(a1–a2) Intradermal injection of a non-bile acid TGR5 agonist compound 15 (C15) does not induce itch in human. (a1) Time courses of the perceived intensity of itch evoked by DCA and vehicle are reproduced from Figure 5a1, and the itch evoked by C15 is from 19 subjects. The equivalent concentration (equiv. conc.) of DCA and C15 means the fold of concentration to the EC50 of activating human TGR5. (a2) The quantification results of area under curve (AUC) of itch intensity shown in (a1) (mean ± s.e.m.). Veh, vehicle. Student’s t-test, ***p<0.001, and n.s. not significant (p>0.05). (b–c) The activation of human MRGPRX4 (b) or human TGR5 (c) by DCA (red), compound 15 (C15, green) and nateglinide in MRGPRX4- or TGR5-expressing HEK293T cells detected by FLIPR and luciferase assay respectively. (d) The relationship between the evoked itch and the relative potency to activate human MRGPRX4 or human TGR5 by the specific agonists of these two receptors. The Y-axis shows the relative activation of certain compound to the receptor, representing the logarithm of (maximal response/EC50). The X-axis shows the human itch intensity, representing the AUC of itch evoked by certain compound. Statistic test was performed between the itch intensity of compound 15 and vehicle, or between the itch intensity of nateglinide and vehicle. Nat, nateglinide; C15, compound 15; Veh, vehicle. Student’s t-test, **p<0.01, and n.s. not significant (p>0.05) versus vehicle treatments. (e) In situ hybridization (ISH) of TGR5 in human DRG sections. (left) The diagram depicting the morphology of DRG neurons and surrounding satellite glial cells. (middle and right) TGR5 was highly expressed in satellite glial cells (indicated by arrows) but not DRG neurons in human DRG. Scale bar, 50 μm. (f) In situ hybridization of TGR5 in mouse DRG sections. TGR5 was highly expressed in DRG neurons (indicated by arrow heads) in mouse DRG. Scale bar, 50 μm. (g–h) Immunohistochemistry (IHC) of human and mouse DRG sections. (g) In human DRG, TGR5 was expressed in satellite glial cells (indicated by arrows) but not in neurons (marked by NeuN, indicated by arrow heads). (h) In mouse DRG, TGR5 was expressed in neurons (marked by NeuN, indicated by arrow heads). Scale bar, 50 μm. (i) Quantification of the percentage of TGR5+ neurons (over NeuN+ neurons) in human and mouse DRG (immunohistochemistry). Chi-square test, **p<0.01. All error bars represent the s.e.m.