Fig. 2: The P2Y₁₄R of intestinal epithelial cell regulates epithelial necroptosis.

a The immunofluorescent images of colon tissues stained with Claudin-1, Occludin and ZO-1, the principal components of tight junction (Scale bar = 200 μm). b The expression of Bcl-2, Bax, GSDMD-NT, Caspase-1 p20, Caspase-3 p17, MLKL, and phosphorylation of MLKL were analyzed by immunoblotting in the IECs from P2Y₁₄R^fl/fl and P2Y₁₄R^△IEC mice after DSS treatment (n = 6 mice per group). c The TUNEL staining of colon tissues from P2Y₁₄R^fl/fl and P2Y₁₄R^△IEC mice after DSS treatment. The immunofluorescent images of colon tissues stained with cleave caspase-3 (Scale bar = 50 μm). d The TEM images showed the typical characteristics of necroptosis in the colon tissues of DSS-treated mice. e Cell viability was determined by CCK8 analysis in HT-29 cells (n = 6 samples per group). f PI-positive cells were analyzed by PI/Hoechst staining in HT-29 cells (n = 3 samples per group). g H&E staining of HT-29 cells. h Phosphorylation of MLKL as well as its protein levels were analyzed by immunoblotting in HT-29 cells (n = 5 samples per group). i PI-positive cells were analyzed by PI/Hoechst staining in HCT-116 cells (n = 3 samples per group). j Phosphorylation of MLKL as well as its protein levels were analyzed by immunoblotting in HCT-116 cells (n = 5 samples per group). k The PI staining and quantification of intestinal organoids from P2Y₁₄R^fl/fl and P2Y₁₄R^△IEC mice treated as indicated with DMSO (veh) or TNF-α adding Smac mimetic and z-VAD (TSZ) for 12 h (n = 3 samples per group). The data represent the mean ± SD, and p-values were determined by One-way ANOVA with Tukey multiple comparison test for e, f, h, i, j, and k, or two-way ANOVA with Sidak’s multiple comparisons test for (b). For a, c, d, and g, each image was acquired independently three times, with similar results. Source data are provided as a Source Data file.