Fig. 4.

Sustained Ca²⁺ influx via P2X4 receptor mediates the inhibition of apoptosis in Gr-1⁺ CD11b⁺ cells. (A) Immunohistochemistry of Ly6G⁺ cleaved-Caspase 3 (Cl-Cas3)⁺ cells in the colon from mice administered 3% DSS for 7 d (Left), the number of Ly6G cells (Right), and the frequency of Cl-Cas3⁺ cells in Ly6G⁺ cells (Middle) in a field (wild-type: 10 fields and Entpd8^−/−: 8 fields). Images are representative of three independent experiments (blue, DAPI; green, Cl-Cas3; and magenta, Ly6G (Scale bar, 50 μm). ****P < 0.001. (B) Flow cytometry–based analysis of ROS production in colonic Gr-1⁺ CD11b⁺ cells stimulated with or without ATP-γS for 3 h (mean values ± SD). *P < 0.05. Data were pooled from six independent experiments. (C) The frequencies of annexin V⁺ cells among Gr-1⁺ CD11b⁺ cells from the colon following stimulation with or without ATP-γS for 5 h. All data are from three independent experiments. **P < 0.01. (D) Flow cytometric dot plots (Left) and the frequencies of annexin V⁺ cells (Right) among Gr-1⁺ CD11b⁺ cells from the colonic lamina propria of wild-type or P2rx4^−/− mice after their stimulation with or without ATP-γS for 5 h. All data are from four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005; n.s., not significant. (E) Cytosolic Ca²⁺ level in Gr-1⁺ CD11b⁺ cells from the large-intestinal lamina propria of wild-type or P2rx4^−/− mice. All data are representative of two independent experiments. (F) Flow cytometric dot plots (Left) and the frequencies of annexin V⁺ cells (Right) in Gr-1⁺ CD11b⁺ cells from the colons of BALB/c mice following treatment with or without ATP-γS in the presence or absence of BAPTA-AM for 5 h. All data are from four independent experiments. *P < 0.05; n.s., not significant.