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. Author manuscript; available in PMC: 2019 Mar 22.
Published in final edited form as: Nature. 2018 Jul 18;559(7715):637–641. doi: 10.1038/s41586-018-0350-5

Extended Data Fig. 8 |. In vitro characterization of the effects of glucose and the AMPK–TET2 axis on tumour suppression.

Extended Data Fig. 8 |

a. MTS proliferation assay shows that A2058-TET2WT cells grew significantly more slowly under normal glucose than under high glucose, but mock cells showed no difference; n = 5 biologically independent repeats, P = 5.68 × 10−5. b, TET2 was effectively knocked down (KD) in TF-1 cells, as confirmed by western blot (left) and RT–qPCR (right). c, TF-1 TET2 KD cells exhibited cytokine-independent growth under no cytokines, consistent with a previous report26. d, Proliferation of TF-1 shControl and shTET2 cells treated with high or normal glucose under the indicated cytokine concentrations. TF-1 shControl proliferation was slower under the lower glucose condition, but TF-1 shTET2 cells showed no differential growth rates. P values (from left to right) are 0.0078, 1.94 × 10−5, 3.21 × 10−4, 0.0011, and 0.0091. a and d suggest that TET2 is a crucial point through which glucose levels affect tumour growth rates. n = 3 biologically independent repeats in b–d. e, Left, dot blot showing 5hmC levels in A2058-TET2WT, A2058-TET2S99A and mock cells with (+) and without (−) metformin (5 mM) treatment. Right, normalization of n = 5 biologically independent dot blot repeats; P = 0.026. f, MTS proliferation assay of A2058 cells expressing TET2WT, TET2S99A or mock treated with (+) or without (−) metformin (5 mM); n = 5 biologically independent repeats, P = 7.18 × 10−5. g, h, Soft agar growth of A2058-TET2WT, A2058-TET2S99A and mock cells treated with 0 mM, 2 mM or 4 mM metformin (g) and respective colony counting (h). P = 6.99 × 10−5 (top), 0.004 (middle), 0.0013 (bottom). i, j, mRNA (i) and protein (j) levels in MDA-MB-231 cells stably expressing mock, TET2WT or TET2S99A. k, l, Soft agar growth (k) and quantification (l) of MDA-MB-231 cells stably expressing mock, TET2WT or TET2S99A; n = 3 biologically independent repeats. The data suggest that TET2WT suppresses anchorage-independent tumour cell growth in vitro. Prevention of TET2 phosphorylation at S99 results in loss of this TET2-mediated suppression. Two-sided Student’s t-test, data shown as mean ± s.d. *P < 0.05, **P < 0.01, ****P < 0.0001.