Extended Data Fig. 4: I3A, IPyA, and IEt are ligands of dopamine receptor D2 (DRD2), which signals via Gαi, and Drd2 is knocked out in intestinal epithelial cells in Drd2^fl/fl x Villin-Cre mice fed Trp diet during C. rodentium infection.

a, c–f, HEK 293 T cells overexpressing either DRD2 and a split luciferase-based cAMP sensor (GloSensor) or b, DRD2-Tango and a β-arrestin-TEV fusion were incubated with dopamine (DA), I3A, IPyA, or IEt (1 mM each in a–b; concentrations indicated in c–f) for 15 min (a, c–f) or 24 h (b), after which luminescence was measured to quantify ligand-induced (a) decrease in cAMP or (b) increase in β-arrestin recruitment. RLU = relative luminescence units. EC50 and Kd values were calculated using GraphPad Prism. (g–h) Drd2^fl/fl x Villin (Vil)-Cre or Drd2^fl/fl mice were fed a conventional (2 g Trp/kg diet, ad libitum) or Trp (42 g Trp/kg diet, ad libitum) diet for 7 d and then infected with C. rodentium (CR, oral gavage, 10⁸ CFU) with continued Trp feeding. Ten days post-infection, intestinal cryosections were stained with DAPI and an anti-DRD2 antibody, followed by an anti-mouse Alexa Fluor 594 antibody. (g) Shown are representative z-slices. Scale bar: 20 μm. (h) Image brightness was quantified using FIJI. Statistical analysis was performed using one-way ANOVA, followed by post-hoc Tukey multiple comparison test; bars = mean, error bars = standard deviation, (a–f) n = 3 biological replicates and (g–h) n = 10 mice per group examined over 3 independent experiments.

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