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. 2023 Aug 4;299(9):105133. doi: 10.1016/j.jbc.2023.105133

Figure 2.

Figure 2

RBM12 loss leads to increased PKA activity and supraphysiological CREB-dependent transcriptional responses.A, PKA sensor (ExRai-AKAR2) activity in response to 10 nM Iso (n = 24–47 cells from 3-4 independent transfections per cell line). # and ∗ denote statistically significant time points between RBM12 KO1 versus WT and KO2 versus WT, respectively. B, flow cytometry measurement of fluorescent CREB transcriptional reporter (CRE-DD-zsGreen) in response to 1 μM Iso and 1 μM Shield, 4 h (n = 17). C, RT-qPCR analysis of the endogenous β2-AR transcriptional target mRNAs, PCK1 (n = 10) and (D) FOS (n = 9) in untreated cells or in cells treated with 1 μM Iso for 1 h. E, flow cytometry measurement of the fluorescent CREB transcriptional reporter (CRE-DD-zsGreen) in response to a panel of endogenous (10 μM norepinephrine/NE, 10 μM epinephrine/Epi) or synthetic (10 μM salbutamol/Sal, 10 μM terbutaline/Terb, 50 nM formoterol/Form) β2-AR agonists and 1 μM Shield for 4 h (n = 5). F and G, PCK1 mRNA expression in untreated or 1 μM Iso-treated cells transfected with empty plasmid (n = 3), (F) plasmid construct expressing lower levels of β2AR (n = 3), or (G) plasmid expressing β2AR from a CMV promoter (n = 3). H, PCK1 mRNA expression in untreated or 1 μM Iso-treated cells for 1 h in the presence of either vehicle (DMSO) or 10 μM of the PDE4 inhibitor Rolipram (n = 4–5). I, RT-qPCR of PCK1 mRNA in untreated cells or cells treated with 1 μM Iso-treated cells or 10 μM forskolin for 1 h (n = 12–13). J, PCK1 mRNA expression in cells pretreated with either vehicle (DMSO) or 30 μM Dyngo-4A for 20 min, then treated with 1 μM Iso for 1 h (n = 5). All data are mean ± SD. Statistical significance was determined using multiple unpaired t-tests with Benjamini, Krieger and Yekutieli false discovery rate correction (A), one-way ANOVA with Dunnett’s correction (B), or two-way ANOVA with Tukey’s correction (C–J). See also Fig. S2. ∗∗∗∗ = p < 0.0001, ∗∗∗ = p < 0.001, ∗∗ = p < 0.01, ∗ = p < 0.05. β2-AR, beta-2-adrenergic receptor; PDE, phosphodiesterase; RBM, RNA-binding motif.