Cytotoxic Effect induced By The HotSpot Peptide Ligands In SK-N-BE Cells. SK-N-BE cells were seeded in 96 well plates using SK-N-BE medium and allowed to grow to 75–80% confluency. Cells were starved overnight in media containing 1% serum before each cell experiment. Briefly, cells were first treated with a TNFR1 specific receptor blocking antibody, mAb225 (10 μg/mL), for at least 30 min at 37°C prior to the addition of test agents. All agents used in the cell-based experiments were serially diluted and mixed in a separate 96 well plate before transferring into the 96 well plate containing SK-N-BE cells. Cells were then returned into the incubator for 40–45 hrs before the addition of 0.1 vol. of WST-1 (Roche Diagnostics Corporation, Indianapolis, IN) to quantitate cell number. The plates were further incubated and developed according to the manufacturer's instruction. Panel A) Synthetic HotSpot peptide ligands, KcF12, KcC7, and KcF6 act as inverse agonists by inhibiting TNFR2-linked cellular survival in SK-N-BE cells. TNF-alpha causes cellular proliferation via a TNFR2-linked mechanism in SK-N-BE cells. Addition of a control peptide with a non-relevant sequence does not alter cell survival. Panel B) Blockade of TNFR2 binding pocket by Anti-TNFR2 antibody, mAb226, inhibits the cytotoxic action of KcC7 and KcF6, but not KcF12. Peptides were added at their ED80 values while mAb226 was at 10 μg/mL. IC80 (10 μM) concentrations, while KcD11 was added at either 10 μM or 100 μM.