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

Figure 3.

Figure 3

FFA2 Internalizes to Endosomes Exhibiting Properties of VEEs

(A) Representative confocal microscopy images of HEK 293 cells expressing FLAG-FFA2 or FLAG- β2AR or STC-1 cells expressing FLAG-FFA2 or FLAG-β2AR imaged live with confocal microscopy before and after ligand treatment. FFA2 was stimulated with sodium propionate (Pro) (1 mM), and β2AR with isoproterenol (Iso, 10 μM) for 20 min. Scale bars, 5 μm; scale bar in inset, 1 μm.

(B) Bar graph showing diameter of FFA2 or β2AR in HEK 293 or STC-1 cells containing endosomes. Endosome diameter was assessed by measuring the diameter of 20 endosomes, n = 10 cells per condition, collected across three independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001.

(C) Representative confocal microscopy images of fixed HEK 293 cells stably expressing FLAG-FFA2 or β2AR or STC-1 cells transiently expressing FLAG-FFA2 treated with ligand for 20 min prior to “stripping” by PBS/EDTA (to remove surface bound FLAG antibody), fixation and stained with anti-EEA1 antibody. Scale bars, 5 μm; scale bar in inset, 1 μm.

(D) Numbers of FFA2 or β2AR-containing endosomes positive for EEA1 quantified from (C); 200 endosomes per condition, 10 cells quantified per condition. Data represent mean ± SEM, n = 10 cells per condition, collected across three independent experiments. Two-sided Mann-Whitney U test, ∗∗p < 0.01, ##p < 0.01. (E, F) FFA2 colocalizes with APPL1.

(E) Representative confocal microscopy images of fixed HEK 293 cells stably expressing FLAG-FFA2 or LHR or STC-1 cells transiently expressing FLAG-FFA2 treated with ligand (LH for LHR). Cells were treated as in (C) except that cells were stained with anti-APPL1 antibody. Scale bars, 5 μm; scale bar in inset, 1 μm.

(F) Numbers of FFA2 or LHR-containing endosomes positive for APPL1 quantified from (E); 200 endosomes per condition, 10 cells quantified per condition. n = 10 cells per condition, collected across three 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 S6.