Figure 11. Inhibition of apoptosis in HUVEC correlates with production of NO by retroviral-derived peptides.

A. HUVEC were grown to 70–80% confluence in 96-well tissue culture plates. Media was removed from the wells and to each of quadruplicate wells was added 200 µl of either fresh media (untreated) or fresh media containing 50 µM of the indicated peptides (treated) and the cells incubated ON at 37°C. Supernatant was removed from each well as assayed for total NO by measurement of nitrite with a Colorimetric Nitric Oxide Assay Kit (Oxford Biomedical Research; Oxford, MI) which employs nitrate reductase to convert nitrate to nitrite. B. HUVEC were grown to confluence in 96-well tissue culture plates. Media was removed from the wells and to each of quadruplicate wells was added 200 µl of either fresh media (untreated) or fresh media containing 50 µM of the indicated peptide (treated) and the cells incubated 3 h at 37°C. To quadruplicate wells of both untreated and treated cells were added 20 µl of media or 20 µl of media containing 500 nM staurosporine. The plates were incubated overnight at 37°C and the cells processed for measurement of apoptosis using the Cell Death Detection Assay (Roche Diagnostics) per the manufacturer's instructions.