Differential proteome analysis of normal and osteoarthritic chondrocytes reveals distortion of vimentin network in osteoarthritis S. Lambrecht, M.Pharm.,

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Differential proteome analysis of normal and osteoarthritic chondrocytes reveals distortion of vimentin network in osteoarthritis S. Lambrecht, M.Pharm., G. Verbruggen, M.D., Ph.D., P.C.M. Verdonk, M.D., Ph.D., D. Elewaut, M.D., Ph.D., D. Deforce, Ph.D. Osteoarthritis and Cartilage Volume 16, Issue 2, Pages 163-173 (February 2008) DOI: 10.1016/j.joca.2007.06.005 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Image of articular cartilage stained with Indian ink. Both samples were isolated from the femur condyle from the same patient. On the upper slice (OAOA) the fibrillated surface is clearly visible. The lower slice (NoOA) shows a smooth, non-fibrillated surface. Osteoarthritis and Cartilage 2008 16, 163-173DOI: (10.1016/j.joca.2007.06.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Match set's standard image of 2-D separated proteins of the soluble fraction (derived from OAOA sample). Numbered spots are identified by MS. Osteoarthritis and Cartilage 2008 16, 163-173DOI: (10.1016/j.joca.2007.06.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Match set's standard image of 2-D separated proteins of the hydrophobic fraction (derived from OAOA sample). Numbered spots are identified by MS. Osteoarthritis and Cartilage 2008 16, 163-173DOI: (10.1016/j.joca.2007.06.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Two spots, identified as vimentin, showed a higher expression in OAOA samples. Bars represent relative average expression levels (±s.e.m.) of spots 1404 and 1437 in each group. Black: NoNo, white: NoOA, and shaded: OAOA. Osteoarthritis and Cartilage 2008 16, 163-173DOI: (10.1016/j.joca.2007.06.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 1-D Western blot analysis of the soluble fraction of 12 samples (from the left to the right: four NoNo samples, four NoOA samples and four OAOA samples). (A) Equal amounts of soluble fractions of chondrocytes were loaded on 10% SDS-PAGE and immunoblotted. Immunoblotting was performed using monoclonal anti-vimentin (clone V9), where NoNo and NoOA samples show a more intense band at the native Mw of vimentin (upper box), three OAOA samples show a more intense band near 43kDa (lower box). (B) and (C) Results of densitometric analysis. Bars represent relative average density (±s.e.m.) of bands per group. Black: NoNo, white: NoOA, and shaded: OAOA. Osteoarthritis and Cartilage 2008 16, 163-173DOI: (10.1016/j.joca.2007.06.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 2-D Western blot of the soluble fraction of OAOA sample. Thirty micrograms of total protein was loaded on 4–7 IPG strip, transferred to 10% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and vimentin was detected using monoclonal anti-vimentin (clone V9). 1, 2 and 3 indicate the protein spots that were excised from a gel, run in parallel to the gels used for Western blotting. Osteoarthritis and Cartilage 2008 16, 163-173DOI: (10.1016/j.joca.2007.06.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Representative confocal microscopy images of NoNo (upper panel), NoOA (middle panel) and OAOA chondrocytes (lower panel). Chondrocytes were immunostained for vimentin (green), F-actin (red) and the nucleus (blue), shown as individual channels and in superposition from the left to the right. Scale bar indicates 5μm. Green panel (vimentin-staining): the majority of NoNo chondrocytes clearly shows a narrow zone of fluorescence, in contrast to the spread fluorescence of OAOA chondrocytes. Red panel (F-actin staining): no clear differences in F-actin staining could be detected between the sample groups. All sample groups show a fluorescence signal distributed over the cytoplasm. Blue panel: nuclear DAPI stain. Right panel: superimposition of the three images. Osteoarthritis and Cartilage 2008 16, 163-173DOI: (10.1016/j.joca.2007.06.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 Percentage of chondrocytes showing a narrow intense zone of fluorescence at the periphery of the cytoplasm (expressed as mean±SD). Cells were incubated with monoclonal anti-vimentin, followed by the appropriate secondary antibody, labelled in Alexa Fluor 488. Black: NoNo, white: NoOA, and shaded: OAOA. Osteoarthritis and Cartilage 2008 16, 163-173DOI: (10.1016/j.joca.2007.06.005) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions