Figure 4: PGC-1β regulated the expression of SIRT3 during osteoclastogenesis.

(a) BMMs were induced to differentiate into osteoclasts with RANKL for indicated days. Relative mRNA levels of Sirt3, Nfatc1, and NFATc1-induced genes Atp6v0d2 were analyzed by qRT-PCR. (b) Immunoblot analysis of SIRT3, NFATc1, and Atp6v0d2 expression during osteoclast differentiation. Actin served as a loading control. (c) BMMs infected with retroviruses expressing PGC-1β or empty vector (EV) were stimulated with RANKL for 2 days. Total RNA was subjected to qRT-PCR analysis to assess Ppargc1b, Sirt3, Nfatc1, and Atp6v0d2 mRNA levels. *P < 0.01 between the indicated groups. (d) As in (c), except that cell lysates were subjected to immunoblot analysis with the indicated antibodies. Immunoblot analysis showed that SIRT3 protein level was induced by PGC-1β. (e) BMMs were transiently transfected with either irrelevant siRNA (si-control) or PGC-1β siRNA and were stimulated with RANKL. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. (f) Schematic representation of the mouse SIRT3 promoter-luciferase reporter constructs. pSIRT3-636-Luc containing a consensus ERRE site (Wt) and mutated ERRE site (Mut), respectively. (g) Expression plasmids for PGC-1β, ERRα or vector control were transfected into the HEK293T cells, along with the indicated pSIRT3-Luc constructs. The relative luciferase activity was corrected for Renilla luciferase activity and normalized to the vector control activity. (h) HEK293T cells were transiently co-transfected with a luciferase reporter plasmid containing a wild-type SIRT3 promoter construct pSIRT3-636-Luc or a mutant construct of the ERRE binding site together with the PGC-1β and ERRα expression plasmids. After 24 h incubation, cells were then assayed for relative luciferase activity. All values represent at least three independent transfections, each conducted in triplicate. Data represent means ± SD *P < 0.01, **P < 0.005.