Journal: Advanced Science

Article Title: Matrix‐Rigidity Cooperates With Biochemical Cues in M2 Macrophage Activation Through Increased Nuclear Deformation and Chromatin Accessibility

doi: 10.1002/advs.202403409

Figure Lengend Snippet: High matrix rigidity promotes IL4/13‐mediated M2 macrophage activation. A) Representative images of CellMask (red) and Hoechst‐33342 (blue) staining in mouse bone marrow‐derived macrophages (BMDMs) cultured on either a 1 or 100 kPa polyacrylamide (PA) gel with or without 20 ng mL −1 IL4/13 treatment (scale bars = 50 µm). The 2D cellular surface area was quantified ( n = 30 cells/group) and presented as mean ± SEM (*** p < 0.001; Kruskal‐Wallis test). ns, not significant. B) Heatmap depicting differentially expressed genes (DEGs) from QuantSeq 3′ mRNA‐Seq analysis (|fold change (FC)|≥1.5). DEGs are categorized into six clusters based on expression patterns: up/downregulated by IL4/13 treatment regardless of substrate rigidity (C1_1/C1_2), up/downregulated by matrix rigidity regardless of IL4/13 treatment (C2_1/C2_2), and up/downregulated only in BMDMs cultured on 100 kPa with IL4/13 treatment compared to the other groups (C3_1/C3_2). Line plots show the mean ± standard deviation of Z‐score of normalized log 2 values from QuantSeq 3′ mRNA‐Seq dataset. C) Heatmap representing 20 biological processes in each cluster, with –log 10 raw binomial p ‐values determined by DAVID Gene Ontology (GO) term enrichment analysis. D) Heat map showing log 10 ‐converted FC ratios of Cluster 3_1 DEGs associated with lipid metabolic process, positive regulation of cell migration, positive regulation of angiogenesis, TGFBR signaling pathway, JAK‐STAT cascade, engulfment of apoptotic cell, actin filament organization, and actin filament‐based movement in the 100kPa_IL4/13(+) condition compared to 1kPa_IL4/13(+) condition. E) Representative Western blots and quantification of phospho‐STAT6 (pSTAT6), STAT6, ARG1, and β‐actin in BMDMs cultured on 1 or 100 kPa PA gels with or without IL14/13 (20 ng mL −1 ) treatment for 12 h. Results are presented as mean ± SEM ( n = 3 replicates; ** p < 0.005, and *** p < 0.001; one‐way ANOVA). A. U.: Arbitrary Unit. F) qRT‐PCR analysis of M2‐associated genes ( Arg1 , Mrc1 , Chil3 , and Retnla ) in BMDMs cultured on 1 or 100 kPa PA gels with IL4/13 treatment ( n = 5 replicates; * p < 0.05, ** p < 0.005, and *** p < 0.001; unpaired Student t ‐test). ns, not significant. G) Representative images of phalloidin (red)‐ and DAPI (blue)‐stained BMDMs on 1 or 100 kPa PA gels. Efferocytotic BMDMs were identified by co‐localization with PKH67‐labelled apoptotic Jurkat cells (AC, green), and percentage of PKH67+ BMDMs in total BMDMs was quantified and presented as mean ± SEM ( n = 4 replicates; * p < 0.05; unpaired Student t ‐test). (H) qRT‐PCR analysis of Il10 , Tnf , and Il6 in BMDMs cultured on 1 kPa or 100 kPa gels with or without ACs. ( n = 3 replicates; * p < 0.05, and ** p < 0.005; one‐way ANOVA).

Article Snippet: Multiplex immunofluorescence targeting M2 macrophages (CD206+), M1 macrophages (CD68+, iNOS+, CD206‐), cytotoxic T cells (CD8+), and DAPI, along with the quantification of each cell densities (cells/mm 2 ), was performed by BioActs Co. (Incheon, South Korea) and data were provided.

Techniques: Activation Assay, Staining, Derivative Assay, Cell Culture, Expressing, Standard Deviation, Migration, Western Blot, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Matrix‐Rigidity Cooperates With Biochemical Cues in M2 Macrophage Activation Through Increased Nuclear Deformation and Chromatin Accessibility

