Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Roles of β-catenin signaling in phenotypic expression and proliferation of articular cartilage superficial zone cells.

doi: 10.1038/labinvest.2011.144

Figure Lengend Snippet: Figure 4 Effects of Wnt3a on proliferation and gene expression in superficial zone (SFZ) cultures. SFZ cells were isolated from epiphyseal cartilage of wild-type mice and serially passaged at the density of 4200/cm2 up to six times. (a–f and i), The cultures were continuously treated with Wnt3a-containing conditioned medium (30%) or control conditioned medium after the first passage in the presence or absence of 100 ng/ml of DKK-1. Phase contrast pictures were taken on day 4 in the second passage cultures (a–d). Cell numbers were counted at indicated passages; values are average and s.d. from three independent samples (e). Total RNAs were prepared from Wnt3a-treated or control SFZ cell cultures at indicated passages to analyze expression levels of Proteoglycan 4 (Prg4), Ets-related gene (Erg), CD105 and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) by RT-PCR as described in Materials and Methods (f). Sixth passage cultures, treated with or without Wnt3a, were transferred into pellet cultures and cultured for 7 days. Total RNAs were then prepared to analyze expression level of aggrecan (Acan) or Prg4 (i). (g) The SFZ cells were transfected with Topflash Wnt reporter plasmid, treated with Wnt3a-containing conditioned medium (30%) or control conditioned medium in the presence or absence of 6BIO (BIO, 1 mg/ml), 100 ng/ml of DKK-1 or 10 mM IWR-1 for 24 h, and then the luciferase activity was measured. (h) The cultures were continuously treated with Wnt3a-containing conditioned medium (30%) or control conditioned medium after 1st passage in the presence or absence of 6BIO (BIO, 1 mg/ml), 100 ng/ml of DKK-1, 10 mM IWR-1, 10 mM SP600125 (JNK inhibitor) or 10 mM KN-93 (Ca2 þ /calmodulin-dependent protein kinase II inhibitor, CaMK inhibitor). Total RNAs were prepared from the SFZ cultures at passage 6 to analyze expression levels of Prg4 by real-time PCR. *Po0.05.

Article Snippet: Chondrocytes were isolated by additional overnight collagenase digestion of residual epiphyseal cartilage tissue as previously described.27 Cultures were treated with recombinant mouse Wnt3a (rWnt3a) (Chemicon, Temecula, CA, USA), conditioned medium containing Wnt3a,27 Wnt/b-catenin signaling inhibitors (recombinant mouse Dkk-1 (R&D systems), IWR-1-endo (Santa Cruz Biotechnology), KN-93 (an effective inhibitor of Ca2þ / calmodulin-dependent protein kinase II, Santa Cruz Biotechnology) or SP600125 (a selective inhibitor of c-Jun N-terminal kinases, Santa Cruz Biotechnology).

Techniques: Gene Expression, Isolation, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction