Fig 2: OGR1 mRNA expression is significantly increased in inflamed mucosa of IBD patients. (A) OGR1 mRNA expression levels in colonic resections from five non-IBD subjects, and paired samples (non-inflamed and inflamed) from eight CD patient and 11 UC patients. Data are presented as mean ± S.E.M. and statistical analysis was performed using one-way ANOVA. ** p < 0.01; *** p < 0.001. (B,C) Correlation of disease severity (Harvey–Bradshaw index for CD patients, Truelove and Witts severity index for UC patients) to OGR1 mRNA expression. Statistical analysis: Spearman’s rank correlation coefficient. (B) Non-inflamed mucosa: rs = 0.5364, p = 0.0218, n = 18. * p < 0.05. (C) Inflamed mucosa: rs = 0.6279, p = 0.0053, n = 18. ** p < 0.01. (D,E) In situ hybridization (ISH) identifying OGR1 mRNA (brown dots) in colonic tissue. Representative images are shown from a CD patient. N = 2 non-IBD subjects, paired (non-inflamed and inflamed) samples n = 2 CD patients, n = 2 UC patients. Scale bars: (D,E) overview images 100 µm; (D1,E1). Detail images, 25 µm. Controls: negative control, positive RNAscope control (peptidyl-prolyl cis-trans isomerase B). CD: Crohn’s disease; IBD: inflammatory bowel disease; OGR1: ovarian cancer G-protein-coupled receptor 1; UC: ulcerative colitis.

Fig 3: The OGR1 inhibitor blocked OGR1-associated downstream events in Caco-2 cells and human CD14+ monocytes. (A) OGR1 (GPR68) antibody 16H23L16 for immunocytochemical detection of OGR1 was verified in previously validated tools. Caco-2 clone stably overexpressing OGR1 (clone U1); negative control, Caco-2 vector control (VC) cells. Cells were examined by confocal microscopy (Leica, Germany). (B–F) Functional assays confirmed pH-dependent OGR1-mediated signalling. (B) U1 and VC cells were subjected to an acidic pH shift and intracellular inositol phosphate (IP) formation was measured. Data are presented as mean ± SD and statistical analysis was performed using one-way ANOVA. *** p < 0.001. (C) Activated GTPase RhoA was measured in U1 and VC cells upon an acidic pH shift. (D,E) Efficacy of the OGR1 inhibitor (GPR68-I) and the enantiomer of the inhibitor were measured using electric cell-substrate impedance-sensing (ECIS) technology. OGR1 clone U1 and VC grown to confluent monolayers were subjected to an acidic pH shift with or without the OGR1 inhibitor (0.5, 1.0, 2.5, 5, 10, 25 µM). Changes in resistance of cell monolayers were monitored in real time. Representative graph of eight independent experiments shown for the inhibitor or the enantiomer. (F,G) Human CD14+ monocytes from healthy volunteers and active IBD patients were subjected to an acidic pH shift, with or without the OGR1 inhibitor (0.5, 1.0, 2.5, 5 µM). IP1 production was measured from healthy volunteers (n = 5) and active IBD patients (n = 2); active IBD patient (n = 1). Samples in (F,G) were read on the Tecan Infinite or Biotek Synergy instrument, respectively. Data are presented as mean ± SD and statistical analysis was performed using one-way ANOVA. * p < 0.05. IBD: inflammatory bowel disease; OGR1: ovarian cancer G-protein-coupled receptor 1; vector control: VC.

Fig 4: Immunohistochemical detection of OGR1. (A,B) OGR1 (GPR68) recombinant rabbit monoclonal antibody (16H23L16) for immunohistochemical (IHC) detection of OGR1 was verified in previously validated tools. Caco-2 clone stably overexpressing OGR1 (clone U1); negative control, Caco-2 vector control (VC) cells. (A1,A2) Strong positive OGR1 staining (brown colour), predominantly in the cytoplasm, is present in OGR1-clone U1. (B1,B2) No specific staining for OGR1 in VC cells. Counterstaining with haematoxylin. Scale bar: (A1,B1) 100 µm and (A2,B2) 25 µm. (C) Localisation of OGR1 in healthy human duodenal mucosa. Scale bars: C. Insert overview image 1 mm; detail images, (C1) 250 µm; (C2) 100 µm; (C3,C5) 50 µm; (C4,C6) 25 µm. (D) Localisation of OGR1 in duodenal tunica muscularis. Scale bars: (D1,D2) 1 mm; detail images, (D3,D4) 25 µm. OGR1: ovarian cancer G-protein-coupled receptor 1.

Fig 5: OGR1 protein expression is significantly increased in the inflamed mucosa of IBD patients. Immunohistochemical detection of OGR1 using the anti-OGR1 antibody 16H23L16. Paired samples: (A1–A3,B1–B3) non-inflamed; (A4–A6,B4–B6) inflamed. Representative images shown are from two CD patients. Scale bars: (A2,B2) and (A5,B5) detail images, 100 µm; (A3,B3) and (A6,B6) 25 µm. (C,D) Representative images shown are from two non-IBD subjects. Scale bars: (C1,D1) 100 µm; (C2,D2) detail images, 25 µm. Intestinal resections were taken from five non-IBD subjects and paired (non-inflamed and inflamed) samples from five CD patients and five UC patients. (E) Total protein was isolated from colonic tissue samples and Western blotting was performed using an OGR1 (GPR68) custom mouse monoclonal antibody (AbMart). (F) Densitometry after normalization of OGR1 to β-actin: five non-IBD subjects, four CD patients and four UC patients. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-test (** p < 0.01; *** p < 0.001). CD: Crohn’s disease; IBD: inflammatory bowel disease; OGR1: ovarian cancer G-protein-coupled receptor 1; UC: ulcerative colitis.