JCS03296 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Quantitative RT-PCR analyses of Smad7 in mES cells. (A) Expression of Smad7 transcripts in MGZ5 ES cells supertransfected with pCAG-Empty (control), Smad7 or Smad6 (Fig. 1). Smad7 transcript level is about ninefold greater in pCAG-Smad7 transfected ES cells than in control. (B) Expression of Smad7 transcripts in fSmad7-10⁺ and fSmad7-10⁻ ES cells (Fig. 3). Smad7 transcript level is about 4.5-fold higher in fSmad7-10⁺ ES cells than in EB3 and fSmad7-10⁻ ES cells. Expression of Smad7 was normalized to that of Gapdh expression at the same time. Each value represents the mean ± s.e.m. (n=4). *P<0.01 (vs. control).

• Supplemental Figure 2 -

  Fig. S2. Quantitative RT-PCR analyses of stem cell marker genes, Oct3/4 and Rex-1, in fSmad7-10+, fSmad7-10- and EB3 (control) ES cells. Each ES cells were cultured in FCS-containing medium for 5days. Rex-1 transcript level is about threefold higher in fSmad7-10+ ES cells than in EB3 and fSmad7-10- ES cells. Expression of each gene transcripts were normalized to those of Gapdh expression at the same time. Each value represents the mean ± s.e.m. (n=4). *P<0.05 (vs control); **P<0.01 (vs control).

• Supplemental Figure 3 -

  Fig. S3. Expression of TGF-β superfamily genes in ES cells. Expression of TGF-β superfamily ligands, receptors, and Smads in MGZ5 and EB5 cells was examined by RT-PCR. MGZ5 and EB5 ES cells were cultured in FCS-containing medium (FCS-containing) or serum-free medium (serum-free). As negative controls, RNAs from EB5 and MGZ cells were examined for β-actin expression without prior generation of cDNA RT (−), and a PCR reaction for each set of primers was run against H₂O. RNAs from NIH3T3 cells and mouse brains were used as positive controls for activin-βA, TGF-β3, ALK-5, TGFβR-II and ALK-7, to prove that primers used are functional.

• Supplemental Figure 4 -

  Fig. S4. Activin and Nodal induced Lefty-1 and Lefty−2 in ES cells. Expression of Lefty-1 (upper panel) and Lefty-2 (lower panel) was determined after treatment for 2, 5, 24 and 72 hours with the indicated factors by quantitative RT-PCR. Activin, Nodal and SB-431542 were added at concentrations of 30 nM, 1 μM and 3 μM, respectively. At each time point, levels of Lefty-1 and Lefty-2 expression were normalized to those of ß-actin expression at the same time. Each line represents the mean ± s.e.m. (n=3).

• Supplemental Figure 5 -

  Fig. S5. Endogenous BMP signals do not contribute to mES cell propagation. (A) Effects of noggin and BMP-4 on Smad1/5/8 phosphorylation in EB5 ES cells. EB5 cells were treated with the indicated factors, and subjected to western blot analysis using anti-phospho-Smad1/5/8 antibody (upper panel) and anti-α-tubulin antibody (lower panel, as loading control). (B) Colony morphologies of EB5 ES cells in serum-free medium supplemented with Noggin. EB5 colonies were grown for 7 days in serum-free medium supplemented with no factor (as control, left) or 90 ng/ml Noggin (right). Numbers indicate numbers of colonies appearing (n=3). (C,D) Relative numbers of cells treated with no factor or 90 ng/ml noggin present on Day 3 (C) and Day 7 (D) of culture compared with Day 0. Each bar represents the mean ± s.e.m. (n=4).

• Supplemental Figure 6 -

  Fig. S6. Activin resulted in decrease in G1 ratio and increase in G2-M ratio. Cell-cycle analysis of the effect of activin on EB5 ES cells. Semi-confluent cells were treated with 30 ng/ml activin for 24 hours. Cell cycle distribution was analyzed after propidium iodide staining of the nuclei using FACS. Each bar represents the mean ± s.e.m. (n=3).

• Supplemental Figure 7 -

  Fig. S7. Activin and Nodal induced Eras, Utf1 and repressed B-Myb in ES cells. Expression of ERas, Utf1, B-Myb, Myc and Cdk4 was determined after 2 days of treatment with the indicated factors at clonal density by quantitative RT-PCR. Activin, Nodal and BMP-4 were added at concentrations of 30 nM, 2 μM and 30 nM, respectively. Expression of ERas, Utf1, B-Myb, Myc and Cdk4 was normalized to that of Gapdh expression at the same time. Each value represents the mean ± s.e.m. (n=4)). *P<0.01 (vs control).