Figure 3

Caspase-3-mediated processing of pre-IL-16 in apoptotic neutrophils. (a and b) Neutrophils were cultured for 0, 5, 10, 20, 30, 40 and 60 h at 37 °C. (a) Lysates were prepared at the indicated time points and analyzed for IL-16 and GAPDH (loading control) using western blot analysis. Shown are representative blots of three independent experiments. Neutrophil viability was assessed with flow cytometry using Annexin-V-FLUOS and PI staining. Bars represent the mean of the proportion of apoptotic cells (Annexin-V-FLUOS⁺/PI⁻)±S.E.M. of three independent experiments. Individual data points are shown as black dots. (b) The correlation between pre-IL-16 (left)/IL-16C (right) levels (normalized to GAPDH) and the proportion of apoptotic cells. The data were analyzed by the nonparametric Spearman correlation, n=21, P (two-tailed). (c) Neutrophils were irradiated with 200 mJ/cm² UV light (256 nm). Immediately after radiation, the neutrophils were treated with the pan-caspase inhibitor (Q-VD-OPh, 1 μM), the caspase-3-specific inhibitor (Z-DEVD-FMK, 100 μM) and the solvent DMSO (1 : 200). Lysates were prepared after 6 h of incubation at 37 °C and analyzed for IL-16, caspase-3 and GAPDH (loading control) using western blot analysis. Shown are representative blots of three independent experiments (left panel), which were quantified and normalized to GAPDH (right panel). Neutrophil viability was assessed by flow cytometry using Annexin V-FLUOS and PI staining. Bars represent the mean of the percentage of apoptotic cells (Annexin V-FLUOS⁺/PI⁻)±S.E.M. of three independent experiments (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 compared with the DMSO sample by one-way ANOVA followed by Holm Sidak multiple comparison correction). Individual data points are shown as black dots. AU, arbitrary units.