Figure 4.

Activation of autophagy at the early stage of differentiation is required for optimal osteoblast differentiation. A, Lysates from MC-3T3 cells obtained following exposure to DM for the indicated days were immunoblotted with anti-pAMPK (T172), pULK (S555), LC3 I/II, Beclin-1, or β-actin. B and D, Lysates from LacZ- (Ctrl) or IGFBP-2-overexpressing cells (B) or from MC-3T3 cells expressing shRNA sequence targeting LacZ (Ctrl Si) or IGFBP-2 (IGFBP-2 Si) (D) obtained on indicated day after DM exposure were immunoblotted with an anti-pULK (S555), LC3 I/II, Beclin-1, or β-actin antibody. C, Lysates from MC-3T3 cells after 3 days of DM exposure in the absence or the presence of compound C (CC) (1uM) were immunoblotted with an anti-pAMPK (T172), pULK (S555), LC3 I/II, Beclin-1, or β-actin. E, Lysates from MT-3T3 cells expressing the shRNA-targeting IGFBP-2 (IGFBP-2 Si) after 3 days of DM exposure in the absence or the presence of metformin (Met) (0.5mM) were immunoblotted with an anti-pAMPK (T172), pULK (S555), LC3 I/II, Beclin-1, or β-actin. F, Lysates from MC-3T3 cells after 3 days of DM exposure in the absence or the presence of IGF-I (100 ng/mL) or IGFBP-2 (1 ug/mL) or both were immunoblotted with an anti-pULK (S555) or β-actin. G, Lysates obtained from cells overexpressing AMPK T172D after 9 or 12 days of DM exposure in the absence or in the presence of compound C (1uM) were immunoblotted with an anti-pULK (S555), LC3 I/II, Beclin-1, or β-actin. H, MC-3T3 cells were stained by Alizarin Red on day 21 after DM alone (Ctrl) or DM plus bafilomycin-1A (2nM) added on the indicated day.