Journal: The Journal of Biological Chemistry

Article Title: Loss of integrin alpha7-mediated signaling induces a dendritic cell-like phenotype in macrophages cultured on laminin-211/221 isoforms

doi: 10.1016/j.jbc.2025.110419

Figure Lengend Snippet: Localization of laminin-related integrins and the effect of functional blocking of integrin α7 in THP-1 macrophages cultured on laminin-211/221/221E8 or laminin-511 for 5 days . A , morphology and multiple immunofluorescent staining for laminin-related integrins (α7, α6, and α3) and phalloidin in THP-1 macrophages cultured on laminin (LM)-211/-511. Nuclei were counterstained with DAPI. Immunostaining was performed four times independently, imaging a total of 300 to 350 cells per antibody. B , morphology of THP-1 macrophages cultured on laminin-211/-221/-221E8 in the presence of integrin α7 function-blocking antibodies (anti-α7) or isotype control IgG. In the presence of integrin α7 function-blocking antibodies, dendritic-like branching and extended cellular processes are observed. These experiments were repeated at least five times independently. C , morphology of THP-1 macrophages cultured on laminin-511 in the presence of integrin α7 function-blocking antibodies. D , western blotting of integrin α7 under conditions of integrin α7-targeted small interfering RNA (siITGA7) on day 5. siITGA7 #9 and #1 reduced integrin α7 levels on laminin-211. Nonspecific siRNA was used as control (siCtrl); β-actin was used as a loading control. E , gene expression levels of integrin α7. Data are presented as the mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001 as determined by Tukey’s post hoc test. F , morphology of THP-1 macrophages treated with integrin α7-targeted small interfering RNA #9, #1 or nonspecific siRNA on day 5. The knockdown experiment was repeated three times independently. Integrin α7 knockdown induced dendritic processes in THP-1 macrophages cultured on laminin-211 ( arrows ). Nonspecific siRNA did not induce obvious morphological alterations. The scale bars represent 10 μm. DAPI, 4′,6-diamidino-2-phenylindole; IgG, immunoglobulin G.

Article Snippet: Neutralizing antibodies specific for integrin α7 (DSHB Hybridoma Product 9.1 ITGA7, deposited by Kramer R. H.) were added to the culture medium at 10 μg/ml from the start to the end of the experiment.

Techniques: Functional Assay, Blocking Assay, Cell Culture, Staining, Immunostaining, Imaging, Control, Western Blot, Small Interfering RNA, Gene Expression, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Loss of integrin alpha7-mediated signaling induces a dendritic cell-like phenotype in macrophages cultured on laminin-211/221 isoforms

doi: 10.1016/j.jbc.2025.110419

Figure Lengend Snippet: Dendritic cell markers are upregulated by siRNA-mediated downregulation or functional blocking of integrin α7 in THP-1 macrophages cultured on laminin-211 or 221E8 for 5 days . A , volcano plot illustrating differentially regulated gene expression from RNA-Seq analysis in THP-1 macrophages transfected with integrin α7 siRNA compared with nonspecific siRNA. B , gene ontology (GO) enrichment analysis of genes upregulated for molecular function. The top five most significantly affected categories are shown. C , heatmap depicting the expression of genes in the antigen-binding signature. Integrin α7-targeted small interfering RNA (siITGA7) transfected cells compared with the nonspecific control siRNA (siCtrl) (n = 2 for each group). D , KEGG pathway analysis of cell adhesion molecules between antigen-presenting cells (APCs) and T cell receptor signaling. E , analysis of gene expression in THP-1 macrophages cultured under the conditions indicated. Data are presented as the mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001 as determined by Tukey’s post hoc test. LM-221E8: laminin-221E8; Anti-α7: anti-integrin α7 antibody. F , morphology of THP-1 macrophages treated with the conditions indicated. Arrows represent spike-like projections. The scale bar represents 10 μm. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Neutralizing antibodies specific for integrin α7 (DSHB Hybridoma Product 9.1 ITGA7, deposited by Kramer R. H.) were added to the culture medium at 10 μg/ml from the start to the end of the experiment.

Techniques: Functional Assay, Blocking Assay, Cell Culture, Gene Expression, RNA Sequencing, Transfection, Expressing, Binding Assay, Small Interfering RNA, Control

Journal: The Journal of Biological Chemistry

Article Title: Loss of integrin alpha7-mediated signaling induces a dendritic cell-like phenotype in macrophages cultured on laminin-211/221 isoforms

