HDAC inhibitors increase acetylation and self-renewal. (A) E14 cells were treated with vehicle (DMSO, left) or with TSA for the indicated time, and histone acetylation levels were assayed using antibodies specific for pan-acetylated H3 (H3ac, top) and H3 acetylated on lysine 9 (H3K9ac, bottom). (B) Quantification of (A). (C) Western blots of H3K9ac in E14 ESCs treated with VPA or NaBu for 16 h. Quantification is shown on the right. (D) Increased acetylation of E14 ESC increases teratoma size in vivo. E14 ESC were treated with VPA (0.5 mM) or TSA (5 nM) for 14 h and transplanted under the kidney capsule in SCID mice. Teratomas were analyzed three weeks following transplantation. (E–G) Hematoxylin-Eosin staining of representative sections of teratomas formed by control E14 ESC (E), VPA-treated E14 ESC (F) and TSA-treated E14 ESC (G). All teratomas displayed evidence of endoderm, mesoderm and ectoderm.