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. 2020 Aug 12;23(9):101449. doi: 10.1016/j.isci.2020.101449

Figure 2.

Figure 2

Propionate-Dependent Gαi/o Signaling Requires Receptor Internalization

(A) Representative confocal microscopy images of HEK 293 cells expressing FLAG-FFA2 were pre-treated with either DMSO (vehicle) or Dyngo-4a (50 μM, 45 min), fed with M1 anti-FLAG antibody prior to stimulation with either NaCl or sodium propionate (Pro) (1 mM, 20 min). Fixed cells were imaged via confocal microscopy.

(B and C) Intracellular cAMP levels (B) or calcium mobilization (C) measured in HEK 293 cells expressing FLAG-FFA2 pre-treated with either DMSO (vehicle) or Dyngo-4a (50 μM, 45 min). For (B), cells were pre-treated with IBMX (0.5 mM, 5 min) and then stimulated with forskolin (FSK, 3 μM) or a combination of FSK with either NaCl or sodium propionate (Pro) (1 mM, 5 min). n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. For (C), cells were incubated with calcium indicator Fluo4-AM for 1 h and imaged live via confocal microscopy for 1 min before the addition of either NaCl or sodium propionate (Pro) (1 mM). Average maximal intensities of n = 20 cells in duplicate per four independent experiments.

(D) Representative confocal microscopy images of WT or β-ARR KO HEK 293 cells expressing FLAG-FFA2. Cells were treated with FLAG antibody and ligands and imaged as in (A).

(E and F) Intracellular cAMP levels (E) or calcium mobilization (F) measured in WT or β-ARR KO HEK 293 cells transiently expressing FLAG-FFA2. Samples were treated and assayed as in (B) and (C). n = 3 independent experiments for either WT or β-ARR KO HEK 293 cells transiently expressing FLAG-FFA2. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001.

(G) Representative confocal images of STC-1 cells transiently expressing FLAG-FFA2 pre-treated with either DMSO (vehicle) or Dyngo-4a (50 μM, 45 min) then stimulated as in (A).

(H) Intracellular cAMP levels of STC-1 pre-treated with either DMSO (vehicle) or Dyngo-4a (50 μM, 45 min). Scale bar, 5 μm; scale bar in inset, 1 μm. n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. For confocal images, representative images are shown of ~10 cells/experiment. Data represent mean ± SEM.

See also Figure S5.