Extended Data Figure 4. Activating EC cells increases afferent output and VMR to colorectal distension.

a, DREADD agonist DCZ (1.7μM) elicits Ca²⁺ responses in EC^hM3Dq intestinal organoids as detected by a change in GCaMP fluorescence ratio. b, Representative examples of pelvic mucosal afferents firing action potentials in response to stroking with 10mg or 500mg von frey hairs (vfh) in the presence of vehicle (black/grey) or CNO (100μM; green) for control (upper panels) and EC^hM3Dq (lower panels) mice. c,d, Group data showing before and after CNO (100μM) application response to increasing mechanical stimulation with vfh for males (c) and females (d) for control (Tac^Cre upper panels) and EC^hM3Dq (lower panels) mice. e, Group data showing total area under the curve for all colonic distension pressures showing VMRs significantly increased in Tac^Cre and EC^hM3Dq male mice (N = 6, 7) following DCZ (75μg/kg i.p.). f, Colonic compliance is unchanged in EC^hM3Dq animals. Wilcoxon matched-pairs signed-rank 2-tailed test in panels c, d; Student’s t-test (unpaired, 2-tailed) in panel e; two-way ANOVA in panel f. *p< 0.05, **p< 0.01, ns = not significant, error bars represent mean ± SEM.