Fig. 8: The Dok1/ITGB1 complex recruits Cofilin and other invadopodia components to adhesion sites to mediate efficient cancer dissemination.
From: Dynamic regulation of integrin β1 phosphorylation supports invasion of breast cancer cells
a–d, Representative images (left) and quantification of apparent FRET efficiency (right) for intermolecular FLIM–FRET of the following tagged protein pairs, Dok1–Clover/Cofilin–mRFP (a), CTTN–mEmerald/Dok1–mScarlet (b), Dok1–Clover/mScarlet–TKS5 (c) and CTTN–mEmerald/Cofilin–mRFP (d). FRET between mScarlet and the donor-tagged protein was used as a negative control for all pairs (for a, n = 85 (Dok1/mScarlet) and 95 (Dok1/Cofilin) cells pooled from five biological replicates; for b, n = 65 (CTTN/mScarlet) and 68 (CTTN/Dok1) cells pooled from three biological replicates; for c, n = 62 cells for each condition pooled from three biological replicates; for d, n = 65 (CTTN/mScarlet) and 70 (CTTN/Cofilin) cells pooled from three biological replicates; unpaired two-tailed Student’s t-test with a Welch’s correction). Scale bars, 20 μm. e, Representative images of mice with MM231 ITGB1(WT or YYFF) cells stably expressing the luciferase/EGFP construct. Oral gavage of Vehicle or SHP099 (100 mg kg−1) proceeded for 5 days from the day of injection. f, A box and whisker plot highlighting the endpoint metastatic burden as an average (Avg) radiance value from the luciferase signal of the MM231 cells in e (n = 9 mice tracked per group). g, Representative lung sections stained for EGFP-positive MM231 cells. Scale bars, 2 mm; insets: 200 μm. h, Quantification of pulmonary nodule number (that is, clusters of greater than ten cells) in lungs from EGFP-positive MM231 cells (n = 10 mice per group). i, Quantitative real-time PCR of the RNA samples collected from the MM231 ITGB1(WT or YYFF) cells stably expressing the luciferase/EGFP construct. The mice were designated as either ‘metastatic’ or ‘low signal’ after setting a threshold for ‘metastatic’ as having an expression fold change >1 compared with the mean of the WT/vehicle control with human GAPDH normalized to mouse/human GAPDH (n = 10 mice/group). The boxplots represent the median and IQR. The whiskers extend to min and max values. The grey areas highlight the IQR of the control conditions. ***P < 0.001.
