cells that migrated to the bottom of the filter was counted and the data are reported as fold induction over the pLK0.1 control cells. The data presented are the result of triplicate analyses and the error bars indicate SEM. **P < 0.001, two-tailed Student’s t test.