Alkaline phosphatase activity measurement ALP activity was measured as previously described using a p- nitrophenyl phosphate substrate and an incubation temperature of 37uC by a method modified from that of Lowry et al. [41]. In brief, the assay mixtures contained 0.1 M of 2-amino-2-methyl-1- propanol, 1 mM of MgCl2, 8 mM of p-nitrophenyl phosphate disodium (Sigma), and cell homogenates. After 3 minutes of incubation, the reaction was stopped with 0.1 N NaOH and the absorbance was read at 405 nm. A standard curve was prepared with p-nitrophenol (Sigma). Each value was normalized with the value in DNA content. DNA in pellets was measured according to the method of Schneider [42]. Alizarin red staining and von Kossa staining To measure extracellular matrix Ca deposits for bone nodule formation, cellular matrix was stained using Alizarin red dye which combines with Ca in the matrix as previously described [43,44]. At 14 days, cells were washed with PBS twice and then fixed with 2.0% formaldehyde. The cells were stained with 40 mmol/L of Alizarin red solution (pH 4.4) for 40 minutes at room temperature and rinsed with deionized water twice. The images of stained cells were captured using a phase contrast microscope with a digital camera (IM50, Leica, Germany). The Alizarin red solution was removed by aspiration, and the cells were rinsed five times with deionized water. The water was removed by aspiration, and the cells were incubated in PBS for 15 min at room temperature on an orbital rotator. The concentration of Alizarin red staining in the samples was determined by comparing the absorbance values with those obtained from Alizarin red standards. The cells were destained for 15 min with 10% (w/v) cetylpyridinium chloride(sigma) in 10 mM sodium phosphate (pH 7.0). The extracted stain was then transferred to a 96-well plate, and the absorbance at 562 nm was measured using spectrophotometer. The mineralization values were normalized to the relative number of PLB as determined directly in the 96-well plates [45]. Because Ca coprecipitates with the phosphate ion in the matrix, von Kossa staining (which stains phosphate ions) was also used for the determination of mineralization in the cultures [46]. Cells were washed with PBS and fixed with 2.0% formaldehyde for 15 minutes. After washing with deionized water 3 times, cells were incubated with 3% silver nitrate at room temperature under ultraviolet light for 1 hour. After washing with deionized water, the images of the stained cells were photographed using phase-contrast microscope. Immunostaining The cells were stimulated with PAPSS2 for the indicated times, plated on 24 well plates and incubated overnight. The cells were References 1. Hoang QQ, Sicheri F, Howard AJ, Yang DS (2003) Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 425: 977–980. 2. 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Function of PAPSS2 in Osteoblasts sequentially fixed with 100% methanol for 10 minutes, blocked with 10% normal rabbit or mouse serum in PBS for 60 minutes, then correspondingly incubated in mouse anti-PAPSS2 antibody (1 mg/ml, Sigma) rabbit anti-type I collagen (Col I) specific polyclonal antibody, rabbit anti-osteopontin (OPN) polyclonal antibody (1 mg/ml, Invitrogen), and mouse anti-phospho-Smad 2/ 3 antibody (1 mg/ml, Santa Cruz Biotechnology) in TBS contain- ing 1.5% normal rabbit or goat serum, respectively, for 60 minutes. Cells were then incubated in FITC- or HRP- conjugated anti-rabbit, or mouse IgG (1 mg/ml, Santa Cruz Biotechnology) with 1.5% normal rabbit, donkey, or goat serum, respectively, for 60 minutes. Cells were treated with FITC- conjugated antibody, then washed three times in PBS, and examined with microscopes [47]. The cell nuclei were stained using DAPI to facilitate determination of the number of cells. Western blot analysis Cells were lysed with NP40 buffer (1%NP-40, 0.15 M NaCl, 50 mM Tris, pH 8.0) containing protease inhibitors (Invitrogen). Cell lysates were centrifuged at 12,000 g for 20 minutes at 4uC, and the supernatants were stored at 280uC. Protein quantitation was performed using a BCA protein assay reagent (Pierce, Rockford, IL, U.S.). Equal amounts of protein were denatured in SDS sample buffer and separated on 10% polyacrylamide-SDS gel. Proteins were transferred in 25 mM of Tris, 192 mM of glycine, and 20% methanol to produce polyvinylidene difluoride. Blots were blocked with Tris-buffered saline (TBS; 20 mM of Tris- HCl [pH 7.5] and 137 mM of NaCl) plus 0.1% Tween20 containing 3% dried milk powder. We used anti-phospho-Smad 2/3 antibody (Santa Cruz Biotechnology, CA, U.S.) and anti- PAPSS2 antibody (Abnova, Taiwan) to detect the signals. 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