Extended Data Fig. 5: Mechanical characterization of nuclei in control and fluidized tissues.
From: Tissue fluidification promotes a cGAS–STING cytosolic DNA response in invasive breast cancer
a. Nuclear elasticity (Young’s modulus) using atomic force microscopy (AFM)-based force indentation of the nuclear surface through the cell cortex of control and RAB5A-MCF10.DCIS.com monolayers seeded at jamming density. Data are the mean ± s.e.m. (100 cells/condition in n = 3 independent experiments), two-tailed Mann–Whitney non-parametric test. Scale bar, 200 µm. b, c. Quantification of cytoplasmic rheology using genetically encoded multimeric nanoparticle (GEM)37. b) Time- and ensemble- averaged Mean Square Displacement (MSD) plots for control (blue line) and RAB5A (red line) in MCF10.DCIS.com monolayers seeded at jamming density. c) Cumulative distribution function showing the coefficient of diffusion, CDF, (Deff) data of for control (blue line) and RAB5A (red line) MCF10.DCIS.com monolayers seeded at jamming density. d. Nuclear deformation of control and RAB5A monolayers subjected to a 22% biaxial stretch. Control and RAB5A-MCF10.DCIS.com monolayers were seeded onto elastic PDMS membranes in a biaxial stretching device63. Thenuclear area strain, Δa was measured in various, randomly picked fields of view. Bottom, middle and top lines of boxes show the 25th, 50th and 75th percentiles, respectively, and whiskers show 1.5 times the interquartile range from hinges (>29 FOV/experimental condition in n = 2 independent experiments, indicated by different size symbols, respectively), two tailed Mann–Whitney non-parametric test. e. The rates of aspect ratio (A/R) changes of mCherry-H2B control (CTR) and RAB5A-MCF10.DCIS.com cells that are passively going through a constricted 6 micrometre-wide channel schematically depicted on the left (Supplementary Video 7), were calculated for several cells in the time interval needed to pass from an undeformed (A/R0, at t0), just before entering into the constriction, to a completely squeezed configuration (A/Rf, at tf). The rates of nuclear AR variation were extrapolated through a robust fit in R using the ‘robustbase’ package http://robustbase.r-forge.r-project.org/, where the rate of AR changes as function of time is the slope of the fitted curve. Scatter plot data of mean ± S.D. (114 cells/control and 61 cells/RAB5A of n = 3 independent experiments) of the rate of A/R changes, two-tailed Mann–Whitney non-parametric test. f. Images of the SUN2-stained NE of control and RAB5A-MCF10.DCIS.com cells (n = 3 experiments), seeded at jamming density. Scale bar 2 µm. g. Scatter plot of EOP quantifications expressed as mean ± s.d. (> 181 cells/condition in n = 3 independent experiments), two-tailed Mann–Whitney non-parametric test. h. Quantification of NE fluctuations in EGFP-Nesprin-expressing, control and RAB5A-MCF10.DCIS.com cells. Nuclei of live cells transfected with EGFP-Nesprin were recorded at 250 msec per frame for 5 min (Supplementary Video 8). NE fluctuations were obtained from the standard deviation of the NE from its mean position (see Methods and in the inset scheme). The box extends from the 25th to 75th percentile, the whiskers show minimum and maximum values, and the line within the box represents the median (n = 60 position along the NE/condition in >10 cells/ condition), two-tailed Mann–Whitney non-parametric test. i–k. Nuclear mechanics of live cells obtained by Brillouin micro-spectroscopy. i) Representative Brillouin spectra (anti-Stokes peaks) of nuclei of RAB5A (upper panel) and control cells (lower panel). j) Mean and median of Brillouin shifts (black line) measured in RAB5A (n = 9) and control cells (n = 11). The box extends from the 25th to 75th percentile, the whiskers show the mean ± s.d., and the line within the box represents the median, unpaired one-tailed t-test with Welch’s correction. k) From Brillouin shifts, we derived the apparent longitudinal elastic modulus. The box extends from the 25th to 75th percentile, the whiskers show the mean ± s.d., and the line within the box represents the median, unpaired one-tailed t-test with Welch’s correction. P values are indicated in each graph.
