NPI-0052 suppresses TNF-induced invasive activity and RANKL-induced osteoclastogenesis. (A) H1299 cells (2.5 × 104) were seeded into the upper wells of a Matrigel invasion chamber overnight in the absence of serum, pretreated with 50 nM NPI-0052 for 4 hours, treated with 1 nM TNF for 24 hours in the presence of 5% serum, and then subjected to invasion assay. The value for no NPI-0052 and no TNF was set to 1.0. (B) RAW 264.7 cells (104) were plated overnight, pretreated with 50 nM NPI-0052 for 4 hours, and then treated with 5 nM RANKL. At 5 days later, cells were stained for TRAP and evaluated for osteoclastogenesis. Cells were analyzed under a phase contrast microscope (Nikon, Tokyo, Japan) and photographs were taken using Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX). (C) Photographs were taken after 5 days of incubation with RANKL. The numbers of TRAP-positive multinucleated osteoclasts (> 3 nuclei) per well were counted. Error bars indicate SD of triplicate value.
