Fig 2: The schematic diagram of the mechanism by which the intestinal motility of WAS mice changes the colonic mucus barrier by acting on Piezo1 to regulate downstream methylation. Left side: Piezo1 on goblet cells of WAS mice decreased, and the decline of intestinal motility in WAS mice led to the downregulated signal of Piezo1 reception, which together led to the weakening of the inhibitory effect of piezo1 on downstream SUV39h1. Enhancement of SUV39h1 promoted H3K9me3 modification on the mucin2 promoter of goblet cells, resulting in the decrease of mucin2, mucus thickness and the increase in bacterial permeability. Right side: Addition of Yoda1 activates Piezo1 protein on intestinal goblet cells, inhibits downstream SUV39h1 and Hdac3, and reduces the binding of H3K9me3 to mucin2 promoter. After H3K9me3 isolation from mucin2 promoter, mucin2 gene was activated. Intestinal goblet cells synthesize and secrete more mucin2. The mucus thickness of WAS mice increased and the mucus barrier permeability improved

Fig 3: Mechanical stimulation reduced methylation in LS174T cells. A–D Western blots and statistical analysis of Suv39h1, Hdac3, GAPDH, H3K9me3 and T-H3 in LS174T cells before and after mechanical stimulation in different strength (n = 4). E ELISAs of mucin2 in LS174T cells before and after mechanical stimulation of 24 and 48 h (n = 5)

Fig 4: H3K9me3 Regulates Chromatin Rheology and Nuclear Mechanics, Related to Figure 4(A) Representative western blots and quantification of GFP-tagged wild-type (WT) Lamin A or Lamin A mutants (Serine 22 to Alanine (S22A) or S22A/S392A double mutant) show equal levels of expression. Lamin A band intensities are normalized to GAPDH (n = 3 independent experiments).(B) Representative immunofluorescence images of nuclei (DAPI), GFP, Lamin A/C, and H3K9me3 in cells expressing WT Lamin A-GFP or Lamin mutants S22A-GFP; S22A/S392A-GFP.(C and D) Quantification of Lamin A intensity and nuclear shape in WT Lamin A and mutant expressing cells (n = 3 independent experiments with > 100 nuclei/condition/experiment; *p = 0.034, ANOVA/Dunnett’s).(E) AFM force indentation experiments of cells expressing WT Lamin A-GFP or Lamin mutants S22A-GFP; S22A/S392A-GFP (n = 3 independent experiments with > 40 nuclei/condition/experiment; no statistically significant difference found, Friedman/Dunn’s).(F) Representative immunofluorescence images and quantification of Suv39H1 levels in cells exposed to uniaxial 40% stretch for 30 min (n = 3 independent experiments with > 100 nuclei/condition/experiment; *p = 0.05, Mann-Whitney)(G) Quantification of H3K9me3 intensity from cells in (B) (n = 3 independent experiments with > 30 nuclei/condition/experiment; no statistically significant difference found, Friedman/Dunn’s).(H) Representative immunofluorescence images and quantification of H3K9me3 levels in cells treated with JIB4 to inhibit H3K9me3 demethylation and exposed to 30 min uniaxial 40% stretch (n = 3 independent experiments with > 200 nuclei/condition/experiment; *p = 0342, Friedman/Dunn’s).(I) Representative immunofluorescence images and quantification of Suv39H1 levels in cells transfected with Suv39H1-IRES-GFP or GFP only (n = 3 independent experiments with > 24 nuclei/condition/experiment).(J) Quantitative RT-PCR analyses of Suv39H1 mRNA expression, normalized to B2M in EPC monolayers where Suv39H1 has been silenced by siRNA (siSUV) (n = 3 independent experiments).(K) Representative immunofluorescence images and quantification of H3K9me3 in siSUV cells cells (n = 3 independent experiments with > 200 nuclei/condition/experiment; *p = 0.05, Mann-Whitney).(L) AFM force indentation experiments of siSUV cells (n = 3 independent experiments with > 40 nuclei/condition/experiment; ****p < 0.001, Mann-Whitney).(M) Quantification of chromatin rheology by mean square displacement versus lag time t of CRISPR-rainbow labeled telomeres. From linear data, material properties of chromatin can be calculated from MSD = Defft, where a higher Deff corresponds to less condensed chromatin (n = 40 (GFP)/30 (Suv39-GFP) cells from 3 independent experiments; ***p = 0.0001, Student’s t test).(N) Representative immunofluorescence images and quantification of cGAS-transfected cells to detect nuclear rupture in cells exposed to 30 or 360 min uniaxial 40% stretch. Note lack of perinuclear accumulation of cGAS indicating absence of nuclear rupture upon stretch. Inset shows accumulation of cGAS to chromatin upon mitotic breakdown of NE as positive control (n = 3 independent experiments with > 150 nuclei/condition/experiment). Bar graphs show mean ± SD, boxplots show 95% confidence interval, scale bars represent 10 µm, white arrows indicate stretch direction, AU = arbitrary units.

Fig 5: H3K9me3 Heterochromatin Regulates Nuclear Mechanics and Chromatin Mobility to Prevent DNA Damage(A) Representative western blots and quantification show reduced levels of Suv39H1 in response to stretch (mean ± SD; n = 3 independent experiments; *p = 0.0438, ANOVA/Dunnet’s).(B–D) Representative images (B) and quantification of H3K9me3 (C) and nuclear wrinkling (D; white arrows) in GFP or Suv39H1-IRES-GFP expressing cells. Note increased H3K9me3 and lack of nuclear wrinkling in Suv39H1 expressing cells (n = 3 independent experiments with > 300 cells/condition/experiment; **p = 0.0053, Friedman/Dunn’s).(E) Force indentation experiments show that decreased nuclear stiffness in 40% stretch is prevented by expression of Suv39H1 (n > 100 cells/condition pooled across three independent experiments; ****p = 0.0015, Kruskal-Wallis/Dunn’s).(F) Quantification of chromatin rheology by mean square displacement (MSD) versus lag time t of CRISPR-Rainbow labeled telomeres. Note transient increase in mobility after 30 min of stretch, nonlinear with lag time, consistent with energy-dependent reorganization of chromatin (n > 21 cells/condition with >200 tracks/condition from three independent experiments; control and 360 min are statistically similar; **p < 0.001 at every lag time, Student’s t test).(G) Representative images and quantification demonstrating increased ?H2AX-positive cells in Suv39-expressing cells upon 40% stretch (n = 4 independent experiments with >250 cells/condition/experiment; *p = 0.018, Friedman/Dunn’s).Bar graphs show mean ± SD, boxplots show 95% confidence interval, white arrows indicate stretch direction, and scale bars represent 10 µm. AU, arbitrary units.See also Figure S4.