Fig. 6. Conserved role for the mouse Tgif2. (A) BRE-luciferase activity in mammalian cells. Mouse C2C12 cells were transfected with Renilla luciferase and BRE-Luc. reporter constructs alone or in combination with mTgif2, as indicated. Transfected cells were stimulated 48 hours after transfection with BMP4 recombinant protein, as indicated. Renilla luciferase was used for normalization. (B) Eight-cell stage embryos were injected into both ventral (VV) or dorsal vegetal (VD) blastomeres with xTGIF2 or mTgif2 mRNAs. Explants were collected at tailbud stage and assayed for expression of the indicated markers by RT-PCR analysis. (C) BTC6 cells were transfected with increasing amount of shRNA targeted against mTgif2 or with control shRNA, and analyzed 48 hours later by real-time RT-PCR for the expression of endogenous mTgif2 level and indicated markers. The plot shows the regulation (expression ratio) of target genes in shTGIF2-transfected cells versus shControl-transfected cells. All the values were normalized to the reference gene SDHA (succinate dehydrogenase), and calculated using the software REST (Pfaffl et al., 2002). Data were determined in triplicate.