Fig. 3. Synergistic association of phospho-AMPK and RUNX2 protein levels in differentiation and transdifferentiation models.

a Western blot analysis showing differentiation of myoblasts (C2C12) to osteogenesis was associated with increased RUNX2 and p-AMPK (T172) and b RT-PCR analysis confirms expression of RUNX2 downstream target, osteocalcin. c Adipogenesis was confirmed by staining for oil droplets using Oil Red O stain (3T3-L1) at different time points during differentiation. d Western blots showing decreased phosphorylated AMPK, which synergistically correlated with low levels of RUNX2 protein. Time-dependent increased expression of PPARγ was seen with adipogenesis. RT-PCR analysis showing e increased RNA levels of RUNX2 along with AdipoQ, PPARγ and C/EBPβ. f, g Activation of AMPK by metformin for 48 h before treating with adipogenic differentiation medium abrogated the effects of adipogenic inducers when compared with control (NM), and adipogenic medium (AM) alone in MSCs was evident from the reduced oil droplets (dark brown). h, i Representative picture of quantitative measurements of eluted Oil Red O stain precipitates (n = 3). j, k Western blot analysis for p-AMPK, RUNX2 and PPARγ expression. l Immunoprecipitation analysis of differentiated MSCs (C3H10t1/2) to adipogenesis with and without metformin showing the reduced levels of RUNX2, p-AMPK and RUNX2-S118 phosphorylation. m Oil Red O staining and western blot confirms the transdifferentiation of osteocytes (U2OS) to adipocytes after induction with adipogenic medium containing charcoal stripped FBS (10%) and rosiglitazone (1 µM) for 14 days. Mean ± S.E.M.; n = 3, *p < 0.1 versus control, **p < 0.05 versus control; ***p < 0.001 versus control