D130 3

Cell layers were harvested in lysis buffer containing 150mM NaCl, 50mM TrisCl, 1mM EDTA, 1% Triton, 1% sodium deoxycholate, 0.1% SDS and protein inhibitor cocktail (Cell Signaling) and frozen at −80°C. Samples were thawed, homogenized on ice and centrifuged at 12,600 rpm for 10 min at 4°C, and protein levels were quantified from the supernatants using the BCA assay (Pierce). 10ug or 25ug of protein (for osteopontin or Runx2 respectively) were mixed with Laemmli loading buffer, boiled and cooled, and separated using SDS-PAGE. Proteins were transferred onto PVDF membranes (Millipore) and blocked in 5% non-fat milk. Membranes were first probed with primary antibodies in 5% non-fat milk or bovine serum albumin and then with horse radish peroxidase (HRP)-conjugated secondary antibodies in 5% non-fat milk. Bands were visualized by chemiluminescence using SuperSignal West Pico substrate (Thermo Scientific). The following primary antibodies and dilutions were used: goat anti-osteopontin (AF808, R&D Systems) at 1:5000, mouse anti-Runx2 (D130-3, MBL International) at 1:500 and rabbit anti-GAPDH (14C10, Cell Signaling Technology) at 1:1000. The following HRP-conjugated secondary antibodies and dilutions were used: anti-goat (A8919, Sigma Aldrich) at 1:20000, anti-mouse (W402B, Promega) at 1:7500 and anti-rabbit (A0545 Sigma Aldrich) at 1:5000.

3 µm-thick paraffin sections were baked at 60 °C overnight. Slides were then deparaffinised, and rehydrated. Antigen retrieval of collagen II and collagen X was performed using 0.2 g pepsin in 50 mL of 0.01 N HC1 at 37 °C for 25 min. Antigen retrieval of Runx2 was performed using a 0.01 M citrate buffer pH 6.0 at 95 °C for 2 h. Dako endogenous blocking reagent (S2003, Dako, Carpinteria, CA, USA) was then used to quench endogenous peroxidase for 10 min. Non-specific binding sites were blocked with 1:20 normal horse/goat serum (S-2000, Vector Laboratories, Burlingame, CA, USA) for 20 min at room temperature. 1:80 dilution (2.5 µg/mL) of collagen II (MS-235-P Thermo Scientific, Rockford, IL, USA), 1:50 dilution of collagen X (2031501005, Quartett, Berlin, Germanv), or 1:100 dilution (10 µg/mL) of Runx2 (D130-3, MBL International, Woburn, MA, USA) primary antibodies were added and the slides were incubated at 4 °C overnight. Secondary biotinylated horse anti-mouse antibody (BA-2000, Vector Laboratories) or goat anti-mouse antibody (BA-9200, Vector Laboratories) at the dilution of 1:200 was added for 30 min on the second day, followed by incubation with 1:250 streptavidin (21130, Pierce, Rockford, IL, USA) for 30 min. Positive staining was detected by Romulin AEC Chromagen (Biocare Medical RAEC810L, Concord, CA, USA).