Figure 8.

Purified CD2v protein induces apoptosis in swine PBMCs. (A) Caspase-3 and PARP1 cleavage. Swine PBMCs were treated with purified CD2v, purified control or staurosporine (positive control) and whole cell lysates were obtained at various times post treatment, resolved by SDS-PAGE, blotted and probed with antibodies against Caspase-3, PARP1 and GAPDH (loading control). (B) Densitometric analysis showing the fold change in caspase-3 activation relative to the purified control treatment. The caspase-3 results are the mean values of six independent experiments (18 h, p = 0.042). (C) Densitometric analysis showing fold changes in PARP1 cleavage relative to the purified control treatment. Results are the mean values of six independent experiments (18 h, p = 0.026; 24 h, p = 0.018). (D) TUNEL assay in lymphocytes. Swine PBMCs treated as in A were fixed at 18 h post treatment and processed for the TUNEL assay as described in Materials and Methods. Approximately 5000 lymphocytes (6–9 μm in diameter) were counted/slide and results are shown as mean values from three independent experiments. p-values for purified CD2v relative to the purified control and mock were 0.004 and 0.006, respectively. (E) TUNEL assay in macrophages. Swine macrophages (16–23 μm in diameter) treated as in (A) were fixed at 18 h post treatment and processed for the TUNEL assay as described in Materials and Methods. Approximately 1600 cells/slide were counted and results are mean values of three independent experiments. p-values for purified CD2v relative to the purified control and mock were 0.004 and 0.003, respectively. (F) Representative confocal images of the TUNEL assay under conditions outlined in (E). Green, TUNEL; Blue, DAPI. (*, p < 0.05 and **, p < 0.01.)