Figure 4: NFATc1 regulated Sirt6 expression during osteoclastogenesis.

(a) (Left) BMMs were cultured with DMSO (control) or cyclosporine A (CsA, 10 μM) in the absence or presence of RANKL for 3 days and subjected to immunoblot analysis with Sirt6, NFATc1 and Atp6v0d2 antibody. GAPDH was used as a loading control. (Right) Quantitative real-time PCR was performed to detect expression of the Sirt6. *P < 0.01. Data are represented as mean ± S.D. (b) (Left) BMMs infected with pMX-puro (EV, empty vector) or constitutively active NFATc1 (caNFATc1) retroviruses were cultured for 6 days with M-CSF alone, and then cell lysates were subjected to immunoblot analysis with Sirt6, NFATc1 and Atp6v0d2 antibody. GAPDH was used as a loading control. (Right) Quantitative real-time PCR was performed to detect expression of the Sirt6. *P < 0.01. Data are represented as mean ± S.D. (c) Schematic representation of Sirt6 promoter luciferase reporters, which have different sizes, is shown. Black box indicates two putative NFATc1-binding sites (N1 and N2). +1 indicates the transcription start sites. Sirt6 promoter luciferase reporter vectors (0.35 kb-Luc and 0.03 kb-Luc) were transfected into RAW 264.7 cells with increasing concentrations caNFATc1(100, 200 and 300 ng). Data are presented as the mean ± S.D. (d) Recruitment of NFATc1 to the Sirt6 promoter in the presence or absence of RANKL was detected by ChIP assay. Samples were subjected to PCR with N1 and N2 specific primers for the NFATc1 binding sites of the Sirt6 promoter region.