Fig. 2

TET1 and TET2 knockdown differentially regulated BMSC differentiation. Cultured BMSC were transfected with siRNA targeting either TET1 (siTET1), TET2 (siTET2), TET3 (siTET3) or scramble control siRNA (siScram). a Knockdown efficiency was analysed by qPCR relative to β-actin. b Knockdown of TET1, TET2 and TET3 protein levels by two siRNA per molecule was analysed by Western blot, using Raptor and Hsp90 protein as loading controls. c BMSC cultured under osteogenic inductive conditions were transfected with siTET1, siTET2, siTET3 or siScram. Representative images depict levels of Alizarin red-stained mineral deposits. d Extracellular calcium levels were measured and then normalised to DNA content per well. e Analysis of RUNX2, BMP2, OSTEOPONTIN (OPN) and OSTEOCALCIN (OCN) gene expression levels was assessed by qPCR per condition, relative to β-actin. f BMSC cultured under adipogenic inductive conditions were transfected with siTET1, siTET2, siTET3 or siScram. Representative images depict levels of Nile red-stained lipid droplets. g The number of adipocytes was expressed as a percentage relative to total DAPI-positive cells per unit area. h Analysis of PPARγ2, C/EBPα, ADIPSIN and LEPTIN 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