Journal: Advanced Science

Article Title: Matrix Rigidity Mechanoprimes Microglia for Inflammation Through Cytoskeletal‐to‐Nuclear Signaling and 3D Spatio‐Epigenomic Remodeling

doi: 10.1002/advs.77736

Figure Lengend Snippet: Matrix rigidity drives microglial nuclear chromatin modifications, mediated by actin cytoskeletal development. (a,b) Confocal imaging of microglial (BV2) cells cultured on compliant (1 kPa) and rigid (23 kPa) matrices for 12 h. Images are maximum‐intensity projections of confocal z‐stacks: (a) Representative images of nuclear DNA stained with DAPI (blue) alongside a rainbow intensity map, and (b) quantification of nuclear condensation, defined as the coefficient of variation (standard deviation/mean intensity of DAPI staining) ( n = 132). (c–e) Super‐resolution N‐SIM analysis of nuclear architecture: (c) Representative images of nuclei stained for DNA (DAPI, blue) and histone H3 acetylation (H3ace, red), detailing nanoscale chromatin patterns. Images are single optical sections acquired at the nuclear mid‐plane by N‐SIM super‐resolution microscopy. (d) Normalized fluorescence intensity profile of H3ace distributed along the indicated vector (white arrow). (e) Semi‐quantification of H3ace occupancy relative to total nuclear area ( n = 5). (f,g) Temporal monitoring of H3ace levels over 12 h using a dynamically stiffening MeHA gel platform (transitioning from 1 to 20 kPa). The violet dotted line indicates the time point of UV crosslinking. Stiffening was induced by UV exposure at t = 0 (arrowhead). Time points span from −2 h (before stiffening) to 12 h, with UV‐induced stiffening applied at t = 0 (arrowhead). The −2 h time point represents the pre‐stiffening baseline, and subsequent time points represent the adaptation phase after stiffening. (f) Immunofluorescence images of H3ace co‐stained with F‐actin, and (g) corresponding semi‐quantification of H3ace intensity ( n = 20). (h,i) Impact of cytoskeletal tension disruption via cytochalasin D (CytoD) treatment on H3ace levels: (h) Confocal images of H3ace (red), F‐actin (green), and nuclei (blue) following 11 h of culture and a subsequent 1 h CytoD treatment, and (i) semi‐quantification of average H3ace intensity across experimental conditions ( n = 64).

Article Snippet: Digested chromatin was labelled with biotin‐14‐dCTP (Invitrogen) and 40 U of Klenow (NEB), and re‐ligated with 2000 U of T4 DNA Ligase (NEB) for 4 h at 23°C.

Techniques: Cytoskeletal, Confocal, Imaging, Cell Culture, Staining, Standard Deviation, Super Resolution, Analysis, Super-Resolution Microscopy, Fluorescence, Plasmid Preparation, Immunofluorescence, Disruption

Journal: Advanced Science

Article Title: Matrix Rigidity Mechanoprimes Microglia for Inflammation Through Cytoskeletal‐to‐Nuclear Signaling and 3D Spatio‐Epigenomic Remodeling

doi: 10.1002/advs.77736

Figure Lengend Snippet: Matrix rigidity, in conjunction with biochemical signals, enhances chromatin accessibility to drive microglial inflammatory activation. (a,b) Representative high‐resolution images of DAPI‐stained microglial nuclei, displaying 3D nuclear morphology and DNA loci, cultured on compliant (1 kPa) or rigid (23 kPa) matrices under LPS stimulation. Semi‐quantification of chromatin condensation (visualized via a rainbow intensity scale) indicates that cells on 23 kPa exhibited a significantly more decondensed chromatin state compared to 1 kPa ( n = 21). (c,d) Representative immunofluorescence images and semi‐quantification of acetylated histone H3 (H3ace) intensity, demonstrating increased levels of this representative euchromatin mark in the nuclei of LPS‐treated cells on 23 kPa versus 1 kPa ( n = 105). (e,f) Western blot analysis and semi‐quantification of H3ace levels. Matrix rigidity increased nuclear H3ace levels, an effect that was reversed by CytoD treatment. (g) Analysis demonstrating that pharmacological inhibition of histone acetyltransferases (HATs) using C646 (20 µ m ) or anacardic acid (AA, 50 µ m ) reduced the rigidity‐induced nuclear accumulation of both MRTF‐A and NF‐κB. (h) Quantitative real‐time PCR (RT‐qPCR) analysis showing the rigidity‐mediated upregulation of inflammatory ( iNOS , IL‐6 ) and HAT ( Ep300 , Crebbp ) genes. Treatment with the F‐actin disruptor CytoD or the HAT inhibitors (C646 or AA) reversed this upregulation ( n = 3). (i) Western blot analysis and semi‐quantification of iNOS protein expression. (j) RT‐qPCR analysis showing that pretreatment with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA, 30 n m ) for 1 h prior to a 12 h LPS stimulation further augmented the rigidity‐driven upregulation of inflammatory genes. Expression levels were normalized to the TSA‐untreated 1 kPa control condition ( n = 3). Data are presented as mean ± SD from three independent biological experiments. Sample sizes ( n ) represent the number of cells or ROIs analyzed per condition for imaging data. Statistical significance was determined using one‐way ANOVA followed by Tukey's post‐hoc test: **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05; ns, not significant ( p > 0.05).

Article Snippet: Digested chromatin was labelled with biotin‐14‐dCTP (Invitrogen) and 40 U of Klenow (NEB), and re‐ligated with 2000 U of T4 DNA Ligase (NEB) for 4 h at 23°C.

Techniques: Activation Assay, Staining, Morphology, Cell Culture, Immunofluorescence, Western Blot, Analysis, Inhibition, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Histone Deacetylase Assay, Control, Imaging