Valproic acid reduces expression of selected pluripotency genes but does not trigger long-term changes in cell growth or differentiation. (A) Embryonic stem (ES) cells (CCE/R) were treated for 8 h with 1 mM valproic acid (VPA) followed by culture for one or five days in the absence of the drug (wash out, WO). The expression of four pluripotency-associated genes was quantified by real time-quantitative (RT-q)PCR, relative to the expression of β-actin, in untreated, VPA-treated and WO cells. Error bars represent the standard error (SE) of the mean of independent experiments (n = 3). To assess the significance of a change, two-tailed unpaired t-tests were performed using GraphPad software; ***P < 0.001. (B) Levels of OCT4 and KLF4 proteins were checked before (left lane) and after (right lane) VPA treatment by western blotting. A measure of the secondary antibody fluorescent signal, normalised to the β-actin signal, allowed a quantitative representation of changes. Error bars indicate SE from 3 independent experiments. (C) Light microscopy was used to assess the cell morphology at each timepoint (upper panels). Alkaline phosphatase activity (red staining) was used as a marker of pluripotency (lower panels, pictures taken using 20× magnification). (D) ES cells were stained with propidium iodide (PI) and analysed by flow cytometry to obtain the mean percentage of cells in each phase of the cell cycle before and after VPA treatment and WO (n = 3).