Fig. 6: Src or Shp2 inhibition of ITGB1 phosphorylation dynamics results in equivalent phenotypes.
From: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells
a,b, Representative FLIM images (a, left) and apparent FRET efficiencies (a, right) and cell area (b) of MM231 cells stably expressing Illusia and seeded on either glass or hydrogels (60, 2 or 0.5 kPa) (for a, n = 110 (glass), 117 (60 kPa), 116 (2 kPa) and 113 (0.5 kPa) cells pooled from four biological replicates; for b, n = 121 (glass), 137 (60 kPa), 157 (2 kPa) and 165 (0.5 kPa) cells pooled from four biological replicates; one-way analysis of variance (ANOVA) with a Tukey correction for multiple comparisons). Scale bars, 10 μm. c, Representative images (left) of MM231 ITGB1(WT or YYFF) cells invading into 3D fibroblast-contracted collagen I treated with DMSO or SHP099 (100 nM). Pan-cytokeratin (PanCK) staining was used to mark cancer cells and exclude fibroblasts from the analysis. Quantification of invasion beyond 100 μm (right), normalized to the total number of cells/region, or proliferation, normalizing the number of Ki67-positive nuclei to the total number of cells/region (n = 24 regions per cell line pooled from four biological replicates; one-way ANOVA with a Tukey correction for multiple comparisons). Scale bars, 100 μm. d, A scheme for the basement membrane invasion assay. e, Left: representative images of MM231 and MM468 cells invading into the basement membrane matrix for 4 or 5 days, respectively, in the presence of SHP099 (100 nM) or Sara (1 μM) (collagen I is labelled with HaloTag-CNA35 (magenta), fibroblasts by mScarlet expression (red) and all nuclei stained with DAPI (cyan); the cancer cells are apparent by nuclei staining alone (cyan-positive, mScarlet-negative cells)). Quantification of basement membrane invasion (MM231 cells, n = 31 (from 11 basement membranes; DMSO), 24 (from 9 basement membranes; Sara) and 23 (from 8 basement membranes; SHP099) regions pooled from three biological replicates (right); MM468 cells, n = 31 (from 12 basement membranes; DMSO), 32 (from 12 basement membranes; Sara) and 27 (from 9 basement membranes; SHP099) regions pooled from four biological replicates; one-way ANOVA with a Dunnett’s correction for multiple comparisons). Scale bars, 50 μm. f, Illusia-expressing MM231s (green) embedded in collagen I (magenta) and treated with SHP099 (100 nM) or Sara (1 μM) for 24 h (cell size, n = 119 (DMSO), 93 (Sara) and 85 (SHP099); solidity, n = 122 (DMSO), 91 (Sara) and 94 (SHP099) cells from four biological replicates; one-way ANOVA with a Dunnett’s correction). The boxplots represent the median and IQR. The whiskers extend to the minimum and maximum values. The grey areas highlight the IQR of the control conditions. NS, not significant. *P < 0.05, ***P < 0.001.
