Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Immunofluorescence staining of alpha-smooth muscle actin (αSMA) and SUN2 in lung tissue samples from healthy human patients and human patients with idiopathic lung fibrosis (IPF). ( B ) SUN2 immunofluorescence staining intensity at the nuclear envelope in healthy and IPF patient lung tissue was quantified in ImageJ, averaging the intensity of >3 fields of view per patient, N=5 patients per condition. No significant difference was found, determined by unpaired two-tailed t test; error bars are SD. ( C ) αSMA+/- cells in IPF patient tissue were identified using QuPath. SUN2 immunofluorescence staining intensity was then measured in both αSMA+/- cells, averaging intensity at the nuclear envelope in cells from >3 fields of view per patient, N=5 patients. There was a significantly higher intensity of SUN2 immunofluorescence staining at the nuclear envelope of αSMA+ cells when compared to αSMA- cells within IPF patient lung tissue. Statistical significance was determined by unpaired two-tailed t test; ***p < 0.001; error bars are SD.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Schematic demonstrating the time course of bleomycin inhalation induced fibrosis. ( B ) Immunofluorescence staining of Sun2 in wildtype mouse lung tissue 2 days post bleomycin inhalation and 14 days post bleomycin inhalation. ( C ) Sun2 nuclear staining intensity is significantly increased at day 14 post bleomycin inhalation during peak fibrosis when compared to day 2 post bleomycin inhalation. N=3 mice per condition. >2 fields of view analyzed for each mouse. Statistical significance was determined by unpaired two-tailed t test; **p < 0.01; error bars are SD. ( D-E ) Immunofluorescence staining of Nesprin-1 in wildtype mouse lung tissue 2 days and 14 days post bleomycin inhalation. Nesprin-1 nuclear staining intensity is significantly increased at day 14 post bleomycin inhalation during peak fibrosis when compared to day 2 post bleomycin inhalation. N=3 mice per condition, >3 fields of view analyzed for each mouse. Statistical significance was determined by unpaired two-tailed t test; ***p < 0.001; error bars are SD. ( F ) Dot plot of Sun2 , Syne1 (encoding Nesprin-1), secretory fibroblast marker Cthrc1 , and collagens Col1a1 and Col3a1 expression in the fibrotic fibroblast cluster of Col1-GFP expressing cells (Supplemental Fig. S2A) from scRNA-seq data in mouse lungs 7, 14, and 21 days after bleomycin inhalation. Data from GEO: GSE210341. ( G ) Gene Ontology (GO) biological process analysis of genes differentially upregulated in cells with high Sun2 expression compared to those with low Sun2 expression within the fibrotic cluster.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Immunofluorescence, Staining, Two Tailed Test, Marker, Expressing

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Immunofluorescence staining of Sun2 in primary wildtype lung fibroblasts seeded on glass and 2 kPa Matrigen substrates for 24 hours. ( B ) Superplot of Sun2 nuclear staining intensity, which is significantly increased in fibroblasts plated on glass compared to a 2 kPa substrate. n=>100 cells, N=3 replicates. Statistical significance was determined by unpaired two-tailed t test; ****p < 0.0001; error bars are SD. ( C ) Plot showing fold change in mRNA expression of Sun2 in primary wildtype lung fibroblasts cultured on 2 kPa versus glass substrates. There is no significant difference in Sun2 expression between substrate stiffnesses as determined by two-tailed t test. N=3 replicates. Error bars are SD.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Immunofluorescence, Staining, Two Tailed Test, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Loss of Sun2 showed reduced collagen as assessed by Sircol assay at 14 and 21-days post-bleomycin administration. N=>4 mice per condition, male and female mice. Statistical significance was determined by performing multiple t-tests; * p<0.05, ** p<0.01. ( B ) Trichrome staining to visualize collagen (blue) was analyzed to determine disease severity using a ( C ) Modified Ashcroft Score, showing less disease severity in Sun2 -/- mice. N=10 mice per genotype, includes both male and female mice. Statistical significance was determined by performing a two-tailed Student’s t test; * p<0.05; Error bars are SD.