Figure 1. Deletion of β1-integrins or ILK disrupts acinar morphogenesis.

(a) Immunofluorescence staining of MECs isolated from β1^fx/fx;CreER™ mice and cultured in 3D on BM-matrix. 4OHT added at the time of plating cells, caused β1-integrin deletion and absence of lumens. Bar: 10μm.

(b) No lumen disruption in acini from non-transgenic ICR mice, treated with 4OHT. Bar: 10μm.

(c) Quantification of ICR, β1-KO, Rac1-KO, ILK-KO acini with lumens, n=100 for each condition, 3 independent experiments.

(d) H+E staining of lactation day 2 (L2) mammary glands isolated from β1−/− mice (β1^fx/fx;Blg- Cre) and their WT littermates (β1^fx/fx ). Bar: 40 μm.

(e) L2 WT and β1−/− glands, immuno-stained for β1-integrin, and WGA to detect apical surfaces and lumens. Note that cells protrude into the luminal space of β1−/− glands. Bar: 15 μm.

(g) Immunofluorescence staining of MECs isolated from Rac1^fx/fx:LSLYFP:CreER™ mice and cultured in 3D on BM-matrix. 4OHT added at the time of plating cells, caused Rac1 deletion but no lumen loss. Bar: 10 μm.

(h) H+E staining of L2 mammary glands isolated from Rac1−/− mice (Rac1^fx/fx:LSLYFP:WAPi- Cre) and their WT littermates (Rac1^fx/fx ). Bar: 40μm

(i) L2 WT and Rac1−/− glands, immuno-stained for β1-integrin, βcatenin and WGA-488 to detect basolateral and apical surfaces, respectfully. Bar: 30μm.

(k) Immunofluorescence staining of MECs from Ilk^fx/fx:CreER™ mice and cultured in 3D on BM-matrix. 4OHT added at the time of plating cells, caused ILK deletion and lumen loss. Bar: 10μm.

(l) H+E staining of L8 mammary glands from Ilk−/− mice (Ilk^fx/fx:Blg-Cre) and their WT littermates (Ilk^fx/fx ). Note the activation of the Blg-Cre promotor is asynchronous in vivo, thus some lumens may already exist before the Ilk gene was ablated. Bar: 40μm.

(m) L8 WT and Ilk−/− glands, immuno-stained for Scribble, Smooth muscle actin (SMA) to detect myoepithelia, and WGA to detect apical surfaces and lumens. Bar: 20μm.

(f, j, n) β1−/−, Rac1−/− and Ilk−/− glands respectively, stained for SMA and Laminin1. Note Laminin1 assembly around the acini of all transgenic glands. Bar: 20μm.

In this and subsequent figures: a) WT refers to in vivo acini from β1/ ILK/ Rac1^fx/fx;Cre-ve mice or cultured acini from β1/ ILK/ Rac1^fx/fx;CreER™ MECs with no 4OHT treatment; b) in IF studies, nuclei were detected with Hoechst; c) confocal images of cultured 3D acini were taken through their centres.

See also Supplementary Figs. 1, 2.