Fig 2: Carboxy-terminal sequence of human, mouse and rat GPR68 and negative GPR68-immunostaining in mouse and rat pancreatic islets. Immunohistochemical staining (red-brown colour), counterstaining with haematoxylin. Scale bar: 100 µm (A,B).

Fig 3: Immunohistochemical detection of GPR68 localisation in different normal human tissues (I). Immunohistochemical staining (red-brown colour), counterstaining with haematoxylin. Scale bar: 100 µm (A,C) and 30 µm (B,D–F). Insets in (A–C): For adsorption controls, the anti-GPR68 antibody 16H23L16 was incubated with 10 µg/mL of the peptide used for immunisations. Arrowheads in (B): positive neuroendocrine cells; arrowhead in (D): positive intramural ganglion in the duodenum; arrows in (E): positive immune cells in the spleen; arrowheads in (F): positive immune cells in the liver, from the shape of their nuclei probably representing granulocytes. Inset in (E): double-labelling immunohistochemical analysis of human spleen tissue. Samples were incubated with the rabbit monoclonal anti-GPR68 antibody 16H23L16 together with a mouse monoclonal anti-CD68 antibody. Labelling for GPR68 was visualised using a Cy3-conjugated anti-rabbit antibody (red), and immunostaining for CD68 was envisioned using an Alexa Fluor 488-conjugated anti-mouse antibody (green). White arrows indicate the double-labelled cells.

Fig 4: Specificity analysis of the monoclonal rabbit anti-human GPR68 antibody 16H23L16. (A) Western blot analysis of whole-cell preparations from mock- or stably GPR68-transfected HEK-293 cells. (B) Western blot analysis of BON-1 cells endogenously expressing GPR68 after transfection with scrambled siRNA (scrambled, right lane) or with specific GPR68 siRNA (left lane), causing downregulation of GPR68 expression. Ordinate, migration of protein molecular weight markers (kDa). Representative results from one of three independent experiments are shown.

Fig 5: Expression profile of GPR68 in different bronchopulmonary and gastroenteropancreatic neuroendocrine tumor entities. (A) Number of GPR68-positive (black) and GPR68-negative cases (grey) within the different neuroendocrine tumour entities. Tumours were only considered positive at Immunoreactivity Score (IRS) values ≥3. (B) GPR68 expression levels (IRS values) in the different neuroendocrine tumour entities. Depicted are median values, upper and lower quartiles, minimum and maximum values, and outliers. Outliers were defined as follows: circles, mild outliers (data points between 1.5- and 3-times above the upper quartile or below the lower quartile); asterisks, extreme outliers (data that fell more than 3 times above the upper quartile or below the lower quartile). TC, typical carcinoid of the lung; AC, atypical carcinoid of the lung; SCLC, small-cell lung cancer; LC-NEC: large-cell neuroendocrine carcinoma; gut, gastroenteropancreatic neuroendocrine tumor (GEP-NEN) from the gut; duod/jejun, GEP-NEN from the duodenum or jejunum; ileum, GEP-NEN from the ileum; colon, GEP-NEN from the colon; rectum, GEP-NEN from the rectum; panc, pancreatic neuroendocrine tumour.