Fig. 1: The 5-hydroxymethylcytosine (5hmC) levels dependent on ten-eleven translocation 1 (TET1) are necessary for the proliferation and differentiation of early mesenchymal stem cells (MSCs).

a Immunofluorescence shows that early MSCs (P2-4) exhibited increased 5hmC level compared to late MSCs (P10-12). b–d Early MSCs were treated with 2-hydroxyglutarate (2-HG) (5 mM) or vehicle control for 3 d, followed by (b) immunofluorescence assay to determine the 5hmC level, (c) WST-1 assay for growth curve, or (d) the induction of cells for osteogenic and adipogenic differentiation for 14 d. Cells induced for osteogenesis were stained by alizarin red S (ARS) staining, followed by measurement of extracted dye at an optical density (OD) of 550 nm, while the cells induced for adipogenesis were stained with Oil Red O staining, followed by measurement of extracted dye at OD 510 nm. We found that 2-HG treatment decreased the 5hmC level, inhibited cell proliferation, and suppressed the differentiation into osteoblasts and adipocytes. e–h Early MSCs were lentivirally transduced with scrambled (CTR), TET1 (TET1-KD) or TET2 short hairpin RNAs (shRNAs) (TET2-KD), followed by (e) western blotting for the detection of TET1 or TET2 levels and (f) immunofluorescence for the detection of 5hmC level. TET1 KD, rather than TET2 KD, decreased the 5hmC level. Cells without or with TET1 KD were subjected to (g) growth curve analysis by WST-1 assay, or (h) induced for osteogenic and adipogenic differentiation for 14 d. TET1 KD inhibited the cell proliferation, and suppressed the differentiation into osteoblasts and adipocytes. Results are expressed as the mean ± standard deviation (SD) of three independent experiments. (**p < 0.01, ***p < 0.001; ****p < 0.0001; unpaired two-tailed t-test, or ordinary one-way ANOVA) Exact p-values are shown in the Source Data file. Scale bar: 50 μm (a, b, f) and 500 μm (d, h).