Fig. 5
Distinct mechanisms in Feingold syndrome mouse models. a Forelimbs of mice with indicated genotypes at postnatal day 16. Wildtype control (Ctrl), Mycn conditional Knockout (Prx1-Cre:Mycnfl/fl, Mycn cKO), Mycn cKO in which one allele of Tgfbr2 is deleted (Prx1-Cre:Mycnfl/fl:Tgfbr2fl/+, Mycn cKO:Tgfbr2 Het), Mir17-92 and Mir106b-25 doubly conditional knockout (Prx1-Cre:Mir17-92fl/fl:Mir106b-25−/−, 17 dKO), and 17 dKO in which one allele of Tgfbr2 is deleted (Prx1-Cre:Mir17-92fl/fl:Mir106b-25−/−:Tgfbr2fl/+, 17 dKO:Tgfbr2 Het). Unlike 17 dKO mice, the digit abnormalities of Mycn cKO mutants are not rescued by Tgfbr2 heterozygous deletion. b Alizarin red and alcian blue staining of forelimbs of corresponding mice in a. Scale bars, 0.5 cm. More than three rescued mice per each model were analyzed to confirm the reproducibility. c Immunoblot analysis for indicated proteins upon treatment with 10% fetal bovine serum (FBS) at indicated time points (top panel). Primary mesenchymal fibroblasts from limb buds of E10.5 Mycnfl/fl embryos were transduced with adenoviruses expressing a Cre recombinase (KO) or a yellow fluorescent protein (Ctrl) to delete Mycn in vitro. Cells were serum starved for 1 h before stimulation. The efficient reduction in the Mycn protein level was confirmed. Levels of p-Akt (Thr308) and p-S6k are decreased in Mycn-deficient limb bud cells (KO). Quantification of p-Akt relative to total Akt (t-Akt) and p-Smad2 to total Smad2 (t-Smad2) at time 0 based on the western blot data (bottom panels) (n = 3, *p < 0.05). d Relative expression of previously reported targets of miR-17-92 miRNAs in Mycn cKO limb bud mesenchymal cells isolated from embryos at age E10.5. Y axis, arbitrary units
