TWEAK/Fn14 interaction induces RANKL expression in MC3T3-E1 cells. (a) MC3T3-E1 cells were stimulated with 100 ng/ml TWEAK for the indicated times in the absence or presence of 1 μg/ml Fn14-Fc chimera, 10 μM LY2940002, or 10 μM PD98059. RNA was then extracted from the cells, and real-time PCR was performed using specific primers for RANKL and GAPDH (C, no treatment). The ratio of each gene to that of GAPDH was calculated, and the value of 1.0 was assigned to MC3T3-E1 cells that were incubated without TWEAK. (b) MC3T3-E1 cells were grown on slide chambers, stimulated with 100 ng/ml TWEAK for 24 and 48 hours, and then stained for RANKL as described in Materials and methods. RANKL-positive cells are shown in green. Goat IgG antibody was used as a negative control for the immunofluorescence staining. Fn14, fibroblast growth factor-inducible 14; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IgG, immunoglobulin G; RANKL, receptor activation of nuclear factor-κB ligand; TWEAK, tumour necrosis factor-like weak inducer of apoptosis.