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Staining, Modification, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Apoptotic cells were stained using TUNEL and the percentage of apoptotic cells were quantified for tissue 2 days post-bleomycin administration. N=6 male and female mice. There is no statistically significant difference (ns) in these levels upon loss of Sun2 based on a Student’s t test. ( B ) The number of cells present in the BAL is not impacted by the loss of Sun2 during the inflammatory response phase of the model (prior to Day 14). N=>4 mice per condition, male and female mice. Statistical significance was determined by performing multiple t-tests; ns no significance; * = p<0.05. The Holm–Sidak method was used to correct for multiple comparisons. Error bars are SD. ( C ) Sun2 is dispensable for the recovery of the alveolar barrier as assessed by the kinetics of the increase and subsequent decrease in albumin leaking from the blood into the BAL. ( D ) Wildtype and Sun2 -/- mouse lung tissue from mice euthanized 14 days post bleomycin inhalation were stained with an antibody against phospho-Smad2/3. ( E ) Superplot of the ratio of nuclear to cytoplasmic levels of pSmad2/3 in fibrotic mouse lung tissue shows a significant increase in pSmad2/3 translocation to the nucleus in Sun2 -/- tissue during peak fibrosis. N=3 male mice per genotype, >1 field of view analyzed per mouse. Determined by two-tailed unpaired t test, ****p < 0.0001. Data from three replicates superimposed in blue, orange, and pink. ( F ) Immunofluorescence staining of lung tissue sections from mice 2- or 14-days post-bleomycin administration was performed using an antibody against alpha smooth muscle actin (αSMA) to visualize hypercontractile cells. Regions marked by the dashed box are shown at higher magnification below. ( G ) There is an expected increase in αSMA positive tissue over time but no significant difference in αSMA positive cell levels in WT and Sun2 -/- tissue, as was determined by an ordinary one-way ANOVA followed by Sidak’s multiple comparisons test. Error bars are SD. ( H ) There is no significant difference between the size of the gel contracted by wildtype lung fibroblasts and Sun2 -/- fibroblasts after 24 hours as determined by two-tailed unpaired t-test. N=6 replicates. Error bars are SD.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Staining, TUNEL Assay, Translocation Assay, Two Tailed Test, Immunofluorescence

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Heat map showing expression patterns of genes characteristic of fibrotic fibroblasts in primary wildtype and Sun2 -/- fibroblasts cultured on 50 kPa in the presence and absence of exogenous TGFβ1 (5ng/ml)(GEO: GSE325284). Overall, compared to WT, Sun2 -/- fibroblasts exhibit increased expression of Cthrc1 , and decreased expression of extracellular matrix genes in the absence or presence of TGFβ1. ( B ) Immunostaining of pro-collagen in primary lung fibroblasts isolated from wildtype and Sun2 -/- mice after growing on glass coverslips for 24 hours. ( C ) Less Sun2 -/- fibroblasts produce pro-collagen. Pro-collagen positivity was determined by the automatic thresholding feature on Fiji. The number of cells positive for pro-collagen staining was counted for each genotype and divided by the total number of cells per replicate and multiplied by 100 to determine a percentage. n=50< cells, N=3 replicates. Determined by two-tailed unpaired t test; ***p < 0.001. ( D ) Cartoon diagram showing our working model in which Sun2 acts as a component of a key nuclear mechanotransduction cascade coupling myofibroblast contractility to collagen production in response to lung injury. In response to injury, subsets of fibroblasts express organized αSMA fibers, contracting surrounding tissue and altering the mechanical environment in both wildtype and Sun2 -/- tissue. Mechanical signals transduced via Sun2-containing LINC complexes in αSMA+ and/or αSMA-fibroblasts prime an increase in transcription of ECM genes. This nuclear mechanosensation is disrupted in Sun2 -/- fibroblasts, which fail to produce pathogenic ECM.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Expressing, Cell Culture, Immunostaining, Isolation, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: Cartoon diagram showing our working model in which Sun2 acts as a component of a key nuclear mechanotransduction cascade coupling a stiffening tissue microenvironment to new collagen deposition in response to lung injury. In the absence of injury, integrins are not strongly engaged, the cytoskeleton is more relaxed, and there is low tension on LINC complexes and the nucleus, corresponding to repression of TGFβ targets including ECM genes and α-SMA. In response to injury, TGFβ binds to the TGFβ receptor and phosphorylated Smads translocate to the nucleus, representing the canonical biochemical cascade. In addition, tension from integrins is propagated through the actin cytoskeleton to LINC complexes. LINC complexes are up-regulated, inducing high tension on the nucleus. Genes contributing to pathogenic ECM production and contractility (ECM genes and α-SMA) are induced, with ECM genes requiring coincidence detection of both nuclear tension and pSmads. The abrogation of nuclear mechanosensation in Sun2 -/- fibroblasts disrupts force propagation into the nucleus, leading to a failure to produce pathogenic ECM but normal contractility (e.g. α-SMA expression) and protection from fibrosis.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Expressing