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. 2023 May 15;82(8):1035–1048. doi: 10.1136/ard-2022-223625

Figure 4.

Figure 4

MYH9 expression in RA-FLSs and RA synovium. (A and B) Induction of MYH9 mRNA expression (A) and secretion (B) by pro-inflammatory cytokines and ER stimuli in RA-FLSs. IL-1β (10 ng/mL), TGFβ (10 ng/mL), TNFα (10 ng/mL), IL-6 (10 ng/mL), TM (10 μg/mL for A; 50 μg/mL for B) and TG (10 μM) were used to treat RA-FLSs (n=6 to 9) for 6 hours (A) or 24 hours (B). MYH9 mRNA expression in the cells and protein levels in culture supernatants were determined via real-time PCR analysis and ELISA, respectively. (C) Intracellular MYH9 protein levels in RA-FLSs (n=4) treated with TGFβ (1 ng/mL) or IL-6 (1 ng/mL) for 24 hours were determined by immuno-blotting. (D–F) MYH9 induction in human peripheral blood monocytes. CD14+ cells (n=6) isolated from blood mononuclear cells using magnetic beads were treated with LPS (100 and 1000 ng/mL), TNFα (10 ng/mL) or IL-1β (1 ng/mL) for 12 hours for mRNA induction or 24 hours for analysis of protein expression. MYH9 mRNA expression in the cells was determined by qRT-PCR (D). Levels of MYH9 in the culture supernatants and in the cells were determined by ELISA (E) and western blot analysis (F), respectively. (G) Immunohistochemical staining of MYH9 in RA and OA synovial tissues. An isotype control antibody was used as a control. Scale bar, 100 μm. (H) Immunofluorescence staining of RA synovium using anti-MYH9 Ab (green), anti-CD55 Ab for RA-FLSs (red) and anti-CD68 Ab for macrophages (red). Co-localisation of MYH9 with CD55 (top panel) or with CD68 (bottom panel) is visualised in yellow in the merged images. Nuclei were stained with DAPI (blue). The rectangular area with a white dashed line was magnified to the merged image. Scale bars, 100 μm (for a magnified image, 20 μm). Data show representatives of more than three independent experiments or the mean±SEM. *P<0.05 and **p<0.01. The p values were determined via the Mann-Whitney U test. DAPI, 4′,6-diamidino-2-phenylindole; FLS, fibroblast-like synoviocyte; IL, interleukin; LPS, lipopolysaccharide; MYH9, myosin heavy chain 9; ns, not significant; OA, osteoarthritis; RA, rheumatoid arthritis; TG, thapsigargin; TGF, transforming growth factor; TM, tunicamycin; TNF, tumour necrosis factor.