Fig. 2.

aPKCi overexpression triggers basal epithelial cell protrusions breaching the BM and cell segregation from the MCF10A WT cell counterparts. (A) Confocal images of mouse mammary organoids with a few epithelial cells overexpressing GFP or GFP-aPKCi. Myoepithelial cells were detected with anti-SMA antibodies (red). Nuclei were stained with DAPI (blue). (B) Quantification of the total number of GFP⁺ epithelial cells breaching the myoepithelial cell layer (***P < 0.001). Five independent experiments were performed. (C) Confocal images of mouse mammary organoids. Luminal cells and the BM are labeled with anti–cytokeratin-8 and anti–laminin-5 antibodies, respectively. (D) Quantification of the total number of GFP⁺ epithelial cells breaching the BM (*P < 0.05). Two independent experiments were performed. Quantification of all GFP⁺ cells was pooled from the independent experiments, and χ² tests were performed for all data. (Scale bars, 10 µm.) n, number of cells. (E) Confocal images of MCF-10A spheroids mixed with GFP⁺ or GFP-aPKCi⁺ cells and mCherrry⁺ cells. (Scale bars, 50 µm.) (F) Quantification of GFP⁺ cell distribution. The graph shows the ratio between the distance of GFP⁺ cells from the center of the spheroid and the radius of the spheroid (from 3 independent experiments). n, number of spheroids. A Mann–Whitney test was performed (***P < 0.001). (G) Images of micropipette aspiration of MCF-10A spheroids at the resting pressure P₀ (Top) and at the critical pressure P_c, for which the deformation of the spheroid reaches the micropipette radius (Bottom). (Scale bars, 50 µm.) (H) Individual (dots) and mean ± SEM (bars) surface tension measurements on spheroids containing a mix of WT and GFP⁺ or GFP-aPKCi⁺ cells. A Student t test was performed (***P < 0.001). Three independent experiments were performed. n, number of spheroids.