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HA from myofibroblasts induces macrophage M2 polarization via the <t>CD44/STAT6</t> axis. (A) RAW264.7 macrophages were stimulated with HA for 24 h, and flow cytometry was performed to assess the expression of CD206 (FITC) and CD86 (PE) (n=3 for each group). (B) The content of TGF-β1 in macrophages following HA treatment over 48 h was quantified using ELISA (n=3 for each group). (C) Western blotting analysis of p-STAT6 and total STAT6 expression at various time points following 100 μ g/ml HA stimulation (n=4 for each group). (D) Flow cytometric analysis of CD86 and CD206 expression in macrophages after 24-h treatment with 100 μ g/ml HA and CD44 inhibitor at indicated concentrations (n=3 for each group). (E) TGF-β1 quantification by ELISA in culture supernatants (n=3 for each group). (F) Western blotting analysis of p-STAT6 and total STAT6 in macrophages following 2-h treatment with HA and CD44 inhibitor at varying concentrations (n=3 for each group). (G) Western blotting analysis of p-STAT6 and total STAT6 in macrophages following 2-h treatment with HA and STAT6 inhibitor (AS1517499) at varying concentrations (n=4 for each group). (H) Schematic workflow: Conditioned medium from TGF-β1-stimulated NIH/3T3 fibroblasts was used to culture RAW264.7 macrophages for 24 h. Where indicated, RAW264.7 cells were co-treated with a CD44 inhibitor or a STAT6 inhibitor. Macrophage polarization was subsequently analyzed by flow cytometry. (I) Flow cytometry was used to measure the expression levels of CD206 (FITC) and CD86 (PE) (n=3 for each group). * P<0.05, ** P<0.01, *** P<0.001. HA, hyaluronic acid; FITC, fluorescein isothiocyanate; PE, phycoerythrin; p-, phosphorylated.
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HA from myofibroblasts induces macrophage M2 polarization via the CD44/STAT6 axis. (A) RAW264.7 macrophages were stimulated with HA for 24 h, and flow cytometry was performed to assess the expression of CD206 (FITC) and CD86 (PE) (n=3 for each group). (B) The content of TGF-β1 in macrophages following HA treatment over 48 h was quantified using ELISA (n=3 for each group). (C) Western blotting analysis of p-STAT6 and total STAT6 expression at various time points following 100 μ g/ml HA stimulation (n=4 for each group). (D) Flow cytometric analysis of CD86 and CD206 expression in macrophages after 24-h treatment with 100 μ g/ml HA and CD44 inhibitor at indicated concentrations (n=3 for each group). (E) TGF-β1 quantification by ELISA in culture supernatants (n=3 for each group). (F) Western blotting analysis of p-STAT6 and total STAT6 in macrophages following 2-h treatment with HA and CD44 inhibitor at varying concentrations (n=3 for each group). (G) Western blotting analysis of p-STAT6 and total STAT6 in macrophages following 2-h treatment with HA and STAT6 inhibitor (AS1517499) at varying concentrations (n=4 for each group). (H) Schematic workflow: Conditioned medium from TGF-β1-stimulated NIH/3T3 fibroblasts was used to culture RAW264.7 macrophages for 24 h. Where indicated, RAW264.7 cells were co-treated with a CD44 inhibitor or a STAT6 inhibitor. Macrophage polarization was subsequently analyzed by flow cytometry. (I) Flow cytometry was used to measure the expression levels of CD206 (FITC) and CD86 (PE) (n=3 for each group). * P<0.05, ** P<0.01, *** P<0.001. HA, hyaluronic acid; FITC, fluorescein isothiocyanate; PE, phycoerythrin; p-, phosphorylated.

Journal: International Journal of Molecular Medicine

Article Title: Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2

doi: 10.3892/ijmm.2026.5764

Figure Lengend Snippet: HA from myofibroblasts induces macrophage M2 polarization via the CD44/STAT6 axis. (A) RAW264.7 macrophages were stimulated with HA for 24 h, and flow cytometry was performed to assess the expression of CD206 (FITC) and CD86 (PE) (n=3 for each group). (B) The content of TGF-β1 in macrophages following HA treatment over 48 h was quantified using ELISA (n=3 for each group). (C) Western blotting analysis of p-STAT6 and total STAT6 expression at various time points following 100 μ g/ml HA stimulation (n=4 for each group). (D) Flow cytometric analysis of CD86 and CD206 expression in macrophages after 24-h treatment with 100 μ g/ml HA and CD44 inhibitor at indicated concentrations (n=3 for each group). (E) TGF-β1 quantification by ELISA in culture supernatants (n=3 for each group). (F) Western blotting analysis of p-STAT6 and total STAT6 in macrophages following 2-h treatment with HA and CD44 inhibitor at varying concentrations (n=3 for each group). (G) Western blotting analysis of p-STAT6 and total STAT6 in macrophages following 2-h treatment with HA and STAT6 inhibitor (AS1517499) at varying concentrations (n=4 for each group). (H) Schematic workflow: Conditioned medium from TGF-β1-stimulated NIH/3T3 fibroblasts was used to culture RAW264.7 macrophages for 24 h. Where indicated, RAW264.7 cells were co-treated with a CD44 inhibitor or a STAT6 inhibitor. Macrophage polarization was subsequently analyzed by flow cytometry. (I) Flow cytometry was used to measure the expression levels of CD206 (FITC) and CD86 (PE) (n=3 for each group). * P<0.05, ** P<0.01, *** P<0.001. HA, hyaluronic acid; FITC, fluorescein isothiocyanate; PE, phycoerythrin; p-, phosphorylated.

Article Snippet: Antibodies for Collagen type I α 1 chain (COL1A1; cat. no. 72026; 1:1,000), Phospho-STAT6 (Tyr641; cat. no. 56554S; 1:1,000) and α-smooth muscle actin (α-SMA; cat. no. 19245; 1:1,000) were obtained from Cell Signaling Technology, Inc.

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot