Fig. 4: GPR3 promotes quiescence by inhibiting SIK2. | Communications Biology

Fig. 4: GPR3 promotes quiescence by inhibiting SIK2.

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

Fig. 4

a Western blot showing an increase in the phosphorylation status of SIK2 target protein CRTC2. The effect of pan-SIK inhibitor (SIK-in) is shown. b Western blot showing the effect of SIK-in on levels of CDKN1A and CDKN2B in human islet cells. c Barplot showing the effect of SIK-in on proliferation in GPR3-silenced human β cells. p-value = 0.00047. d Western blots showing silencing of SIK2 with shRNA2. e Effect of co-silencing SIK2 in cells silenced for GPR3 on β cell proliferation. p-values vs NT: GPR3sh = 0.0021, GPR3sh + SIK2sh1 = 0.018, GPR3sh + SIK2sh3 = 0.009. f Western blot showing levels of SIK2 wild type (WT) and kinase-dead (KM mutant) proteins. g, h Barplots showing the effect of overexpression of SIK2 WT and kinase-dead (KM) mutants of SIK2 on β cell proliferation in the presence and absence of TAg. EV vs. KM = not significant. The p-value for g = 0.00042. p-values for (h) are 3.35 × 10−9 and 1.35 × 10−8. i Western blots showing levels of CIP/KIP proteins in GPR3 silenced β cells. Effect of pan-SIK inhibitor HG-9-91-01 (SIK-in) shown. j Western blots showing CDKN1B levels in cells following GPR3 silencing or overexpression of SIK2 WT or kinase-dead mutant (KM). k Barplot showing quantification of CDKN1B levels. p-values are 0.0002 and 0.0103. Error bars are defined as the standard error of the mean of three biological replicates. All western blots are representative blots from three donors. For (c), (e), (g), and (h), the error bars are representative of three donors and are defined as the standard error of the mean of three technical replicates. MWs for Western blot markers are in kDa.

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