Fig. 3. P416R SH3BP2 mutation augments osteoclast-associated genes expression and nuclear translocation of NFATc1.

(A) Quantitative-PCR analysis of osteoclast associated genes. Bone marrow cells were isolated from Sh3bp2^+/+ and Sh3bp2^KI/+ mice. After 2-day preculture with M-CSF (25 ng/ml), BMMs were stimulated with TNF-α (100 ng/ml) for 96 hours. RNA samples were collected at indicated time points. Gene expression levels relative to Hprt were calculated and normalized to the expression level of Sh3bp2^+/+ BMMs at 0 hour. The data are representative of three independent experiments. (B) Immunofluorescent staining of actin, nuclei, and NFATc1 visualized by phalloidin, DAPI, and anti-NFATc1 antibody (clone: 7A6), respectively. BMMs were fixed 72 hours after TNF-α (100 ng/ml) treatment. (C) Quantitation of NFATc1-positive nuclei. The percentage of NFATc1-positive nuclei per total nuclei was measured at the indicated time points. (D) Western blot analysis for NFATc1. NFATc1 expression levels in nuclear and cytoplasmic fractions were determined at the indicated time points. (E) Relative NFATc1 expression. Ratio of NFATc1 to nuclear matrix protein p84 in nuclear fractions were calculated and normalized to the ratio of Sh3bp2^+/+ at 0 hour (n = 3). (F) Quantitation of TRAP+ MNCs. BMMs were stimulated with TNF-α (100 ng/ml) for 96 hours in the presence of FK506. (G, H) TRAP staining images and quantitation of TRAP+ MNCs. Sh3bp2^KI/+ mice were crossed with Nfatc1-floxed (Nfatc1^fl/fl) Mx1-Cre mice. Nfatc1 gene was deleted in hematopoietic cells by the injection of pI:pC. Nfatc1-deleted (Nfatc1^Δ/Δ) bone marrow cells were isolated and stimulated with TNF-α (100 ng/ml) for 96 hours. Data are presented as mean ± SD. +/+: Sh3bp2^+/+, KI/+: Sh3bp2^KI/+. * P < 0.05, NS: not significant.