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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2021 Oct 21;42(1):e27–e43. doi: 10.1161/ATVBAHA.121.316651

Figure 2. Gαq-R183Q alters downstream signaling of PLCβ3.

Figure 2.

(A) Schematic of Gαq downstream signaling. PKC, NF-kB and calcineurin, highlighted in yellow, were suggested to be increased in EC-R183Q by bulk RNA-Seq. (B) qPCR showed the increased ANGPT2, DSCR1.4 and the NF-kB target genes E-selectin, G-CSF and IL-1B in EC-R183Q compared to EC-WT. Data from 13 independent experiments were plotted. P-values calculated by unpaired two-tailed, Student-t tests. (C) EC-WT and EC-R183Q treated with increasing concentrations of Gαq inhibitor YM-254890 showed reduced ANGPT2 and DSCR1.4 (shown by qPCR, N=5). P-values calculated by one-way ANOVA followed by Dunnett’s multiple comparison. In YM-254890-treated EC-WT, one-way ANOVA showed P=0.0160 for ANGPT2 and P=0.1961 for DSCR1.4. In YM-254890-treated EC-R183Q, one-way ANOVA showed P<0.0001 for ANGPT2 and P=0.0006 for DSCR1.4. (D) ELISA (N=4) showed the increased ANGPT2 protein in EC-R183Q compared to EC-WT. ANGPT2 protein level was reduced by 100 nM YM-254890 both in EC-WT and EC-R183Q. P-values calculated by one-way ANOVA followed by Tukey’s test. One-way ANOVA showed P<0.0001. (E) EC-WT and EC-R183Q treated with 3 different PLCB3-siRNA as well as non-targeted control siRNA. ANGPT2 and DSCR1.4 in EC-R183Q were reduced (shown by qPCR, N=3). P-values calculated by one-way ANOVA followed by Dunnett’s multiple comparison. In EC-WT, one-way ANOVA showed P=0.0773 for ANGPT2 and P=0.0873 for DSCR1.4; in EC-R183Q one-way ANOVA showed P=0.0028 for ANGPT2 and P=0.0502 for DSCR1.4. (F) ELISA showed three different PLCB3-siRNA reduced ANGPT2 protein in both EC-WT and EC-R183Q. P-values calculated by one-way ANOVA followed by Dunnett’s multiple comparison. One-way ANOVA showed P=0.0001 for EC-WT and P=0.0002 for EC-R183Q. (G) EC-WT and EC-R183Q treated with increasing concentrations of the PKC inhibitor AEB071; levels of ANGPT2 was decreased while DSCR1.4 was not (shown by qPCR, N=4). P-values calculated by one-way ANOVA followed by Dunnett’s multiple comparison. In AEB071-treated EC-WT, one-way ANOVA showed P=0.0415 for ANGPT2 and P=0.1489 for DSCR1.4; in AEB071-treated EC-R183Q, one-way ANOVA showed P<0.0001 for ANGPT2 and P=0.8156 for DSCR1.4. (H) ELISA showed increased ANGPT2 protein in EC-R183Q compared to EC-WT; 5μM AEB071 reduced ANGPT2 protein in both EC-WT and EC-R183Q. P-values calculated by one-way ANOVA followed by Tukey’s test. One-way ANOVA showed P<0.0001.