Figure 4.

mTORC2 regulates histone acetylation through nuclear translocation of PDH/PKM2 and suppression of class IIa HDACs. A, immunofluorescent staining of PDH and PKM2 in U87-EGFRvIII cells treated by PP242 (mTORC1/C2 inhibitors) for 24 h. Arrowheads indicate each nucleus, and an arrow shows the mitochondrial distribution. Green, PDH staining; red, PKM2 staining; blue, 4′,6-diamidino-2-phenylindole (DAPI) staining. Scale bar, 10 μm. B, subcellular fractionation of cytosolic and nuclear protein from U87 cells with or without Rictor cDNA overexpression, followed by immunoblotting against PDH (E1α) and PKM2. Lamin and GAPDH are loading controls for nuclear and cytoplasmic proteins, respectively. Bar graphs showing the nuclear/cytoplasmic ratio of each protein in control and Rictor-overexpressing U87 cells. C, bar graphs showing the total, cytoplasmic, and nuclear amount of acetyl-CoA from U87-EGFRvIII cells with Scramble or Rictor knockdown. D, ELISA-based detection of global histone H3 acetylation in U87 cells overexpressing Rictor, combined with siRNA-mediated knockdown of PDH or PKM2. E, quantitative immunoblot analyses of histone modifications including methylation and acetylation in U87-EGFRvIII cells with the overexpression of class IIa HDAC (HDAC4–3A: phosphorylation-resistant mutant form). F, immunoblot analysis demonstrates that H3K9ac reduction in Rictor-depleted U87-EGFRvIII cells was partially rescued by the concurrent inhibition of HDACs with TSA for 24 h.