HDACi greatly promote neuroectodermal specification. (A) Heat map of normalized expression levels of the typical neuroectoderm, mesoderm and endoderm genes which were differentially expressed after MGCD treatment for three days or seven days. Low expression levels are indicated by green colors, and high expression levels are indicated by red colors. (B , C) On day 7 of differentiation, real-time PCR was performed to determine the mRNA levels of typical genes for three germ layers after NaB or MGCD treatment for seven days. (D, E) On day 7, neuroectodermal cells were visualized by immunostaining with antibodies against SOX2 (red) and PAX6 (green) (D) or SOX2 (red) and NESTIN (green) (E). Scale bars, 50 μm. Higher magnification views are shown in the inset frames. Scale bars, 150 μm. (F-I) Protein levels of PAX6 and SOX2 were assessed by immunoblotting. The density of the bands was analyzed by the Image J software and expressed as fold of control. (J, K) Acetylation levels of histone H3K9 were detected by immunostaining (J) and immunoblotting (K), respectively. Higher magnification views are shown in the inset frames. Scale bars, 150 μm. (L) H9 cells were cultured in the ESC medium with the addition of NaB or MGCD for seven days and immunoprecipitated with anti-acH3K9 antibody. ChIP DNA levels of PAX6 were analyzed by semi-quantitative PCR. Scale bars, 50 μm. The error bars indicate SEM, *P <0.05; n >/=3. ChIP, chromatin immunoprecipitation; HDACi, HDAC inhibitors; SEM, standard error of the mean.