Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage E.B. Hunziker,

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Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage E.B. Hunziker, M.D., E. Kapfinger, J. Martin, Ph.D., J. Buckwalter, M.D., T.I. Morales, Ph.D. Osteoarthritis and Cartilage Volume 16, Issue 2, Pages 185-194 (February 2008) DOI: 10.1016/j.joca.2007.06.008 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 125IGF-II Western LBs of cell lysates compared to total tissue extracts. Sample 10-04 D was divided into two portions; one was extracted (Methods) and the second was subjected to proteolytic digestion to isolate chondrocytes, which were then lysed by RIPA buffer (Methods). Sample 11-04D was used for isolation and lysis of chondrocytes in the same manner. Twenty-five micrograms of tissue extract or cell lysate protein were applied per well. IGF LB was as described9. The IGFBPs were identified by their molecular weight and previous immunological identification9. BP3=IGFBP-3, BP2=IGFBP-2, and BP4=IGFBP-4. The histological Mankin scores for the samples (from left to right) were: 8.5, 8.5, and N.D. (not determined), 7.5, 3, and 7.5. Osteoarthritis and Cartilage 2008 16, 185-194DOI: (10.1016/j.joca.2007.06.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 IHC for IGFBP-3 of samples with normal and intermediate stage OA. Panels A and B show slices from a normal donor (Mankin grade 1). The superficial zone is facing the top of the figure and approximately the top half of the cartilage section is shown. Panels C and D show tissue from a donor with intermediate grade OA (Mankin grade 6). The photo in C shows approximately the lower 2/3 of the section, and D shows the middle section. Magnification is ∼200×. Sections were counterstained with Meyer's hematoxylin at a dilution of 1:5 for 1min. Osteoarthritis and Cartilage 2008 16, 185-194DOI: (10.1016/j.joca.2007.06.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 IHC for IGFBP-3 of samples with advanced OA. Panels A and B show cartilage from a donor with a Mankin grade 9. Panel C shows a control of the same cartilage treated with antigen-blocked primary antibody (AG:AB molar ratio=12:1), and D shows a second control in which the primary antibody was omitted. Full cartilage depth is shown in the sections, which were counterstained as in Fig. 2. Magnification is ∼200×. Osteoarthritis and Cartilage 2008 16, 185-194DOI: (10.1016/j.joca.2007.06.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 IHC of nuclei for IGFBP-3 in normal cartilage. Panels A, B and C show the normal cartilage (Mankin grade 1) immunostained for IGFBP-3. Panel A shows the approximate upper third, panel B shows another view of a similar section just under the superficial zone and panel C shows the approximate lower third of cartilage (deep zone). Antibody was used at 1:100 dilution. Sections were counterstained as in Fig. 2. Magnification is approximately 600×. The right hand inset in panel C shows the full-depth section, stained with safranine-O; the rectangle in this inset shows the approximate location of the IHC section shown in panel C. Osteoarthritis and Cartilage 2008 16, 185-194DOI: (10.1016/j.joca.2007.06.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Immunohistochemical staining for IGF-I. Panel A shows a normal cartilage, panel B shows a severe OA cartilage and panels C and D are intermediate OA samples, with D being the minus antibody negative control. Panel A shows the approximate upper third of the cartilage, B is a full section of very thin residual cartilage in this severe OA sample, and panels C and D are full-depth figures. Sections were counterstained as described in the legend to Fig. 2. Magnification is 400× for panels A and B and 200× for C and D. Osteoarthritis and Cartilage 2008 16, 185-194DOI: (10.1016/j.joca.2007.06.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Fluorescent immunomicroscopy of IGFBP-3 and DAPI stained nuclei in young human cartilage. Panels A and C show the orange fluorescent stain for IGFBP-3 detected with Upstate anti-IGFBP-3 (1:250 dilution) and a Cy2-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch). Panels B and D show adjacent sections stained with DAPI to reveal the nuclei. Panel E shows an overlay of panels C and D demonstrating co-localization of IGFBP-3 and DAPI stain. Osteoarthritis and Cartilage 2008 16, 185-194DOI: (10.1016/j.joca.2007.06.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Immunogold and immunohistochemical staining on thin sections of human cartilage. Panels A through D show light micrographs of 1μm sections. Panels A and C show staining of nuclei in chondrocytes (arrows) in the transitional zone and in the superficial zone, respectively. Panels B and D show negative controls (without antibody). Panel E shows intra-nuclear immunogold labeling (arrow) for IGFBP-3 in a transitional zone chondrocyte imaged by TEM. Osteoarthritis and Cartilage 2008 16, 185-194DOI: (10.1016/j.joca.2007.06.008) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions