Fig. 3 | Nature Communications

Fig. 3

From: Matrix mechanical plasticity regulates cancer cell migration through confining microenvironments

Fig. 3

MDA-MB-231 cells in high plasticity matrices extend invadopodia protrusions. a RFP-LifeAct MDA-MB-231 cells were imaged for 12 h using time-lapse confocal fluorescence microscopy. Transient actin-rich spots were observed in all IPNs (yellow arrows, top), but in HP IPNs, these spots often elongated into oscillatory, actin-rich protrusions (yellow arrows, bottom). Scale bar is 10 μm. b Probability of a cell extending one or more protrusions in LP, MP, and HP IPNs, with either vehicle alone or protease inhibitor (10 μM GM6001) added to the medium. Graph displays the number of cells per condition, taken from R = 3–5 biological replicate experiments. Bars indicate mean probabilities and error bars indicate 95% confidence intervals. Differences in protrusivity as indicated are statistically significant (****P < 0.0001, Fisher’s exact). Probability of extending a protrusion also trends with plasticity (####P < 0.0001, χ2 tests for trend). c and d Histograms of extensions and widths of cell protrusions in LP and HP IPNs. Includes number of cells indicated per condition, pooled from R = 3 biological replicate experiments each. e Tracings of protrusion lengths, for one cell each in LP and HP IPNs, over time during a 6 h timeframe, plotted alongside a sinusoidal fit. fh Confocal immunofluorescence imaging was used to investigate localization of indicated proteins. Main panel scale bar is 10 μm. f and g Imaging of indicated staining on cryosections of MDA-MB-231 cells encapsulated in IPNs for 1 day. In f, main image shows merged maximum intensity projection (MIP) and inset (1.5× zoom for LP and 4× zoom for HP) shows merged image on one z-plane.  In g, inset is 2× zoom. h Live imaging of actin (RFP-LifeAct) and TKS5 (TKS5-EGFP). Inset is 1.5× zoom. i Probability of a cell extending one or more protrusions, for cells transfected with control siRNA, siRNA targeting cortactin (siCTTN), or siRNA targeting TKS5 (siTKS5)

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