Fig. 4.

MMP activity is required for branch extension but not branch initiation. (A) Bright-field images of control and GM6001-treated (20 µM) chicken lung explants after 24 h of culture. (B-D) After the 24 h culture period, fold-change in branch (B) initiation, (C) extension and (D) projected area were measured; n=4-6 independent experiments for B-D. (E-G) Remodeling of basement membrane was monitored by staining for laminin (LAM; green) in (E) control explants, as well as in (F,G) explants cultured in the presence of GM6001. Epithelial cells were stained for E-cadherin (Ecad, magenta). Arrows and arrowheads indicate regions of basement membrane thinning and accumulation, respectively. (H) Relative staining intensities were quantified as described in Fig. 2; n=4-6 independent experiments. (I) qRT-PCR analysis for relative MMP expression in HH28 lungs; n=3 independent experiments. (J) In situ hybridization for MMP2 in HH25-HH28 lungs. (K) Relative staining intensity of laminin in lung explants treated with the MMP2-specific inhibitor ARP100; n=4-6 independent experiments. Data are mean±s.d. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA with Tukey's post-hoc test). Scale bars: 200 µm in A,J; 50 µm in E-G.