Journal: Nature Communications

Article Title: GPR101 drives growth hormone hypersecretion and gigantism in mice via constitutive activation of G s and G q/11

doi: 10.1038/s41467-020-18500-x

Figure Lengend Snippet: a Upper panel: Macroscopic analysis of the pituitary gland from 27-week-old WT (+/+) and Ghrhr Gpr101 (+/T) mice. Lower panel: microscopic visualization of pituitary sections after H&E staining. Scale bar: 150 µm. b High magnification of anterior and posterior pituitaries stained with H&E. Scale bar: 15 µm. c Left panel: Immunohistochemical staining of the anterior pituitary sections with the cell proliferation marker Ki-67 (scale bar: 15 µm). Blue arrows indicate Ki-67-positive nuclei staining. Right panel: quantification of the Ki-67 labeling index in pituitary sections of 27-week-old WT (+/+) and Ghrhr Gpr101 (+/T) mice. The Ki-67 labeling index represents the percentage of positive nuclei stained by anti-Ki-67 antibody. n = 11 independent areas from staining section of WT (+/+) and Ghrhr Gpr101 (+/T) ( n = 4 mice per group, p = 0.9487). d Immunofluorescent staining of GH (green) and Ki-67 (Red). Scale bar: 10 µm. e Reticulin staining of the anterior and posterior pituitaries of WT (+/+) and Ghrhr Gpr101 (+/T) mice (scale bar: 15 µm). f The expression of GH in the pituitary of WT and Ghrhr Gpr101 mice (aged 27 weeks n = 5, p = 0.0079) was quantified by RT-qPCR. GAPDH was used as a control housekeeping gene. g The content of the GH protein was quantified by ELISA and normalized to total protein in pituitary lysates of both males and females of the WT (+/+) and Ghrhr Gpr101 (+/T) genotypes (aged 29 weeks, n = 4, p = 0.0286). h Ex vivo pituitary superfusion analysis. Pituitary glands of WT (+/+) and Ghrhr Gpr101 (+/T) (aged 29 weeks, n = 3 mice) were superfused at 0.1 ml min −1 in superfusion chambers. Effluents were collected every 5 min for GH measurement. GHRH (100 nM) was added to the medium for 15 min and KCl (0.03 M) for 20 min (as it is indicated with arrows). GH secretion was quantified by ELISA at indicated time points. All the experiments were independently repeated three times unless stated otherwise. F Female, M Male. For statistical analysis of all data, a two-sided Mann–Whitney test was used unless stated otherwise. ns not significantly different; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After that, sections were stained with rabbit polyclonal anti-Ki-67 antibody (Merck, Cat. No AB9260) (dilution 1:300) by using the Rabbit Specific HRP/DAB (ABC) Detection IHC kit (abcam, Cat. No ab64264) according to the manufacturer’s protocol.

Techniques: Staining, Immunohistochemical staining, Marker, Labeling, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Ex Vivo, MANN-WHITNEY

Journal: Nature Communications

Article Title: GPR101 drives growth hormone hypersecretion and gigantism in mice via constitutive activation of G s and G q/11

doi: 10.1038/s41467-020-18500-x

Figure Lengend Snippet: a Proliferation was measured with XTT cell proliferation kit on GH3 cells transiently transfected with increasing amounts (0, 25, 50, and 100 ng) of pcDNA3.1 FLAG-GPR101 plasmid (0 ng vs 25 ng p = 0.8182; vs 50 ng p = 0.5887; vs 100 ng p = 0.6991 ) . b Quantification of proliferation (by using the XTT reagent) of GH3 cells co-transfected with GHRHR and GPR101 (or MOCK) and treated with vehicle or GHRH at final concentration of 10 nM. c Comparison of cAMP levels (measured by ELISA) in GH3 cells transfected with GHRHR (100 ng) in the presence of increasing amounts (0, 25, 50, and 100 ng) of GPR101 plasmids, and treated with vehicle (dark grey) or GHRH (10 nM, orange). d , e GH3 cells transfected with GHRHR (100 ng) and increasing amounts of FLAG-GPR101. The cells were treated with increasing concentration of GHRH. d cAMP levels measured by ELISA and normalized to vehicle condition. e Proliferation measured with XTT assay. f Effect of the siRNA-mediated depletion of different G protein α subunits (G αs , G αq/11 , or G α12/13 ) on GH3 proliferation measured Briefly, GH3 cells were incubated with siRNAs (G αs , G αq/11 , or G α12/13 , at a final concentration of 1 µM) for 24 h, and then transfected with MOCK, GPR101 (NTS vs G αs p = 0.0022), GHRHR (NTS vs G αs p = 0.0022), GPR101+GHRHR (untreated NTS vs G αs p = 0.0286, GHRH-treated NTS vs G αs p = 0.0286) or GHSR (GHS-treated NTS vs G αq/11 p = 0.0065). GHRHR- and GPR101+GHRHR-transfected cells were stimulated with GHRH (10 nM) and GHSR-transfected cells with GHS (10 nM). g Determination of the proliferation of GPR101-transfected GH3 cells treated with vehicle or different pharmacological agents, such as FSK (adenylate cyclase activator, 10 µM), 8-Br-cAMP (PKA activator, 10 µM), and H89 (PKA inhibitor, 10 µM). All data are Mean ± S.D. of n = 6 independent experiments (except for the co-transfection of GHRHR with GPR101 in panel f and for experiments of panels d , e where n = 4). For statistical analysis of all data, a two-sided Mann–Whitney test was used. ns not significantly different; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: After that, sections were stained with rabbit polyclonal anti-Ki-67 antibody (Merck, Cat. No AB9260) (dilution 1:300) by using the Rabbit Specific HRP/DAB (ABC) Detection IHC kit (abcam, Cat. No ab64264) according to the manufacturer’s protocol.

Techniques: Transfection, Plasmid Preparation, Concentration Assay, Enzyme-linked Immunosorbent Assay, XTT Assay, Incubation, Cotransfection, MANN-WHITNEY