Fig. 3: The extracellular matrix promotes SHP2 phosphorylation.

a Cell viability of 4T1 cells cultured in 2D for 6 days in the absence or presence of the indicated concentrations of 11a-1 and SHP099. b Representative photomicrographs and quantification of 4T1 cell viability after 16 days in 3D culture in the absence or presence of the indicated concentrations of 11a-1 and SHP099. For a and b data are normalized to untreated controls and are the mean ± s.e.m. of three experiments where **p < 0.01 and ***p < 0.001. Immunoblot analyses showing differential phosphorylation of SHP2 at Y542 in 4T1 cells following 3D culture (c) or in 4T1 cells lysed directly from gel-based 3D culture (d). e Kaplan–Meier analyses of patients from the TCGA breast cancer dataset separated into two groups based on the median mRNA expression value of SHP2 (left) or median phosphorylation levels of SHP2 at Y542 (right). Overall survival was analyzed by a log-rank test resulting in the indicated p values. f RPPA data from the TCGA breast cancer dataset were analyzed for correlation of total expression levels and post translational modifications of the indicated proteins in relation to SHP2-Y542 phosphorylation. The heat map indicates the Pearson correlation coefficient and the size of the circle is representative of the value. g Immunoblot analyses showing differential phosphorylation of Src in 4T1 cells following culture under gel-based 3D conditions as compared to 2D culture. h Immunoblot analyses showing differential phosphorylation of FAK at Y925 in 4T1 cells isolated directly from gel-based 3D cultures as compared to 2D culture. i Representative photomicrographs showing 4T1 cells cultured on FN-coated tessellated scaffolds. j Immunoblot analyses showing differential phosphorylation of FAK, Src, and SHP2 in 4T1 cells cultured on FN-coated scaffolds as compared to 2D culture. k 4T1 cells were cultured on FN-coated scaffolds for 16 days and treated with the indicated concentrations of a Src inhibitor (PP2) or a FAK inhibitor (PF271) for the last 24 h before harvesting cells for immunoblot analyses of SHP2 phosphorylation at Y542. All immunoblots shown are representative of at least three independent experiments.