Figure 7. IL-33 induces PLC-γ1 activation for 5-HT release in EC cells.

(A) Representative trace and (B) Quantification IL-33-induced action potentials were recorded by whole-cell patch-clamp from isolated EC cells of Tph1^CFP mice with or without BAPTA. K⁺ was used as a positive control. Inset: representative action potential. Scale bar, 20 mV, 10 ms. BAPTA (calcium chelator, cell-impermeant).

(C) Tph1^CFP EC-enriched intestinal organoids were treated with PBS or IL-33 for 10 minutes and fixed. Representative immunofluorescence staining for TPH1-CFP, p-PLC-γ1 and DAPI. Scale bar, 100 μm.

(D) Representative trace and (E) Quantification of IL-33-induced action potentials were recorded by whole-cell patch-clamp in isolated EC cells from Tph1^CFP mice treated with U-73343 (inactive analog of PLC-γ1 inhibitor U-73122) or U-73122. Inset: representative action potential; K⁺ was used as a positive control. Scale bar, 20 mV, 10 ms.

(F) Relative 5-HT amounts in the cultured supernatant from EC-enriched intestinal organoids treated with the indicated conditions for 8 hours derived from Tph1^CFP mice were assessed by ELISA. BAPTA-AM (calcium chelator, cell-permeant).

(G) Myd88^−/−Tph1^CFP EC-enriched intestinal organoids were treated with PBS or IL-33 for 10 minutes and fixed. Representative immunofluorescence staining for TPH1-CFP, p-PLC-γ1 and DAPI. Scale bar, 100 μm.

(H) Relative 5-HT amounts in the cultured supernatant from EC-enriched intestinal organoids derived from Tph1^CFP and Myd88^−/−Tph1^CFP mice treated with PBS or IL-33 for 8 hours were assessed by ELISA.

(I) Representative immunofluorescence staining for 5-HT, ST2, TRPA1 and DAPI in the healthy human intestinal tissues. Scale bar, 10 μm.

(J) Human EC cell-enriched intestinal organoids were treated with PBS or IL-33 for 10 minutes and fixed. Representative immunofluorescence staining for 5-HT, p-PLC-γ1 with DAPI. Scale bar, 100 μm.

(K-L) The human EC cell-enriched intestinal organoids were treated with the indicated conditions for 8 hours. Relative 5-HT amounts in the cultured supernatant were assessed by ELISA.

Data are representative of three independent experiments (A, C, D, F-L) or are pooled from two independent experiments (B, E). NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test, error bars represent SD). Please also see Figure S7.