Fig. 1

Forced expression of Ssbp3 induces differentiation of mouse ESCs with a trophoblast-like gene expression pattern. a Western blotting of Ssbp3 protein levels in ESCs transfected with a vector, or an Ssbp3 plasmid. Twenty-four hours after transfection, ESCs were selected by puromycin for an additional 72 h. b Morphology changes and AKP staining of ESCs overexpressing Ssbp3. c–g Expression levels of pluripotency and lineage markers in ESCs overexpressing Ssbp3 determined by qRT-PCR analyses. Pluripotency markers (c), trophoblast markers (d), primitive endoderm and endoderm markers (e), mesoderm markers (f), and ectoderm markers (g). The average mRNA level in cells transfected with the control vector was set at 1.0. Data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01. h Immunofluorescence staining of ESCs 96 h after transfection. Samples were stained with anti-Cdx2 (red) antibody, and DAPI staining highlighted the nuclei (blue). i Western blotting of Cdx2 protein levels in ESCs 96 h after transfection. β-Tubulin was used as an internal control. j The schematic diagram of the Ssbp3 coding sequence and various truncation mutants. k qRT-PCR analysis for the expression levels of trophoblast markers in ESCs 96 h after transfection of plasmids as indicated. The average mRNA level in cells transfected with the control vector was set at 1.0. Data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01. l qRT-PCR analysis for mRNA levels of pluripotency and lineage markers 96 h after transfection of indicated plasmids. The average mRNA level in cells transfected with the control vector was set at 1.0. Data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01. m, n The mRNA and protein levels of Ssbp3 in ESCs and TSCs determined by qRT-PCR (m) and Western blotting (n). ESC embryonic stem cell, OE overexpression, TSC trophoblast stem cell