Fig. 4

mTORC2-induced DNA hypomethylation is dependent on the downregulation of DNMT3A. A Enzymatic activities of fractionated nuclear DNMT in U87-EGFRvIII cells with shScramble or shRictor. OD, optical density; U, unit of DNMT enzyme control. B RNA-sequencing analysis of potential DNMT3A target genes regarding cell proliferation and differentiation in U87-EGFRvIII cells with siScramble or siRictor. Note that mTORC2 activation (Scramble) upregulates proliferation-related genes, but downregulates differentiation-related genes. KD, knockdown. C A shift in global DNA methylation level, represented by LINE-1 methylation in Scramble- or Rictor-depleted U87-EGFRvIII cells, with concurrent knockdown of DNMT3A. D Immunohistochemistry for mTORC2 activation marker (p-Akt S473), DNMT3A and 5-mC in human GBM tissue (n = 21). The scatter plots showed the negative or positive correlation between DNMT3A and mTORC2 marker (p-Akt S473: upper panel) or 5-mC (lower panel) respectively, based on quantitative immunohistochemistry. Scale bars, 40 µm (for pAKT and DNMT3A) and 80 µm (for 5-mC)