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

Figure 4.

Figure 4

FFA2 Trafficking and G Protein Signaling Is Regulated by APPL1

(A) Representative western blot of total cellular levels of APPL1 from cells transfected with either scramble or APPL1 siRNA. GAPDH was used as a loading control.

(B) Representative confocal microscopy images of propionate-induced internalization and recycling following APPL1 siRNA-mediated knockdown. HEK 293 cells stably expressing FLAG-FFA2 were labeled with anti-FLAG antibody and then treated with NaCl (1 mM) or propionate (Pro) (1 mM) for 20 min, then “stripped” and incubated with ligand-free medium for 1 h to allow receptor recycling. Scale bars, 5 μm.

(C) Recycling of HEK 293 cells stably expressing SEP-FFA2 was measured in real time, via TIRFM; cells were transfected either with scramble or APPL1 siRNA and stimulated with NaCl (1 mM) or sodium propionate (Pro) (1 mM) for 5 min n = 20 cells per condition, collected across four independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001.

(D and E) APPL1 negatively regulates propionate-mediated Gαi signaling. HEK 293 cells stably expressing FLAG-FFA2 (D) or STC-1 cells (E) transfected with either scramble of APPL1 siRNA prior to pre-treatment of IBMX (0.5 mM, 5 min) and then stimulated with forskolin (FSK, 3 μM) or a combination of FSK and NaCl or stated SCFAs (1 mM, 5 min). Data are expressed as percent change of FSK and NaCl treatment. n = 4 independent experiments. Two-sided Mann-Whitney U test, ∗p < 0.05; ∗∗p < 0.01.

(F and G) FFA2 colocalizes with Gαi within APPL1 endosomes. Representative TIRFM images of HEK 293 cells stably expressing FLAG-FFA2 (magenta), Gαi (green), APPL1 (blue) in cells stimulated either with NaCl (1 mM) or sodium propionate (Pro) (1 mM) for 5 min (F). Dotted line marks cell boundary. The lower panel of each treatment, highlighted in red, shows higher magnification image of the region of colocalization indicated by the red box in the corresponding upper-panel image. Arrows indicate FFA2 endosomes positive for Gαi only; circle indicates FFA2 endosomes positive for Gαi and APPL1; squares indicate FFA2 endosome positive for APPL1 only. Scale bars of upper-panel images, 10 μm; scale bar of lower-panel images 1 μm. (G) Quantification of FFA2 endosomes positive for Gαi, APPL1, or Gαi and APPL1; n = 12 cells per condition from (F) were quantified across three independent experiments. Two-way ANOVA, Bonferroni multiple comparisons test, ∗∗∗p < 0.001. Data represent mean ± SEM.

See also Figures S7 and S8.