Fig. 6

α3-mediated changes in collagen adhesion and FA formation. a Short-term adhesion assays of HER− MDA-MB-231 and HER2+ BT474, AU565, and SKBR3 on laminin-rich matrix and collagen I. Experiments were performed in triplicate and repeated three times (mean ± SD, unpaired t test, *P < 0.05, **P < 0.005, ***P < 0.0001). b α3KO SKBR3 show significantly decreased cell area, c reduced size of focal adhesions, and d reduced total adhesion area per cell after 30 min of adhesion to collagen I. Experiments were performed three times with 30 cells analyzed per experiment (total n = 90, unpaired t test, *P < 0.05, **P < 0.005, ***P < 0.0005). e Representative images of SKBR3 ITGA3 KO and WT cell, used for quantifications (b–d), stained for actin and vinculin (scale bar, 10 μm). f Representative images of SKBR3 ITGA3 KO and WT cells, allowed to adhere to collagen I-coated coverslips for 30 min and stained for actin and integrins α3, α2, or β1. ITGA3 KO cells show reduced clustering of integrins in adhesion complexes. Note that α3 and α2 signals were enhanced by biotin-conjugated secondary antibody, resulting in unspecific biotin staining in the center of the cell (Additional file 4: Figure S4b) (scale bar, 10 μm). g Invasion assays through the mixture of collagen I and Matrigel-coated membrane. The reduction of α3 increases invasiveness of HER2+ carcinoma cells under interstitial fluid flow conditions, which cannot be recapitulated with blocking adhesion of α3 to laminin by addition of function-blocking J143 antibody (A3-X8: control non-blocking antibody). Left—analysis of the experiments, performed in duplicate and repeated three times (mean ± SD, unpaired t test, *P < 0.05, **P < 0.005). Right—representative images of the part of the membrane, showing DAPI-stained nuclei of invading cells