Fig. 2. Neuropilin 1 is required for SMG branching morphogenesis in mice. (A) Npn1-neutralizing antibody inhibited SMG branching in a concentration-dependent manner. (a) Representative photographs at the indicated time point of the SMG explants treated with either control antibody or different concentrations of anti-Npn1 antibodies. (b) The numbers of terminal buds in each cultured SMG explant were counted and summarized (from five independent experiments). Paired t-test: ^*, P<0.05; ^**, P<0.01. (B) ODNs against Npn1 mRNA specifically blocked SMG branch formation. (a) Top panels: representative photographs of the SMG explants cultured 48 hours after treatment with 2 µM Npn1 antisense ODNs, scrambled ODNs or Npn1-sense ODNs. Middle panels: RNA in situ hybridization indicated that the Npn1 transcript decreased in the explant treated with Npn1 antisense ODNs. Bottom panels: BrdU (bromodeoxyuridine) labeling revealed that the rates of cell proliferation were not significantly changed in each experimental condition. (b) The numbers of terminal buds in each SMG explant cultured for 48 hours were counted and are summarized as a bar graph (from five independent experiments). Paired t-test: ^*, P<0.05. (c) Quantification of the proliferative activity labeled by BrdU is shown by bar graph as the green fluorescence intensity relative to DAPI-stained blue fluorescence intensity per unit area analyzed by MetaMorph software (n=12). (C) Semi-quantitative RT-PCR indicated that Npn1 transcripts were significantly reduced in the SMG treated with Npn1 antisense ODNs. Scale bars: 100 µm. AS, Npn1 antisense ODN; C, scrambled sequence; HGF, hepatocyte growth factor; S, Npn1-sense ODN.