Figure 6.

Biphasic regulation of AMPK is required for calvarial osteoblast differentiation. A and B, Calvarial osteoblasts isolated from IGFBP-2^+/+ WT or IGFBP-2^−/− mice were exposed to differentiation medium (DM) for indicated number of days. A, Lysates were immunoblotted with anti-pAMPK(T172) or β-actin. B, Calvarial osteoblasts isolated from IGFBP-2^−/− mice were exposed to DM for indicated days in the absence or the presence of IGFBP-2 (1 ug/mL). Lysates were immunoblotted with anti-pAMPK (T172), pULK (S555), or AMPK. C, Lysates were immunoblotted with an anti-pULK (S555), LC3 I/II, Beclin-1 or β-actin. D, E, and G, Cells were stained by Alizarin Red on day 21 after DM alone (control) or DM plus bafilomycin A1 (2nM) (D), compound C (1uM) (E), or metformin (0.5 mM) (G) that had been added on the indicated day. F and H, Cell lysates from the same treatments as panel E (F) or G (H) were immunoblotted with an antiosteocalcin or β-actin antibody. I, Lysates from cells that had been exposed to DM for indicated number of days in the absence or presence of metformin (0.5mM) for 2 hours were immunoblotted with anti-pAMPK (T172) or AMPK.