Fig. 2

Increased protein crotonylation upon crotonate treatment promotes hESC differentiation into the endodermal lineage. A H9-LTR7 GFP hESCs were cultured in different concentrations of crotonate for 24 h before being harvested for western blotting as indicated. B H9-LTR7 GFP hESCs were cultured in different concentrations of crotonate for 12 days before analysis by microscopy (left) and flow cytometry (right). The percentage of GFP-expressing cells was plotted below. Cells cultured without crotonate or with 10 mM acetate (Ac) for 12 days served as controls. Scale bar, 100 μm. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01. C Cells from B were analyzed by RT-qPCR for marker gene expression. OCT4 and NANOG, pluripotency markers. GATA6 and SOX17, endoderm markers. MIXL1 and TBXT, mesoderm markers. SOX1 and NESTIN, ectoderm markers. Error bars represent mean ± S.D. (n = 3 independent experiments). D hEPS cells derived from reporter hESCs were cultured and western blotted as described in A. E, F hEPS cells derived from reporter hESCs were treated as described in B and similarly harvested for microscopy and flow cytometry E and RT-qPCR analysis F. Scale bar, 100 μm. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01. G H9-LTR7 GFP hESCs, converted EPS cells, and ectoderm and endoderm cells derived from the reporter hESCs were western blotted with the indicated antibodies