Fig 2: Obestatin and GPR39 are expressed in AGS cells(A) Immunocytochemical expression of obestatin and GPR39. 1) Obestatin immunoreactivity was intense and diffuse in the cytoplasm. 2) The preadsorption control was performed applying the primary antibody plus obestatin (10 nmol/mL per control peptide) to positive samples. 3) The expression of GPR39 presented a perinuclear location. 4) The preadsorption control was performed applying the primary antibody plus GPR39 control peptide (10 nmol/mL per control peptide) to positive samples. Images were taken with objective magnification of x20. (B) Electron micrograph of obestatin and GPR39 expression. 1) Morphology of the AGS cells in culture. 2) The expression of obestatin in the AGS cells showed a diffuse cytoplasmic pattern. 3) The expression of GPR39 was located in the mature face of the Golgi area. 4) The expression of GPR39 was located in the cell membrane, mainly in the microvilli.

Fig 3: Obestatin exerts its actions through the GPR39 receptor in AGS cells(A) Effect of GPR39 knockdown by siRNA on obestatin-activated pAkt(S473) and pERK1/2(T202/Y204) in AGS cells. The AGS cells were transfected with GPR39 siRNA prior to obestatin treatment (200 nM, 10 min). GPR39 was expressed as a fold of its level to control siRNA-transfected cells (n = 3). Activation of Akt [pAkt(S473)] and ERK1/2 [pERK1/2(T202/Y204)] was expressed relative to the control siRNA-transfected cells. (B) Effect of SiRNA depletion of GPR39 on obestatin-activated Ki67 expression in AGS cells. The AGS cells were transfected with GPR39 siRNA prior to obestatin treatment (200 nM, 24 h). Expression of Ki67 was denoted as a fold of the respective levels in control siRNA-transfected cells (n = 3). Equal amounts of protein in each sample were used to assess the expression of GPR39 by western blotting. The level of proteins was expressed as fold change relative to the control siRNA-transfected cells (mean ± SE). The protein expression was normalized relative to actin. The data were expressed as mean ± SE obtained from intensity scans of independent experiments. The asterisk (*) denotes P < 0.05 when comparing the treated control siRNA group with the control siRNA group; the dagger (#) denotes P < 0.05 when comparing the GPR39 siRNA group with the control siRNA group. (C) Effect of GPR39 knockdown by siRNA on the immunocytochemical expression of obestatin-activated Ki67 in AGS cells. The AGS cells were transfected with GPR39 siRNA prior to obestatin treatment (200 nM, 24 h). 1) siRNA control without treatment. 2) siRNA GPR39 without treatment. 3) siRNA control plus OB. 4) siRNA GPR39 plus OB. Images were taken with objective magnification x20. (D) Effect of GPR39 knockdown by siRNA on the cytoskeleton reorganization in AGS cells. The AGS cells were transfected or not with GPR39 siRNA prior to obestatin treatment (200 nM, 24 h). Cells were stained with Phalloidin CruzFluor-594 (red), anti-GPR39 antibody (green), and DAPI (blue). Scale bar 20 µm. Images are representative for at least three independent experiments.

Fig 4: GPR39 and obestatin are expressed in human healthy stomach tissueImmunohistochemical expression of GPR39 and obestatin in normal stomach. (A) There was no GPR39 expression in the mucosal cells of the gastric pits. A weak positivity was observed in parietal cells, whereas an intense immunostaining was present in the chief cells (x4). (B) Higher magnification view (x10) of the GPR39 immunostaning in the chief cells. (C) Obestatin immunoreactivity was only observed in neuroendocrine cells of oxyntic glands (x4). (D) At higher magnification, obestatin producing cells were recognized as small round or spindle cells with brownish staining in the cytoplasm (x10).