DEV01248 Supplemental Figure

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• Supplemental Figure 1 -

  Fig. S1. Functional characterization of the Smad4^Nallele. Smad4^N/N ES cells were generated by targeting the remaining wild-type allele in Smad4^N/+ES cells with the Smad4^TA replacement vector followed by Cre-mediated excision of exon 1 (Fig. 1A). (A) Western blot analysis of ES cell and HepG2 hepatoma cell protein extracts with a Smad4 monoclonal antibody. A 64 kDa immunoreactive band is seen in wild-type CCE ES cells, Smad4^N/+ ES cells and control HepG2 cells but not Smad4^N/N ES cells. (B) Semi-quantitative RT-PCR analysis of Smad4 target genes in embryoid bodies (EBs). RNA was isolated from wild-type (+/+), heterozygous (N/+) and homozygous (N/N) ES cells on days 5, 7, 10 and 12 of differentiation. Genes examined were Msx1 and Msx2, Smad4-dependent targets of Bmp signaling, and the visceral endoderm markers, Hnf4 and transferrin. Gapd was used as a normalization indicator. The marked downregulation of these genes in Smad4^N/N EBs was identical to that described for the previously characterized Smad4^tm1Ari null allele (Sirard et al., 1998; Sirard et al., 2000). (C) Smad4^N/N murine embryonic fibroblasts (MEFs) were generated for reporter assay analysis using a tamoxifen-inducible Cre transgene (Hayashi and McMahon, 2002). Wild-type (black bars) and Smad4-deficient (grey bars) fibroblasts were transfected in triplicate with 3TP-lux and stimulated with Tgfb1 ligand. A Tgfb response was detected in wild-type, but not Smad4^N/N MEFs. Separately, constitutively active Bmp type I receptor (caALK3) was transfected together with Msx2-lux. A Bmp-response was detected only in wild-type cells. Ligand-responsiveness was restored to Smad4^N/N MEFs by co-transfection with a Smad4 expression construct (pCMV-MADR4, white bars). These Smad4-dependent Tgfb and Bmp responses reproduce those observed in Smad4^tm1Ari null MEFs (Sirard et al., 2000).