Fig. 4

Epigenetic reprogramming promoted by αKG dose-dependent TET activity in AKT-activated cells during reprogramming. a A schematic representation of the 5mC-to-5hmC conversion step, including related enzymes (filled shapes) and metabolites (unfilled shapes). αKG is an essential cofactor for TET activity; conversely, 2HG is a TET activity-inhibitory metabolite. b-e Requirement of TET-enzymatic activity for an enhanced reprogramming efficiency in AKT-activated cells, as revealed by a decrease in reprogramming resulting from TET inhibition due to 2HG production by IDH1 R132H. Cellular αKG (b) and 2HG (c) levels in cells undergoing reprogramming induced by OSKA, OSKA + IDH1 and OSKA + IDH1 R132H were quantified by ion chromatography–tandem mass spectrometry at 10 dpi. Genome-wide 5hmC levels were quantified by dot blot analysis (d). GFP-positive colonies were counted at 10 and 14 dpi (e). f and g Correlation between αKG levels and genome-wide 5hmC levels at 10 dpi. AOA administration at 3 dpi reduced αKG levels in AKT-activated cells in a dose-dependent manner, as quantified by ion chromatography–tandem mass spectrometry (f). Genome-wide 5hmC levels of cells undergoing reprogramming with or without AOA administration at 3 dpi were quantified by dot blot analysis (g). h Requirement for upregulated αKG production for an enhanced reprogramming efficiency in AKT-activated cells. GFP-positive colonies were counted at 10 dpi. AOA administration at 7 dpi reduced the reprogramming efficiency, but the simultaneous administration of DM-αKG rescued the reprogramming efficiency. i Effects of DM-αKG on the reprogramming efficiency with or without TET2CD overexpression. GFP-positive colonies were counted at 14 dpi. The dot blot images shown in Figs. 3 and 4 are from the same membrane as shown in Additional file 5: Fig. S4. Therefore, the ‘total’ in Fig. 3b, ‘IDH1-’ in d and ‘AOA-’ in g are the same as ‘OSKA’ shown in Additional file 5: Fig. S4