Extended Data Fig. 1 |. Biochemical and molecular analysis of 5hmC and TET2 in response to glucose in primary blood cells and cultured cell lines.

a, Dot blot comparison of global 5hmC levels between PBMC gDNA from 28 healthy donors and 29 patients with diabetes, **P = 0.0017. b, Genomic DNA extracted from several cell types cultured under high or normal glucose were dot blotted for 5hmC, *P = 0.022 (PBMC), 0.046 (HUVEC), 0.047 (TF-1). c, Western blot revealed that endogenous TET2 protein levels in PBMCs, HUVECs and TF-1 cells were higher when cultured in normal glucose than in high glucose. TET1 and TET3 protein levels were minimally detectable in these cell lines. d, e, RT–qPCR (d) and western blot (e) showed that TET2 dominated amongst the TET family in A2058-TET2WT cells. In A2058 cells, expression of all three genes was extremely low. f, Flag–TET2 protein levels in whole-cell lysates from A2058-TET2WT cells cultured in high glucose or normal glucose. g, Comparable levels of Flag–TET2 mRNA from A2058-TET2WT cells cultured in high glucose or normal glucose. h, Half-lives of Flag–TET2 in A2058-TET2WT cells cultured in high glucose or normal glucose. i, High glucose shock resulted in an acute increase in 5hmC in A2058-TET2WT cells followed by a rapid drop to baseline. j, Long-term culturing of A2058-TET2WT cells in normal glucose resulted in a sustained increase in 5hmC, which could be reversed by switching the medium to high glucose. See Supplementary Information for more details. k, Dot blot quantification of 5hmC levels in A2058-TET2WT, mock, A2058-TET2M, and A2058-TET2CD cell lines in Fig. 1e; **P = 0.0068. Data in b–k represent three biologically independent repeats each. Two-sided Student’s t-test, data shown as mean ± s.d. *P < 0.05, **P < 0.01. For gel source data, see Supplementary Fig. 1.