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Fig. 4. Proteinase inhibitors suppress branching morphogenesis induced by growth factors. (A, part a) Quantification of the percentage of branching organoids treated with (i) KGF or (ii) EGF in the presence of protease inhibitors E64, pepstatin, leupeptin, aprotinin, GM6001 and GM1210. Only the MMP inhibitor GM6001 had a statistically significant inhibitory effect on branching morphogenesis of both basal (*P<=0.05) and growth factor-treated organoids (***P<=0.001). The mean±s.d. of six samples in two independent experiments are shown. (A, part b) Primary organoids after 6 days of treatment with KGF and EGF with or without GM6001. Note the absence of branches in the organoids treated with the MMP inhibitor. Similar results were obtained with bFGF and HGF (not shown). (A, part c) Quantification of branching and (A, part d) appearance of the organoids treated with TIMP1 and TIMP2 in the presence of KGF. The mean±s.d. of six (TIMP1) and five (TIMP2) samples in two independent experiments are shown; (***P<=0.001). (A, part e) Quantification of the effect of proteinase inhibitors on branching morphogenesis induced by co-culturing the organoids with primary fibroblasts from virgin animals (Fv). Only GM6001 had a statistically significant inhibitory effect (**P<=0.01). The mean±s.d. of six samples in two independent experiments are shown. (B) GM6001 inhibits increase in cell number in organoids (B, part a), but does not alter apoptosis. Primary organoids were embedded in collagen I gels and treated for 6 days with KGF in the presence and absence of GM6001. (B, part a) Cell number was measured using Alamar Blue. The mean±s.d. of six samples in two independent experiments are shown. (B, part b) Whole organoids (3D) and cross sections are shown. Apoptotic nuclei were stained in green (FITC). Cross sections were also stained for {alpha}-smooth muscle actin-positive cells (red). There were no statistical differences in the number of apoptotic cells in control and treated groups. DAPI nuclear counterstain is in blue. Scale bars: 400 µm in A; 200 µm in B.