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. 2020 Mar 12;10:4570. doi: 10.1038/s41598-020-61630-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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p22phox interaction with Rubicon in human and CIA mice. (a) Schematic of the collagen-induced RA (CIA) model (upper). p22phox complexes purified from synovial fluid containing synoviocytes from CIA mice were subjected to mass spectrometry analysis. Silver stained gel (bottom left) and peptides identified by mass spectrometry analysis (bottom right) The red-colored letters indicate the peptides identified from mass spectrometry analysis. (b) Representative Hematoxylin-Eosin (H&E) staining of the ankle joints of each group (upper); Scale bar, 500 μm. Clinical arthritis, swelling of paws scores (middle), and histopathology scores (bottom). Results are expressed as means ± SD (5 mice per group). (c) Synovial fluid containing synoviocytes from CIA mice for the indicated times, followed by IP with αp22phox (left) or αRubicon (right), followed by IB with αRubicon, αp22phox, and αgp91phox. The anti-p22phox blot was also tested for αTLR4, αTRAF6 binding. The anti-Rubicon blot was also tested for αBeclin-1, or αUVRAG. WCLs were used for IB with αRubicon, αp22phox, αgp91phox, αTLR4, αTRAF6, αBeclin-1, αUVRAG. Actin Western blot was used as a loading control. (d) OA and RA cells were stained with αp22phox (Alexa Fluor 488; green) and αRubicon (Cy3; red). Nuclei were counterstained with DAPI. Cells were visualized by confocal microscopy (left). The middle panel shows the quantitative data of staining intensity of p22phox and Rubicon (upper) and the colocalization index (%) between p22phox and Rubicon (bottom). Immunohistochemical analysis to examine p22phox-DAB (3,3′-diaminobenzidine) and Rubicon-AEC (3-amino-9-ethylcarbazole) expression (right). Representative images from six independent OA and RA patients are shown. Insets, enlargement of outlined areas. Scale bars: 200 μm. The data are representative of five independent experiments with similar results (a, c and d). Statistical analysis was done using the Student’s t-test with Bonferroni adjustment. Data are considered different at p < 0.05. ***p < 0.001. Experimental procedures were described in Supplementary Information.