Figure 5.

(a) Sirt6 promotes DNA binding of ATF4 to the Ihh gene promoter. (a), (b) ATDC5 cells were treated with Sirt6 siRNA and grown in differentiation medium for 7 days. Total RNA was isolated from the cells and used in real-time RT-PCR with the indicated primers. Ihh, Col2a1, Col10a1 and Gli1 were decreased by Sirt6 knockdown. Reduced expression of Col10a1, but not Col2a1, was rescued by purmorphamine (a). (b) NMN treatment enhanced Col10a1, Ihh and Gli1. These effects were clearly abolished by Sirt6 silencing. (c) Impaired expression of Col10a1, but not Col2a1, induced by Sirt6 silencing was partially restored by the administration of salubrinal with regard to restoration of Gli1 expression. ATF4 expression was enhanced by salubrinal treatment. (d) Control and Sirt6-silenced primary chondrocytes were processed for ChIP analysis using antibodies for ATF4. PCR was then performed with primers flanking the promoter for Ihh. Full-length blots are presented in Supplementary Figure S4. (e) Representative ChIP of Ihh with anti-ATF-4 Ab (*) and anti-Sirt6 Ab (**) and qRT-PCR of ChIP-ed in primary chondrocytes. The gels have been run under the same experimental conditions. The displayed data are representative of three experiments.