Figures and data
Cancer cells adapt to prolonged confinement by decreasing their nuclear volume
(A) 3D representation of our confinement device showing the molded agarose pad. (B) Schematic diagram of the multi-height pad with the theoretical heights of each pillar. (C) Confocal images of cells and the shaped pillar of each confined zone. Scale bar = 250 µm. (D) Quantification of cells and nuclear height by z-stack confocal imaging, under each confined zone. N = 3 experiments with n = 30 cells; Mann-Whitney test. (E) Representative confocal images of nuclei stained with NucGreen in XY (Scale bar = 10 µm) and its orthogonal view (scale bar = 5 µm) at 2 h and 24 h under each level of confinement. (F) Initial nuclear deformation in percent for each degree of confinement defined as 
Nuclear volume adaptation occurs during mitosis setting a new state of homeostasis
(A-B) Quantification of the size of daughter nuclei over their respective mother, under several levels of confinement during the first confined mitosis (A) and the second confined mitosis (B) and representative images of nuclei at the end of the G2 phase (just before mitosis) and its respective daughter nuclei just after mitosis (beginning of G1 phase), for control and strong confinement conditions during the first confined mitosis (A) and the second confined mitosis (B), scale bar = 10 µm. N = 2 experiments with n > 15 cells per condition, unpaired t-test. (C) Time-lapse images of HT-29 nuclei expressing the FUCCI fluorescent cell cycle reporter, without confinement (control) or under strong confinement (60% nuclear deformation). Scale bar = 10 µm. (D-F) Quantification of the duration of HT-29 Fucci cells under confinement during the S-G2 before the first division (D) and during the G1 and S-G2 phase after the first division (F). N = 2 experiments with n = 50 cells (D) and n > 200 cells (F), unpaired t test. (E-G) Quantification of the nuclear relative growth of HT-29 Fucci cells under confinement during the S-G2 before division (E) and during the G1 and S-G2 phase after the first division (G). N = 2 experiments with n = 39 cells, unpaired t test for (E) and n > 200 cells, Mann-Whitney test for (G).
Mitosis regulates nuclear tension under prolonged confinement
(A) Representative confocal images (max intensity projection) of stained nuclei and lamin A/C without confinement (control) or under 60% nuclear deformation (strong confinement) at 2 h and 24 h. Scale bar = 10 µm. (B) Quantification of the positive area of the nuclear envelope (lamin A/C) which represents the folded nuclear envelope. N = 3 experiments with n > 30cells/experiments; mean ± SEM; Mann-Whitney test. (C) Max projection confocal images of stained nuclei and pMLC at 2 h under strong confinement, scale bar = 50 µm. Cropped nuclei correspond to nuclei with blebs, dividing and apoptotic cells and normal nuclei, scale bar = 10 µm. (D, E) Percentage of HT-29 cells presenting nuclear blebs, dividing, apoptotic cells, or normal nuclei in control condition or under confinement, at 2 h (D) and 24 h (E). N = 3 experiments with n > 5,000cells/experiments. (F) Sequential images of a blebbing nucleus under strong confinement before and after mitosis, NEB is set here as a reference time t = 0 h, scale bar = 10 µm and quantification of the number of blebs before and after mitosis under strong confinement. N = 3 experiments with n = 77 cells, Mann-Whitney test. (G) Representative images of nuclei and DNA damage, shown by stained γH2AX foci. Phleomycin is a drug that induces DNA damage used here as a positive control. Scale bar = 10 µm. (H) Quantification of the number of foci/cell, representative of the DNA damages according to an increase in nuclear deformation. N = 2 experiments with n > 2000; mean ± SEM; unpaired t-test. Ordinary one-way ANOVA was also performed on all 24 h confinement conditions and was no significant.
A new state of nuclear homeostasis is determined by the apparent nuclear surface at mitosis exit
(A) Schematic representation of the geometric model, showing the apparent nuclear envelope surface area Sapp and the unfolded nuclear envelope surface area S0 (B) Calculations of nuclear volume using the apparent surface area (green) and the total surface area (pink). The total surface area is obtained from the tensed nuclear envelope measured under intermediate confinement at 2 h. Experimental values are shown in black. (C) Nuclear apparent surface area as a function of the initial nuclear deformation ɛNz at 2 h and 24 h under confinement. The dashed line shows the apparent surface measured for unconfined cells.
The long-term nuclear adaptation is defective with altered actomyosin cortex
(A) Quantification of the nuclear volume changes (in percentage) between 2 h and 24 h without confinement and under strong confinement with several drugs. N = 3 experiments with n>2,500 cells, mean ± SEM, Welch’s t-test. (B) Representative images of stained nuclei and phospho-Myosin Light Chain either with or without Blebbistatin at 2 h and 24 h, without confinement or under very strong confinement. Scale bar = 20 µm. (C) Quantification of nuclei with blebs, dividing, apoptotic, or normal without confinement or under very strong confinement at 2 h and 24 h, with no drug or with Blebbistatin. N = 3 experiments with n = 9,818 cells.
