Figure 5. MafB expression was enhanced in Sirt6-deficient BMMs.

(a) Sirt6^fl/fland Sirt6^fl/flMx1Cre BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for the indicated time periods. Cell lysates were subjected to immunoblot analysis with MafB, Blimp1, Sirt6, NFATc1 and Atp6v0d2 antibody. GAPDH was used as a loading control. (b) Quantitative real-time PCR was performed for the mRNA expression of Sirt6 and osteoclastogenic genes, such as Nfatc1, Atp6v0d2, and Blimp1, and anti-osteoclastogenic genes, such as Mafb, Irf8 and Bcl6. *P < 0.01. Data are represented as mean ± S.D. (c) Recruitment of Sirt6 to promoters of anti-osteoclastogenic genes such as Mafb, Irf8 and Bcl6 in the presence or absence of RANKL was detected by ChIP assay. Samples were subjected to quantitative real-time PCR with specific primers for the Sirt6-binding sites in the Mafb, Irf8 and Bcl6 promoter region. Sirt6 occupancy at the promoter is shown relative to the background signal with IgG control antibody. *P < 0.01. n.s: not significant. Data are represented as mean ± S.D. (d) 293 T cells were transfected with Flag-tagged Blimp1 together with V5-tagged Sirt6 construct. Protein lysates were prepared and subjected to immunoprecipitation (IP) using FLAG antibody. The total amount of transfected DNA was kept constant by addition of empty pFlag-CMV expression vector. (e) BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 3 days. Protein lysates were prepared and subjected to co-immunoprecipitation using the anti-Sirt6 or control IgG antibodies. (f) Working model for the role of Sirt6 during osteoclastogenesis. During osteoclastogenesis, Sirt6 was induced by the RANKL-NFATc1 axis. Sirt6 in cooperation with Blimp1 suppressed anti-osteoclastogenic transcription factor such as MafB.