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. 2018 Sep 21;9:1071. doi: 10.3389/fphar.2018.01071

FIGURE 4.

FIGURE 4

CuB-mediated GLP-1 secretion and activation of AMPK are involved in α-gustducin. (A) The siRNAs of GNAT3 inhibited CuB-mediated GLP-1 secretion in differentiated NCI-H176 cells. Data are presented as a Mean ± SEM, n = 5. ##P < 0.01 vs. control, ∗∗∗P < 0.001 vs. GNAT3 siRNA + CuB. (B) AMPK expression levels were observed using western blotting under knock down condition of GNAT3. (C) The knock down of GNAT3 prevented CuB-mediated activation of AMPK in differentiated NCI-H716. Data are presented as a Mean ± SEM, n = 5. ##P < 0.01 vs. control, ∗P < 0.05 vs. GNAT3 siRNA + CuB. (D) The transfection of GNAT3, TAS2R10, and TAS2R14 siRNA caused to inhibit CuB-mediated GLP-1 secretion in differentiated NCI-H176 cells. Data are presented as a Mean ± SEM, n = 5. ##P < 0.01 vs. control, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Control siRNA. (E) CuB treatment led to time-dependently decrease intracellular cAMP. Data are presented as a Mean ± SEM, n = 8. ###P < 0.001 (Frs) vs. control, ∗∗P < 0.01, ∗∗∗P < 0.001 (CuB) vs. control, $$$P < 0.001 (DB) vs. control. (F) CuB-mediated decreasing intracellular cAMP concentration restored by treatment of PDE4D inhibitor (Rolipram, 50 μM). Data are presented as a Mean ± SEM, n = 8. ###P < 0.001 vs. Fsk, ∗∗∗P < 0.001 (CuB) vs. control. (G) AMPK expression levels were observed using western blotting under treatment of rolipram. (H) Rolipram treatment prevented CuB-mediated activation of AMPK in differentiated NCI-H716. Data are presented as a Mean ± SEM, n = 5. ##P < 0.01 vs. control, ∗P < 0.05 vs. Rolipram + CuB. (I) CuB-mediated decreasing intracellular cAMP concentration maintained under Dorso treatment. Data are presented as a Mean ± SEM, n = 5. ##P < 0.01 vs. control, ∗P < 0.05, ∗∗∗P < 0.001 vs. Dorso + CuB. DB, Denatonium benzoate (10 mM); Fsk, Folskolin (5 μM). Fsk is positive control of activation of cAMP. Dorso, Dorsomorphin. CuB, Cucurbitacin B.