doi: 10.1016/j.jbc.2025.110419

Figure Lengend Snippet: Morphologic evidence of endocytosis by THP-1 macrophages treated with integrin α7 function-blocking antibodies on laminin-221E8 . A , immunofluorescent staining for phalloidin and microtubule in THP-1-derived macrophages cultured without integrin α7 function-blocking antibodies for 5 days. B , immunofluorescent staining for phalloidin, microtubule, and microtubule-associated protein 2 (MAP2) in THP-1-derived macrophages cultured with integrin α7 function-blocking antibodies (anti-α7) for 5 days. Arrows represent the tips of dendritic processes. An enlarged view of the MAP2 box area is shown on the right . C , analysis of gene expression of MAP2 in THP-1 macrophages cultured under the conditions indicated. Data are presented as the mean ± SEM of three independent experiments. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 as determined by Tukey’s post hoc test. D and E , morphologic evidence for endocytosis. THP-1 macrophages treated with integrin α7 function-blocking antibodies ( D ) or not treated ( E ) were incubated with FITC-dextran at 37 °C or on ice (background control). Arrows indicate the same point on the immunofluorescence image or immunofluorescence + phase contrast image ( D ). The boxed area is shown in the inset. A large number of FITC + puncta were evident in dendritic processes ( D ). Original magnification: × 200 (left lane) and × 600. F and G , FITC-dextran uptake of THP-1 macrophages treated with integrin α7 ( F ) or not treated ( G ) were analyzed by flow cytometry and statistical analysis. Data are representative of three independent experiments. Each column represents the mean ± SEM of three independent experiments. ns, not significant. ∗∗∗∗ p < 0.0001 as determined by an unpaired t test. FITC-Dex: FITC-dextran. The scale bars represent 10 μm.

Article Snippet: Neutralizing antibodies specific for integrin α7 (DSHB Hybridoma Product 9.1 ITGA7, deposited by Kramer R. H.) were added to the culture medium at 10 μg/ml from the start to the end of the experiment.

Techniques: Blocking Assay, Staining, Derivative Assay, Cell Culture, Gene Expression, Incubation, Control, Immunofluorescence, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Loss of integrin alpha7-mediated signaling induces a dendritic cell-like phenotype in macrophages cultured on laminin-211/221 isoforms

doi: 10.1016/j.jbc.2025.110419

Figure Lengend Snippet: Proliferation of CD4 + and CD8 + T cells after coculture with DC-like cells induced by integrin α7 function-blocking antibodies on laminin-221E8-coated plates. CD4 + and CD8 + T cells were stained with CFSE before coculture. A , DC-like cells or THP-1 macrophages were stimulated with LPS (1 μg/ml) or left unstimulated for 24 h before coculture. Representative histograms showing the proliferation of CD4 + and CD8 + T cells under the indicated conditions are presented. B , THP-1 cells were treated with rhGM-CSF and rhIL-4 to generate immature model DCs, or with rhGM-CSF, rhIL-4, rhTNF-α, and ionomycin to generate mature model DCs, prior to coculture. Representative histograms showing the proliferation of CD4 + and CD8 + T cells under the indicated conditions are presented. C , percent cell proliferation of CD4 + and CD8 + T cells. Each column represents the mean ± SEM of three independent experiments. ns, not significant. ∗∗ p < 0.01 and ∗∗∗∗ p < 0.0001 as determined by Tukey’s post hoc test. CFSE, carboxyfluorescein succinimidyl ester; DC, dendritic cell; LPS, lipopolysaccharide; rhGM-CSF, recombinant human granulocyte macrophage colony-stimulating factor.

Article Snippet: Neutralizing antibodies specific for integrin α7 (DSHB Hybridoma Product 9.1 ITGA7, deposited by Kramer R. H.) were added to the culture medium at 10 μg/ml from the start to the end of the experiment.

Techniques: Blocking Assay, Staining, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Loss of integrin alpha7-mediated signaling induces a dendritic cell-like phenotype in macrophages cultured on laminin-211/221 isoforms

doi: 10.1016/j.jbc.2025.110419

Figure Lengend Snippet: Functional blocking of integrin α7 induces a decrease in p85α levels and increases AKT activation in THP-1 macrophages on laminin-221E8 . A , gene set enrichment analysis (GSEA) of downregulated genes in integrin α7 siRNA-transfected macrophages compared with control. B , expression heatmap enriched for signaling by PI3K/AKT activation in RNA-Seq data. Integrin α7-targeted small interfering RNA (siITGA7)-transfected cells compared with nonspecific control siRNA (siCtrl) (n = 2 for each group). C , representative western blot image of the regulatory subunit p85α and the catalytic subunits p110α, p110β, and p110δ of class IA PI3K, and AKT phosphorylation (p-AKT) at Thr308 and Ser473, and total AKT on day 5 under indicated conditions. GAPDH was used as a loading control. Levels of phosphorylated proteins were measured by densitometry, normalized to total protein and GAPDH levels. D , representative western blot image of PTEN phosphorylation (p-PTEN) at Ser380 and subunits of PP2A: scaffold ( A ), regulatory ( B ), and catalytic ( C ). GAPDH was used as a loading control. E , gene expression levels of PIK3R1 in THP-1-derived macrophages treated with PIK3R1 siRNA (siPIK3R1) or nonspecific siRNA (siCtrl) on laminin (LM)-221E8 on day 5. F , representative western blot image for class IA PI3K and p-AKT on day 5 under the conditions indicated. GAPDH was used as a loading control. Levels of phosphorylated proteins were measured by densitometry, normalized to total protein and GAPDH levels. G , phase contrast images of THP-1 derived macrophages treated with PIK3R1 siRNA or nonspecific siRNA and immunofluorescent staining for microtubule on laminin-221E8 on day 5. Arrows represent short projections. H , representative western blot image of p-AKT and total AKT under indicated conditions on day 5. AKT inhibitor (MK-2206: MK and 5 μmol/L: MK5) or the PI3K catalytic subunit (p110α/β/δ) inhibitor (LY294002: LY, 5 μmol/L: LY5, and 25 μmol/L: LY25) was added on day 2, and the cells were cultured for 3 days in the presence or absence of integrin α7 function-blocking antibodies. Levels of phosphorylated proteins were measured by densitometry, normalized to total protein and GAPDH levels. I , phase contrast images of THP-1 macrophages treated with MK-2206 (MK) or LY294002 (LY) on laminin-221E8 on day 5. Arrows represent dendritic processes. PIK3R1 knockdown experiments were repeated three times independently for gene and protein expressions, respectively. The experiments using inhibitors, MK-2206 or LY294002, were repeated three times independently. Anti-α7: anti-integrin α7 antibody. Values are the mean and ± SEM of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as determined by Tukey’s post hoc test. The scale bars represent 10 μm. PP2A, protein phosphatase 2A; PTEN, phosphatase and tensin homologue; RNA-Seq, RNA sequencing.

