Extended Data Fig. 1: Mutation of the ITGB1 NPxY tyrosine (Y) residues to non-phosphorylatable phenylalanine (F) does not change surface integrin levels, or cancer cell proliferation.
From: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells
a, Representative western blot (left) and densitometry (right) of ITGB1 levels after shRNA-mediated KD in MM231 cells (shβ1; n = 3 biological replicates; significance assessed using a one-sample two-tailed t-test against the normalised control value; ***p < 0.001). Data are mean ± s.e.m. b & c, Representative curves (b) and doubling times (c) from the relative cell density of parental and shβ1 MM231 cells, and shβ1 MM231s with stable ITGB1(WT or YYFF) reexpression (ITGB1(WT or YYFF)-mRuby2 transposon vectors, described in methods; n = 3 biological replicates; significance assessed using a one-way ANOVA with a Šidák correction for multiple comparisons; NS, not significant). d, Gating strategy for the flow cytometry data presented in e & f. e & f, Representative histograms (e) and quantification (f) of the surface expression of total ITGB1 (P5D2), inactive ITGB1 (mAb13) and total integrin β3 (MCA728) using flow cytometry in shβ1 MM231s with or without ITGB1 reexpression as indicated (n = 4 biological replicates; significance assessed using a one-way ANOVA with a Tukey correction for multiple comparisons; NS, not significant; ***p < 0.001). g & h, TIRF images (g) of MM231 shβ1 cells with ITGB1 WT or YYFF reexpression (cells outlined with pink dashed lines) and quantification of colocalization (h) between active integrin staining (12G10 antibody) and either mRuby2-tagged ITGB1 WT (average Pearson’s r of 0.4853) or YYFF (average Pearson’s r of 0.5012; n = 65 [ITGB1 (WT)] and 63 [ITGB1(YYFF)] cells pooled from three biological replicates; significance assessed using an unpaired two-tailed Student’s t-test with a Welch’s correction; NS, not significant). i, Analysis of paxillin staining in MM231 cells from (g) to compare IAC average number and size per cell (n = 87 [ITGB1 (WT)] and 85 [ITGB1 (YYFF)] cells pooled from four biological replicates; NS, not significant). j, Representative images of invading MM231 ITGB1(WT or YYFF) cells (left) and quantification of their proliferation (right) from the fibroblast-contracted 3D collagen I invasion assays in Fig. 1c. Cells are stained with the proliferation marker Ki67. Quantification was achieved by normalising the number of Ki67-positive nuclei to the total number of cells/region (n = 24 regions for each cell line pooled from three biological replicates; significance assessed using an unpaired two-tailed Student’s t-test with a Welch’s correction; NS, not significant). Scale bars, 100 μm. k, Representative images (left) and quantification (right) of Ki67-stained subcutaneous xenografts of MM231 cells with either ITGB1(WT or YYFF) from Fig. 1e. Quantification of positive (brown) to negative (blue) staining of Ki67 in 400 μm2 regions of interest from subcutaneous xenografts is shown (n = 9 mice (WT) or 11 mice (YYFF); 5 regions/mouse/condition); significance assessed using an unpaired two-tailed Student’s t-test with a Welch’s correction; NS, not significant). Scale bars, 50 μm. Source data and exact p-values are provided in the statistical source data file. Boxplots represent median and interquartile range. Whiskers extend to min and max values. Grey areas on boxplots highlight the IQR of the control conditions.
