Figure 5.

Restoration of osteogenic and adipogenic differentiation potential of ank/ank BMSCs after transfection with expression vector containing ank. ank/ank BMSCs were transfected with empty expression vector (pcDNA) or expression vector containing ank (pcDNA-ank). In addition, ank/ank BMSCs and WT BMSCs were co-transfected with the TOPFlash luciferase reporter. A: Twenty-four hours after transfection cells were cultured in osteogenic differentiation medium for 5 days. mRNA levels of ALP were determined by real time PCR using SYBR Green and normalized to the level of 18S RNA. In addition, cell extracts were analyzed for luciferase activity from the TOPFlash reporter (TOPFlash). Transfection efficiency was monitored by co-transfection with Renilla luciferase vector, which provides constitutive expression of the Renilla luciferase reporter. B: Twenty-four hours after transfection cells were cultured in adipogenic differentiation medium for 5 days. mRNA levels of C/EBPβ and PPARγ were determined by real time PCR using SYBR Green and normalized to the level of 18S RNA. The mRNA levels of ALP, C/EBPβ and PPARγ are expressed as relative to the levels of WT BMSCs cultured in osteogenic or adipogenic differentiation medium for 5 days, which were set as 1. Data in A and B were obtained from three different experiments and are expressed as mean ± S.D. (^ap < 0.01 vs. WT cells, ^bp < 0.01 vs. ank/ank transfected with empty pcDNA vector).