Fig. 4: Tissue fluidification induces nuclear stiffness, heterochromatin reorganization and actin remodelling.
From: Tissue fluidification promotes a cGAS–STING cytosolic DNA response in invasive breast cancer
a, Left: map of the divergence of the velocity field from PIV in RAB5A-MCF10A monolayer. Cold (warm) colours indicate negative (positive) values of divergence. Right: magnified view of a smaller portion (70 × 70 μm) centred on a segmented nucleus (red outline). The velocity field (green arrows) converges to the central cell, corresponding to a local negative value of divergence and compressive deformation. Scale bar, 70 μm. b,c, Root mean square value σ∇ of the divergence of the velocity field from PIV MCF10A (b) and MCF10.DCIS.com (c) monolayers. Different points correspond to different FOVs, each one corresponding to ~1.2 × 104 and ~8 × 103 cells for MCF10A and MCF10.DCIS.com samples respectively. Black lines are the averages ± s.d. two-tailed t-test. d,e, Nuclear strain rate \(\dot \gamma _N\) as a function of the corresponding cell strain rate \(\dot \gamma _C\) for MCF10A (d) and MCF10.DCIS.com (e) monolayers. \(\dot \gamma _N\) is obtained from nuclear segmentation, while \(\dot \gamma _C\) is estimated from the divergence of the velocity field. Data are grouped into evenly spaced bins along the horizontal axis. Symbols and error bars are the mean and standard deviation of the \(\dot \gamma _N\)-values in each bin, respectively. Straight lines are best fitting curves with a linear model \(\dot \gamma _N = s\dot \gamma _C\). Insets: the ratio between the effective elastic moduli EN/ECY of the nucleus and the cytoplasm reported as mean ± s.d. obtained as the slope of best fitting line to the data in the main panel (n = 10 randomly populated subsets of cells), two-tailed t-test. f, Immunofluorescence images of control (CTR) and RAB5A MCF10.DCIS.com monolayers (n = 3 experiments), stained with DAPI and anti-H3K27me3-antibody. Magnified images are shown. Scale bar, 10 μm. g, Relative H3K27me3 intensity of control (CTR) and RAB5A MCF10.DCIS.com monolayers silenced or not for EZH2 or SUZ12. Each dot represents a cell, and the median is indicated (>1,400 cells per experimental condition for CTR and RAB5A, >400 cells per experimental condition in n = 3 independent experiments for siRNA-treated conditions), Kruskal–Wallis/Dunn’s test. h, Ratio of H3K27me3 intensity of the nuclear central region over the periphery in control-(CTR) and RAB5A-(RAB5A)-MCF10.DCIS.com cells. Each dot is a cell and the mean ± s.d. is indicated. (>1,000 cells per experimental conditions in n = 3 experiments), two-tailed Mann–Whitney non-parametric test. i, Immunofluorescence images of control (CTR) and RAB5A MCF10.DCIS.com monolayers (n = 3 experiments), silenced for EZH2 or SUZ12 and stained with DAPI and anti-H3K27me3-antibody. Scale bar, 10 μm. j, Immunofluorescence images of control (CTR) and RAB5A MCF10.DCIS.com monolayers (n = 2 experiments), stained with phalloidin to detect F-actin. Scale bar, 20 μm. k, Percentage of cells with actin rings per FOV expressed as mean ± s.d (dots represent seven FOVs in n = 2 independent experiments), two-tailed Mann–Whitney non-parametric test.
