Figure 5: SIRT3 regulated AMPK stability.

(a) BMMs from WT or Sirt3^−/− mice were stimulated with RANKL for the indicated time periods. Activation of AMPK or ACC and expression levels of NFATc1, Atp6v0d2, and SIRT3 during osteoclast differentiation. (b) The chemiluminescence signals for phospho-AMPK and AMPK were quantified and normalized based on the signals of AMPK and actin, respectively. Data represent means ± SD *P < 0.05, **P < 0.01. (c) RANKL stimulated BMMs transfected with si-control (scramble siRNA) or si-SIRT3 (siRNA against SIRT3) were immunoblotted with the indicated antibodies. (d) AMPK degradation in Sirt3^−/− osteoclasts is inhibited by proteasome inhibitor. BMMs from Sirt3^−/− mice were stimulated with RANKL in the absence or presence of MG-132 for 3 days. AMPK protein level was determined. Actin serves as a loading control. (e) Retroviral AMPKα introduction rescued osteoclast differentiation in Sirt3^−/− BMMs. Sirt3^−/− BMMs were transduced with empty or AMPKα retrovirus and then stimulated with RANKL. TRAP⁺MNCs (>5 nuclei) were counted. Scale bar, 500 μm. *P < 0.01. Data are represented as mean ± SD. (f) BMMs from Sirt3^−/− mice were transduced with retroviruses expressing AMPKα or EV and were treated with RANKL for 3 days. NFATc1 and Atp6v0d2 protein expression was determined by immunoblot analysis. Actin serves as a loading control.