Fig. 5.

Loss of Lkb1 leads to coalescence of BM-defective alveoli in vivo and trypsin-like serine protease-dependent spontaneous branching and aggregation of 3D acini. (A) H&E-stained nontumorous glandular tissue sections from W-Myc;W-Cre, W-Myc;W-Cre;Lkb1^lox/+, and Myc/Lkb1- mice illustrate the tissue ultrastructure and presence or absence of BM by Collagen IV staining. (Scale bar: 50 μm.) Note the alveolar multiplication typical for Myc-overexpressing glands (W-Myc;W-Cre) and intact BM surrounding the alveoli. The images of Myc/Lkb1- glands show merged epithelial structures and absence of BM. (Scale bar: 100 μm.) (B) Phase-contrast images of AdCre-treated W-Myc;Lkb1^lox/+ and W-Myc;Lkb1^lox/lox MMECs grown in 3D culture for 5 d show extensive aggregation and branching of acinar structures. (Scale bar: 100 μm.) (C) Immunostaining of W-Myc;Lkb1^lox/lox 3D acini shows increased apoptosis (active Caspase-3) and mitotic activity (phospho-Histone-H3) and reduced levels of BM components nidogen and Collagen IV in the aggregated Myc/Lkb1-deficient acini. Ultrastructure of acini is revealed by E-cadherin staining. (Scale bar: 20 μm.) (D) Trypsin-like serine protease activity mediates Lkb1-dependent spontaneous branching and aggregation of 3D acini. AdCre-treated Lkb1^lox/lox and W-Myc;Lkb1^lox/lox 3D acini were allowed to form for 3 d and subsequently treated for 48 h with the following effectors/inhibitors: TGF-β1, ROCK inhibitor Y-27632, MMP inhibitor GM6001, MEK inhibitor UO126, and trypsin-like serine protease inhibitor Compound 10a. (Scale bar: 100 μm.) The level of spontaneous branching (Lkb1^lox/lox) or branching/aggregation (W-Myc;Lkb1^lox/lox) of 3D acini in each treatment was evaluated on day 5 by visually comparing treated 3D acini with DMSO control (+++, extensive branching; ++, moderate branching; +, slight or no branching). (E) Quantification of TGF-β1 and Compound 10a-mediated inhibition of spontaneous branching and branching/aggregation. The 3D acini were treated as in A, and the mean and SD values were calculated from three independent experiments. (F) Compound 10a treatment inhibits mitotic activity without significant effect on apoptosis in the Lkb1^lox/lox and W-Myc;Lkb1^lox/lox 3D acini. (Scale bar: 20 μm.)