Figure 2. GPRC5A is hypoxia/HIF‐induced in vivo .

• A, B
  Expression of CA9 and GPRC5A in formalin‐fixed paraffin‐embedded hypoxic SW620 cells by IHC. Reduced CA9 and GPRC5A expressions with siRNA confirm antibody specificity (scale bars: 200 μm).
• C
  IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia (strangulated colon). GPRC5A is co‐expressed with CA9 in the colonic epithelial cells (scale bars: 50 μm).
• D
  Quantitative RT–PCR analysis of mouse intestinal tissue. Gene expression was normalised to housekeeping gene Tbp. Raw data from three independent experiments (n = 3 mice) are shown (error bars ± SEM).
• E
  Tg[fli1:eGFP; vhl ^−/−] and Tg[fli1:eGFP] zebrafish embryos (5 days post‐fertilisation) demonstrate excessive angiogenesis and increased expression of HIF target genes (scale bars: 100 μm).
• F
  gprc5ba was induced in vhl mutant zebrafish embryos and fli1:eGFP zebrafish embryos exposed to 5% O₂ (vs. normoxia) for 24 h (RT–PCR).
• G, H
  Bioinformatic analysis of transcriptomics dataset GSE24551. Gene set analyses reveal GPRC5A mRNA strongly correlated with HIF/hypoxia gene signatures. GSEA datasets used were Semenza_HIF1_Targets (M12299) Broad_Hallmark_Hypoxia (M5891). Analysis was performed using R2 (http://r2.amc.nl).
• I
  Kaplan–Meier curve following analysis of transcriptomics dataset GSE24551. Event‐free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA. Analysis was performed using R2 (http://r2.amc.nl).

Data information: Level adjustments were made to images in Adobe Photoshop post‐acquisition for clarity (equal changes applied to the entire image). Representative examples of n = 3 independent experiments are shown.Source data are available online for this figure.