Fig. 2: GPCRome screen identifies GPR3 as a regulator of human β cell proliferation. | Communications Biology

Fig. 2: GPCRome screen identifies GPR3 as a regulator of human β cell proliferation.

From: Silencing the G-protein coupled receptor 3-salt inducible kinase 2 pathway promotes human β cell proliferation

Fig. 2

a Z-score plot of screen results. Control cells infected with non-targeting virus (black circles, NT), positive control CDKN2C or CDKN1A shRNA virus (gray circles), or GPCRome library shRNAs (red circles) are shown. The x-axis represents Z-scores of double-positive (EdU, insulin) cells based on the absolute number; the y-axis represents Z-scores for the percentage of total b cells. Dotted blue lines mark the threshold of Z-scores = 2. b Validation data for candidates identified in the screen. Percentage proliferation achieved for individual shRNAs against indicated targets was averaged over three screens (n = 3 technical replicates/donor) using islets from three independent donors. c Screen images showing representative NT control or GPR3 shRNA-infected b cells (C-peptide, blue) stained for EdU (red). d Violin frequency distribution plots showing the percentage of EdU+ insulin+ cells following GPR3 silencing in 18 donors over a 10-day assay period compared to non-targeting control (NT) and CDKN2C and CDKN1A positive controls. Dark line = median, light line = quartiles. e Barplot showing the percentage of EdU+ β cells following silencing GPR3 in the presence of the shRNA-resistant GPR3 cDNA tagged with V5 (GPR3-RES-V5) or with NT shRNA control. Western blots showing expression of exogenous GPR3 are shown underneath. f Barplot showing the percentage of EdU+ β cells following silencing of GPR3 and five independent shRNAs targeting GPR6 and four independent shRNAs targeting GPR12, the most closely related GPCRs to GPR3. g GSIS in cells silenced for GPR3 compared to NT control. LG low glucose, HG high glucose, KCl depolarizing stimulus. p-values for GPR3 HG = 0.002; GPR3 KCl = 0.005. n = 6 technical replicates. h Insulin content in cells silenced for GPR3 compared to NT control. n = 6 technical replicates. i Dotplot showing C-peptide intensity in EdU+ and EdU− populations in cultures silenced for indicated targets. Non-targeting (NT) control shown. n = 200 β cells per condition. All cultures were infected with TAg. p-values: NT = 0.0012; CDKN2C = 1.4 × 10−14; CDKN1A = 5.0 × 10−6; GPR3 = 3.4 × 10−12. All plots are representative of three biological replicates. Error bars are defined as the standard error of the mean for n = 3 technical replicates unless stated otherwise. MWs for Western blot markers are in kDa.

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