Gossypin inhibits TNF-induced invasion and RANKL-induced osteoclastogenesis. (A) Gossypin suppressed TNF-induced invasion. H1299 cells were seeded in the top of a Matrigel invasion chamber overnight in the absence of serum, preincubated with 50 μM gossypin for 2 hours, treated with 1 nmol/LTNF for 24 hours in the presence of 1% serum, and then subjected to an invasion assay, as described in “Materials and methods.” (B and C) Gossypin inhibited RANKL-induced osteoclastogenesis. RAW 264.7 cells were incubated alone or with RANKL (5 nmol/L), with or without 50 μM gossypin, for 5 days and stained for TRAP expression. (B,D) Multinucleated (three nuclei) osteoclasts were counted. (C) TRAP-positive cells were photographed (10× 0.25 objective, TMS, Nikon, Tokyo, Japan; Coolpix 950 color camera, Nikon, Lewisville, TX). Original magnification, ×100. (D-E) Gossypin inhibited RANKL-induced osteoclastogenesis. Mouse (C57BL/6J) primary bone marrow cells were incubated alone or with RANKL (5 nmol/L), with or without 50 μM gossypin, for 3 days and stained for TRAP expression. (D) Multinucleated (three nuclei) osteoclasts were counted. (E) TRAP-positive cells were photographed. Original magnification, ×100.