Fig. 1

Crotonate-treated hESCs display transcriptomic changes associated with endodermal development. A H9-LTR7 GFP reporter hESCs were cultured in crotonate (Cr)-containing (10 mM) maintenance medium for 12 days before phase-contrast and fluorescence microscopy. Scale bar, 100 μm. B Cells from A were harvested after 12 days of Cr treatment for RNA-seq. Untreated H9-LTR7 GFP reporter hESCs served as controls. Results from gene ontology (GO) analysis of genes up- or down-regulated with treatment were plotted as shown. C Results from B were compared to RNA-seq data using endodermal cells derived from H9-LTR7 GFP reporter hESCs to calculate the Pearson correlation coefficients. D Cells from A were harvested after 12 days of Cr treatment for RT-qPCR analysis of the indicated endodermal development genes. Untreated cells served as controls. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01. E H9-LTR7 GFP reporter hESCs were cultured in crotonate (Cr)-containing (10 mM) maintenance medium and harvested for RT-qPCR analysis at the indicated time points for pluripotency (top) or endoderm (bottom) markers. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. *p < 0.05, **p < 0.01. F H9-LTR7 GFP reporter hESCs were cultured first in LCDM medium for 15 passages to derive hEPS cells, which were then cultured in crotonate (Cr)-containing (10 mM) maintenance medium for 12 days before phase-contrast and fluorescence microscopy. Scale bar, 100 μm. G Cells from F were harvested after 12 days of Cr treatment for RNA-seq and GO analysis. H RT-qPCR analysis for the indicated marker genes. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01. I H9-LTR7 GFP hESCs (±10 mM Cr for 12 days), converted EPS cells (±10 mM Cr for 12 days), and endodermal cells derived from the reporter hESCs were immunostained with an anti-GATA6 antibody. DAPI was used to stain the nuclei. Scale bars, 10 μm