Figure 4

PDGF-D induced osteoclast differentiation is NFAT dependent. (a) Immunofluorescent staining of NFATc1 in RAW264.7 cells after treatments for 24 h with 12.5 ng/ml PDGF-B, 40 ng/ml PDGF-D or 20ng/ml RANKL. (b) Immunoblot analysis of cytoplasmic (Cyt) and nuclear (Nuc) fractions of NFATc1 in RAW264.7 cells after different treatments for 72 h. The same blot was reprobed with anti-histone H1 and GAPDH antibodies as controls for nuclear and cytoplasmic fractions, respectively. Densitometry of target bands was quantitated using Image J software, and the cytoplasmic and nuclear NFATc1 protein levels were normalized to GAPDH and histone H1, respectively. The NFATc1 band in the cytoplasmic fraction in serum-free condition was given as 1. (c) RAW264.7 cells were treated with recombinant PDGF-D (40 ng/ml) in the presence or absence of NFTA inhibitor III 0.3 μM for 5 days. The relative levels of TRAP mRNA over GAPDH mRNA after treatments were graphed. Statistical analyses were based on three independent experiments performed in triplicates. ^*P<0.01.