Fig. 4
Rescue of cell proliferation defects with TGF-β receptor inhibitors. a Cell proliferation assay on control (Ctrl) and Mir17-92:Mir106b-25-deficient (17 dKO) limb bud mesenchymal cells treated with vehicle (DMSO) or a STAT3 inhibitor (STAT3 inh, S31-201, 100 µM). Ctrl and 17 dKO limb bud cells were prepared by transducing YPF (Ctrl) or Cre (17 dKO) in vitro. STAT3 inhibitor treatment shows no significant improvement in 17 dKO cell proliferation. b Treatment of limb bud cells with S31-201 downregulates the p-Stat3 level; *non-specific band. c Cell proliferation assay on Ctrl and 17 dKO (dKO) cells treated with DMSO or a TGF-β receptor inhibitor (Tgfbr inh, Ly364947, 0.2 µM). Treatment with Ly364947 has no significant effect on control cells whereas it significantly ameliorates the proliferation defect of 17 dKO cells (n = 6, *p < 0.05 vs. 17 dKO + DMSO). d Treatment of limb bud cells with Ly364947 downregulates the p-Smad2 level. e–g Ly364947 treatment efficiently rescues the skeletal defects of 17 dKO mutants. Ly364947 (1 mg/kg/day, i.p.) was injected into pregnant and nursing mothers from E9.5 through P7.5, and then injected directly into individual mice. The shortening of the fifth digit (double arrows) syndactyly (white arrows) (e), missing middle phalanx (M) (f), and microcephaly and frontal bone ossification defect (black arrows) (g) in 17 dKO mutants were ameliorated by Ly364947 treatment. h Quantification of the length of the fifth mesophalanx (5th M), fifth metacarpal bone (5th MC), and the ratio of 5th M to 5th MC. Values are expressed as mean ± SE (n = 6 each group, p < 0.001 vs. dKO). Scale bars: 0.5 cm in e, 1.0 cm in g. D distal phalanx; P proximal phalanx
