Fig. 3

Overexpression of TET1 and TET2 differentially regulates osteogenesis and adipogenesis. a BMSC donors were infected with empty lentiviral vector alone (Vector), or vector overexpressing TET1 cDNA (TET1OE) or TET2 cDNA (TET2OE). Gene expression levels were analysed by qPCR relative to β-actin. b Protein levels of TET1 and TET2 were analysed by Western blot, using Raptor protein as a loading control. c TET1-overexpressing BMSC were cultured under osteogenic (Osteo) inductive conditions control media (Cont) for 3 weeks. Representative images depict mineralised deposits stained using Alizarin red. Extracellular calcium levels were measured and then normalised to DNA content per well. d Analysis of RUNX2 and BMP2 gene expression levels was assessed by qPCR per condition, relative to β-actin. e TET2-overexpressing BMSC were cultured under Osteo or Cont media for 3 weeks. Representative images depict mineralised deposits stained with Alizarin red. Extracellular calcium levels were normalised to DNA content per well. f Analysis of RUNX2 and BMP2 gene expression levels was assessed by qPCR per condition, relative to β-actin. g TET1-overexpressing BMSC were cultured under adipogenic (Adipo) inductive conditions or Cont media for 3 weeks. Representative images depict levels of Nile red-stained lipid droplets. The number of adipocytes was expressed as a percentage relative to total DAPI positive cells per unit area. h Analysis of PPARγ2 and ADIPSIN gene expression levels was assessed by qPCR per condition, relative to β-actin. i TET2-overexpressing BMSC were cultured under Adipo or Cont media for 3 weeks. Representative images depict levels of Nile red-stained lipid droplets. The number of adipocytes was expressed as a percentage relative to total DAPI-positive cells per unit area. j Analysis of PPARγ2 and ADIPSIN gene expression levels was assessed by qPCR per condition, relative to β-actin. Data represent mean S.E.M., n = 3 BMSC donors, *p ≤ 0.05, one-way ANOVA with multiple comparison analyses