doi: 10.1002/advs.202403409

Figure Lengend Snippet: Lamina‐associated domains (LADs) are reduced in macrophages by sensing high matrix rigidity. A,B) Representative immunostaining images of DAPI (blue) and H3K9me2/3 (green) in BMDMs cultured on either a 1 kPa A) or 100 kPa B) PA gel with or without IL4/13 treatment (scale bars = 10 µm). The mean Z‐scores of normalized H3K9me2/3 signal intensity across the relative nuclear distance indicated by dashed arrows ( n = 30 for 1 kPa_IL4/13(−), n = 30 for 1 kPa_IL4/13(+), n = 31 for 100 kPa_IL4/13(−), n = 29 for 100 kPa_IL4/13(+)) are shown. 0 represents the center of the nucleus, and ± 1 represents the nuclear periphery. C) Representative Western blots and quantification of H3K9me2/3 and B‐actin in BMDMs cultured on 1 or 100 kPa PA gels with or without IL4/13 treatment. Results are presented as mean ± SEM ( n = 3 replicates; ** p < 0.005; one‐way ANOVA). D,E) Representative co‐immunostaining images of Lamin A/C (red), H3K9me2 (green), and Hoechst‐33342 (blue) in BMDMs cultured on either a 1 kPa D) or 100 kPa E) PA gel with or without IL4/13 treatment (scale bars = 10 µm). Dashed arrows indicate positions of the line signal intensity profiles. F) A schematic image illustrating the DNA adenine methyltransferase identification (DamID) technique. In the presence of DNA near the nuclear lamina, Lamin B1‐linked Dam induces adenine methylation, enabling the subsequent identification of N 6 ‐methyladenosine through m 6 A tracer conjugated with GFP. G) Representative images of Hoechst‐33342 (blue), m 6 A tracer (GFP, green), and Lamin B1 immunostaining (Red) in HEK‐293 cells cultured on 1 or 100 kPa PA gels and co‐transfected with the DD‐DamWT‐LMNB1‐IRES2‐mCherry plasmid and m 6 A‐Tracer‐NES plasmid for 48 h. The mean Z‐scores of normalized m 6 A tracer GFP intensity are presented across the relative nuclear distance, as indicated by the dashed arrows ( n = 30 nuclei per each condition).

Article Snippet: Multiplex immunofluorescence targeting M2 macrophages (CD206+), M1 macrophages (CD68+, iNOS+, CD206‐), cytotoxic T cells (CD8+), and DAPI, along with the quantification of each cell densities (cells/mm 2 ), was performed by BioActs Co. (Incheon, South Korea) and data were provided.

Techniques: Immunostaining, Cell Culture, Western Blot, Methylation, Transfection, Plasmid Preparation

Journal: Advanced Science

Article Title: Matrix‐Rigidity Cooperates With Biochemical Cues in M2 Macrophage Activation Through Increased Nuclear Deformation and Chromatin Accessibility

doi: 10.1002/advs.202403409

Figure Lengend Snippet: M2‐activated macrophages exhibit enhanced immunosuppressive phenotypes under high environmental rigidity. A) A schematic image illustrating the experimental design to evaluate the immunosuppressive characteristics of M2 macrophages under distinct substrate rigidity conditions. B) Significantly increased (red) or decreased (blue) cytokines released from BMDMs cultured on 100 kPa PA gels, compared to that released from BMDMs cultured on 1 kPa condition ( p < 0.05). The log10 normalized relative expression values are shown. C) KEGG pathway analysis in which increased (red) or decreased (blue) cytokines are involved. The log10 normalized p‐values are shown. D) CFSE histograms detecting the inhibition of T cell proliferation after treatment with the conditioned medium (CM) of BMDMs cultured on 100 kPa, compared to that of BMDMs cultured on 1 kPa. Results are presented as mean ± SEM ( n = 4 replicates; * p < 0.05; unpaired Student t ‐test). E) Representative images of Sirius Red (SR) staining and multiplex immunofluorescence analysis for M2 macrophages (CD206; green), M1 macrophages (CD68; red, iNOS; yellow), cytotoxic T cells (CD8; orange), and DAPI (blue) in tissue microarrays of human head and neck squamous cell carcinoma tissues. Quantification graphs of the cell density (cells/mm 2 ) ratio between cytotoxic T cells (CD8+) and M2 macrophages (CD206+) and between M2 and M1 macrophages (CD68+, iNOS+, CD206‐) are shown. Results are presented as the mean ± SEM (1: n = 142, 2: n = 48, 3: n = 20; * p < 0.05; Mann‐Whitney test or unpaired Student t ‐test, respectively).

Article Snippet: Multiplex immunofluorescence targeting M2 macrophages (CD206+), M1 macrophages (CD68+, iNOS+, CD206‐), cytotoxic T cells (CD8+), and DAPI, along with the quantification of each cell densities (cells/mm 2 ), was performed by BioActs Co. (Incheon, South Korea) and data were provided.

Techniques: Cell Culture, Expressing, Inhibition, Staining, Multiplex Assay, Immunofluorescence, MANN-WHITNEY