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. 2016 Nov 4;7:13287. doi: 10.1038/ncomms13287

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Copyright © 2016, The Author(s)

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(a) Cell cycle analysis of recombinant human NODAL (rhNODAL) or SB431542 treatment compared with negative control (NC) in hESCs. DMSO is used as NC. s.d. of three replicates is indicated, ratio of G2 phase were compared for significance, **P<0.01, t-test, n=3. (b) EdU corporation analysis (n=3) of rhNODAL or SB431542-treated hESCs compared with control. Quantification of EdU corporations using flow cytometry (n=3) were shown in the upper right panel. The lower right panel shows the colony formation assay of hESCs with indicated treatment (n=2). Scale bar, 50 μm. (c) Fetal bovine serum induced differentiation assay in hESCs. The left panel shows the microscopy images of alkaline phosphatase stained hESCs under indicated treatment. Scale bar, 200 μm. The right panel shows the qPCR analysis of the pluripotency genes in the indicated treatment group. SB431542, an ALK4/7 inhibitor. The significance of changes in OCT4 and NANOG expression were compared with GAS5+FBS group. Here Mock represents untreated hESCs, NC represents FBS-induced hESCs with no other treatment. *P<0.05, **P<0.01, t-test, n=3. (d) Luciferase activities of SMAD reporter in different treatment groups in hESCs. *P<0.05, **P<0.01, t-test, n=4. (e,f) Analysis of expression changes of NODAL, OCT4, NANOG and SOX2 in the indicated groups. Westerm Blot analyses are shown in the right panel. NC represents hESCs treated with empty vector transfection. **P<0.01, t-test, n=3. (g) The left panel shows the scheme for co-culture assay. Right panel shows the mRNA level of pluripotency genes and GAS5 of the upper chamber cells in the indicated groups. NC/NC stands for no treatment in both upper and lower chamber, NC/GAS5 stands for GAS5 overexpression in the lower chamber cells, SB431542/GAS5 stands for SB431542 treatment in the chambers and GAS5 overexpression in the lower chamber cells. rhNODAL/shGAS5 stands for rhNODAL treatment in the chambers and GAS5 knockdown in the lower chamber cells. **P<0.01, t-test, n=3. (h) NODAL ELISA testing the level of NODAL secreted in the indicated groups. **P<0.01, t-test, n=4. See also Supplementary Fig. 4. Error bars represent standard deviation of the indicated experiment replicates. RNA level of β-actin served as internal reference for qPCR.(a) Cell cycle analysis of recombinant human NODAL (rhNODAL) or SB431542 treatment compared with negative control (NC) in hESCs. DMSO is used as NC. s.d. of three replicates is indicated, ratio of G2 phase were compared for significance, **P<0.01, t-test, n=3. (b) EdU corporation analysis (n=3) of rhNODAL or SB431542-treated hESCs compared with control. Quantification of EdU corporations using flow cytometry (n=3) were shown in the upper right panel. The lower right panel shows the colony formation assay of hESCs with indicated treatment (n=2). Scale bar represents 50 μm. (c) Fetal bovine serum induced differentiation assay in hESCs. The left panel shows the microscopy images of alkaline phosphatase stained hESCs under indicated treatment. Scale bar represents 200 μm. The right panel shows the qPCR analysis of the pluripotency genes in the indicated treatment group. SB431542, an ALK4/7 inhibitor. The significance of changes in OCT4 and NANOG expression were compared with GAS5+FBS group. Here, Mock represents untreated hESCs, NC represents FBS-induced hESCs with no other treatment. *P<0.05, **P<0.01, t-test, n=3. (d) Luciferase activities of SMAD reporter in different treatment groups in hESCs. *P<0.05, **P<0.01, t-test, n=4. (e,f) Analysis of expression changes of NODAL, OCT4, NANOG and SOX2 in the indicated groups. Westerm blot analyses are shown in the right panel. NC represents hESCs treated with empty vector transfection. **P<0.01, t-test, n=3. (g) The left panel shows the scheme for co-culture assay. Right panel shows the mRNA level of pluripotency genes and GAS5 of the upper chamber cells in the indicated groups. NC/NC stands for no treatment in both upper and lower chamber, NC/GAS5 stands for GAS5 overexpression in the lower chamber cells, SB431542/GAS5 stands for SB431542 treatment in the chambers and GAS5 overexpression in the lower chamber cells. rhNODAL/shGAS5 stands for rhNODAL treatment in the chambers and GAS5 knockdown in the lower chamber cells. **P<0.01, t-test, n=3. (h) NODAL ELISA testing the level of NODAL secreted in the indicated groups. **P<0.01, t-test, n=4. See also Supplementary Fig. 4. Error bars represent s.d. of the indicated experiment replicates. RNA level of β-actin served as internal reference for qPCR.