ERG-associated protein with SET domain (ESET)-depleted embryonic stem (ES) cells differentiate towards the trophectoderm lineage. (a) Western blot shows downregulation of ESET and histone 3 lysine 9 trimethylation (H3K9me3) in Eset knockdown ES cells at day 3 of short hairpin RNA (shRNA) transfection. Tubulin and H3K4me2 served as loading control. (b) Alkaline phosphatase staining of Eset knockdown (right panel) and control (left panel) ES cells after 7 days of shRNA transfection. (c) Morphology of fluorescence-activated cell sorting (FACS)-sorted Eset knockdown (right panel) and control (left panel) ES cells at day 5 of shRNA transfection. Scale bar, 50 μm. (d) Gene expression levels in Eset knockdown ES cells relative to control cells after normalising against Gapdh, a house keeping gene. Error bars, standard deviation (SD) of three technical replicates. (e) Images of three representative colonies from three different wells of control (top panel) and Eset knockdown (bottom) ES cells which were purified by FACS at day 3 of shRNA transfection followed by 4 days of culture in medium that is conducive for development of trophectoderm cells (TS medium). Cdx2-positive cells are labelled in red. Nuclei are labelled in blue. Scale bar, 100 μm.