FIGURE 3.

Minimal hypoxia induction of Ror2 expression. A, Ror2 expression is minimally induced after 48 h of exposure to cobalt chloride. Normoxic (N) and CoCl₂ (C)-treated protein samples were immunoblotted with HIF-2α and Egln3 antibodies to show that HIF-2α and the HIF target Egln3 were induced in VHL(+) cell lines. Ror2 levels remained stable to this manipulation. Ku80 antibody was used as a loading control (LC). B, left, ROR2 mRNA levels are minimally induced under hypoxic-like conditions in RCC4 cells. qRT-PCR analysis of the hypoxia mimetic cobalt chloride (CoCl₂) transcriptional induction after 24 h in the VHL expressing cell line RCC4 3-14 demonstrate induction of the HIF target gene EGLN3 (**, p = 0.0015) in response to treatment with hypoxia mimetic, confirming HIF transcriptional activity. Ror2 mRNA was not significantly induced under these conditions. Right, Ror2 mRNA levels are minimally induced under hypoxic-like conditions in 786-0 WT8 cells. qRT-PCR analysis of the hypoxia mimetic DMOG transcriptional induction after 24 h in the VHL expressing cell line 786-0 WT8 demonstrate induction of HIF target genes EGLN3 (**, p = 0.0091) and GLUT1 (**, p = 0.0015) in response to treatment with the hypoxia mimetic, confirming HIF transcriptional activity. Ror2 transcript levels show minimal enrichment upon treatment. Transcript values are shown as normalized to the β-actin RNA internal standard and relative to the unstimulated cells of each set of paired cells. Error bars represent ± S.E.