Example 2
Human PBMCs were plated in 96 well plate at a density of 150,000 cells per a well in RPMI medium supplemented with 10% FBS. The cells were pre-incubated with compounds for 2 h. Following pre-incubation, the cells were stimulated with 100 ng/mL LPS for 18 hours. The secreted IL-23 was determined by ELISA (Human IL-23 Quantikine ELISA Kit, R&D cat #D2300B). Conclusion: APY0201 and Compound 1 (designated as NSN22769) completely blocked secretion of IL-23 by the LPS-induced PBMCs. See
Acute myelomonocytic leukemia cells ML-2 were plated in 96 well plate at a density of 50,000 cells per a well in RPMI medium supplemented with 10% FBS. The cells were exposed to compounds and the early apoptosis marker, Caspase3/7 activity, was measured in the cells at 15 h, 24 h and 41 h after exposure to compounds. The caspase activity was determined using Caspase-Glo® 3/7 Assay (Promega) and according to the protocol provided by the manufacturer. Conclusion: all three compounds including Compound 1 (designated as NSN22769) triggered Caspase3/7 activation in ML-2 cells—a hallmark of early apoptosis. See