Article Snippet: Neutralizing antibodies specific for integrin α7 (DSHB Hybridoma Product 9.1 ITGA7, deposited by Kramer R. H.) were added to the culture medium at 10 μg/ml from the start to the end of the experiment.

Techniques: Functional Assay, Blocking Assay, Activation Assay, Transfection, Control, Expressing, RNA Sequencing, Small Interfering RNA, Western Blot, Phospho-proteomics, Gene Expression, Derivative Assay, Staining, Cell Culture, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Loss of integrin alpha7-mediated signaling induces a dendritic cell-like phenotype in macrophages cultured on laminin-211/221 isoforms

doi: 10.1016/j.jbc.2025.110419

Figure Lengend Snippet: Laminin isoforms differentially affect GM-CSF-induced human primary macrophage kinetics . A , day 6 morphology of M-CSF or GM-CSF stimulated monocytes cultured on laminin (LM)-221E8/-511E8 or without laminin coat (LM-). M-CSF stimulated monocytes are floating on LM-221E8 or -511E8 coated plates. B , gene expression levels of CD68, integrin β1 (ITGB1), integrin α7 (ITGA7), integrin α6 (ITGA6), integrin α3 (ITGA3) in GM-CSF stimulated monocytes cultured on LM-221E8/-511E8 or LM (−). Data are presented as the mean ± SEM of three independent experiments. ∗ p < 0.05 and ∗∗ p < 0.01 as determined by Tukey’s post hoc test. C , multiple immunofluorescent staining for laminin-related integrins (α7, α6, and α3) in GM-CSF-stimulated monocyte-derived macrophages cultured on LM-221E8 or -511E8. Immunostaining was performed four times independently, imaging a total of 300 to 350 cells per antibody. Nuclei were counterstained with DAPI. The scale bars represent 10 μm. DAPI, 4′,6-diamidino-2-phenylindole; GM-CSF, granulocyte macrophage colony-stimulating factor.

Article Snippet: Neutralizing antibodies specific for integrin α7 (DSHB Hybridoma Product 9.1 ITGA7, deposited by Kramer R. H.) were added to the culture medium at 10 μg/ml from the start to the end of the experiment.

Techniques: Cell Culture, Gene Expression, Staining, Derivative Assay, Immunostaining, Imaging

Journal: The Journal of Biological Chemistry

Article Title: Loss of integrin alpha7-mediated signaling induces a dendritic cell-like phenotype in macrophages cultured on laminin-211/221 isoforms

doi: 10.1016/j.jbc.2025.110419

Figure Lengend Snippet: GM-CSF-stimulated monocyte-derived macrophages cultured on laminin-221E8 are altered to exhibit a DC-like phenotype . A , schematic diagram of the experimental method using human peripheral blood CD14 + monocytes. B , day 10 morphology of GM-CSF derived macrophages cultured on laminin (LM)-221E8/-511E8 or without laminin coat (LM-). Arrows indicate veil-like membrane projections. A closer image of the boxed area is shown in the inset. The scale bars represent 10 μm. C , analysis of gene expression in monocyte derived macrophages cultured under the conditions indicated. Data are presented as the mean ± SEM of three independent experiments. ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as determined by Tukey’s post hoc test. Anti-α7: anti-integrin α7 antibody. DC, dendritic cell; GM-CSF, granulocyte macrophage colony-stimulating factor.

Article Snippet: Neutralizing antibodies specific for integrin α7 (DSHB Hybridoma Product 9.1 ITGA7, deposited by Kramer R. H.) were added to the culture medium at 10 μg/ml from the start to the end of the experiment.

Techniques: Derivative Assay, Cell Culture, Membrane, Gene Expression