Fig. 6. S1P delays neutrophil spontaneous apoptosis dependent on S1PR₂ in vitro.

The apoptotic rate of isolated neutrophils was measured by Annexin V and PI staining. The percentage of apoptotic neutrophils (Annexin V⁺) is shown. Protein expression of cCasp3 and Casp3 was measured by western blot. a–d BM neutrophils were cultured with or without S1P for 0, 2, 4, or 8 h. A representative flow cytometric plot of Annexin V and PI staining of a single sample from each group was provided. e–g Neutrophils were treated with S1P gradients of 1, 10, 100, and 1000 nM for 8 h. h–j Cells were pre-incubated with W146, JTE-013, or CAY10444 for 30 min, and then stimulated with S1P (1 µM) for 8 h. Data are presented as the mean ± SEM (n = 6 per condition in three experiments). One-way ANOVA was used in e, g, and two-way ANOVA was used in b, d, h, j. *P < 0.05 vs. control. ^#P < 0.05 vs. S1P-treated alone.