X-linked gene expression in female XEN cells treated with the EZH2 inhibitor GSK126. (A) Representative images produced by immunostaining followed by fluorescence in situ hybridisation on RNA for H3K27me3 (green) and Xist (red) on female extraendoderm stem (XEN) cells (GHP7/9 cell line) treated or not with 2 μM GSK126 for 5 days. Arrowheads indicate the nuclear position of the inactive X chromosome coated with Xist RNA. The percentage of visible H3K27me3 accumulation on the Xist-coated X chromosome in each condition is indicated. n > 50. Scale bar = 5 μm. (B) Cumulative histograms showing allelic gene expression levels of Xist, Atp7a, Chic1, Hmgn5, Jpx, Ftx and Pbdc1 in female XEN cells (GHP7/9 cell line) treated (+) or not (−) with 2 μM GSK126 for 5 days. Mean values and standard deviations of two independent experiments are shown for each gene. Values have been standardised by the ubiquitously expressed Rplp0 gene. On the left, the diagram shows the position of the genes analysed by reverse transcription followed by quantitative polymerase chain reaction along the X chromosome. No significant difference in the relative levels of paternal X (Xp) chromosome expression could be detected between treated and untreated cells (P > 0.05 by Χ2 test). AU, Arbitrary unit; [K27-high], Enriched in H3K27me3 in female compared to male XEN cells; [K27-low], Low levels of H3K27me3 in female and in male XEN cells; Xm, Maternal X chromosome.