Fig 2: CTR expression in MTC.(A and B) negative control, a MTC case in which the primary antibody was omitted. (C and D) negative CTR-expression in a case of MTC. (E and F) positive CTR-expression in a case of MTC (score 2: extent 25–50%, intensity 1+). (G and H) positive CTR-expression in a case of MTC (score 12: extent 100%, intensity 3+). The dashed square in (A), (B) and (C) photomicrographs taken at 10× magnification represents the area in (D), (E) and (F) pictures taken using the 60× objective. Bar 100 μm.

Fig 3: cAMP Signalling by CTR activated by its cognate agonists. Characterization of cAMP accumulation (30 min) in response to stimulation by sCT, hCT and amylin (rAmy) in WK1 (a), JK2 (b), SB2b (c) and PB1 (d) cell lines. Data are analysed using a three-parameter logistic curve. All values are mean + S.E.M. of 3 to 5 independent experiments conducted in triplicate

Fig 4: The role of CTR in metabolism, proliferation and patient outcome. a metabolic activity of SB2b was assessed by an MTT assay in the presence (+GF) or absence (−GF) of defined growth factors (EGF + bFGF) in combination with 1 μM of CTR agonists sCT or hCT or the CTR specific antagonist sCT(8–32), results are mean + S.E.M. of 3 independent experiments performed in triplicate. In b, the effect of 1 μM CTR ligands on cell proliferation was assessed using live cell imaging (Operetta) over 72 h, cell number per field is expressed as a Log2 value and for clarity only every 3rd data point is shown, with representative experiment (of 3 independent experiments). Kaplan-Meier plots were derived from publicly available data using a threshold for expression of 1.25 FKPM (~ 1 transcript per cell) for all patients for whom RNAseq and survival data was available, CALCR +ve means over 1.25 FKPM, CALCR -ve means less than 1.25 FKPM, in c is the data from the IVY-GAP database and d shows data from TCGA

Fig 5: Morphology and gene expression in the four GBM cell lines used in this study. a SB2b and PB1 of classical GBM subtype; JK2 of proneural subtype and WK1 of mesenchymal GBM subtype as adherent cultures on matrigel, scale bar represents 100 μM. b Expression of CALCR, CALCRL, RAMP1, RAMP2, RAMP3, CALCA, and IAPP in SB2b, PB1, WK1 or JK2 cells. Data represent mean + SEM of 3 independent experiments, performed in duplicate, relative to β-actin expression. ND (not detected in all 3 samples), 2/3 (in 2 out of the 3 samples mRNA was detected), 1/3 (in 1 out of the 3 samples mRNA was detected). c Western blot for CTR, 50 μg of membrane protein probed with anti-CTR 1H10, representative of 3 independent experiments