Figure 3. Analysis of xylosyltransferase activity and GAG chain composition. (A) xylosyltransferase activity from normal, early OA and late-stage OA cartilage of three representative patients. Data are mean 6 SD of 3 experiments per parameter, per joint, per patient. **Significantly (P,0.01) higher than all groups; *significantly (P,0.01) higher than normal and late groups; 1significantly (P,0.05) lower than normal. (B) GAG chain composition in human cartilage explants as determined by FACE. Proteinase K-digested cartilage explants contain- ing equal amounts (5 mg) of GAGs, as determined by uronic acid assay from each group, were digested with chondroitinase ABC and the disaccharides obtained were fluorotagged and derivatized. Disaccha- rides were run on a monocomposition gel and gels are representative images from 3 experiments per parameter, per joint, per patient. Markers are DDi0S, nonsulfated chondroitin-unsaturated disaccharide; DDi4S, chondroitin-4-sulfated unsaturated disaccharide; DDi6S, chon- droitin-6-sulfated unsaturated disaccharide. doi:10.1371/journal.pone.0034020.g003 late-stage OA, suggesting that a functional link may exist between the expression of this glycosyltransferase and sulfated-GAG content in human OA cartilage. Down-regulation of XT-I expression impairs sulfated-GAG synthesis in cartilage To determine the importance of XT-I in synthesis of sulfated- GAG in cartilage shRNA was used to transfect chondrocytes and cartilage explants. Quantitative PCR analyses indicated a down- regulation of XT-I mRNA expression of about 70% in shRNA- XT-I and Proteoglycan Synthesis in Osteoarthritis transfected chondrocytes compared to control groups (Fig. 4A). Interestingly, analysis of sulfated-GAG anabolism at 72 h post- transfection showed a reduction of about 50% in shRNA- transfected chondrocytes compared to control groups (Fig. 4B) indicating that XT-I plays an important role in sulfated GAG synthesis in chondrocytes. Similarly, transfection of cartilage explants from three different patients with shRNA led to a decrease of sulfated GAG synthesis ranging from 30% to 58% whereas no inhibition was seen in cartilage explants non- transfected or transfected with shRNA control (Fig. 4C). Altogeth- er, these results demonstrated the key role of XT-I in sulfated GAG synthesis in human cartilage. Gene delivery of XT-I in chondrocytes and cartilage explants stimulates GAG synthesis To directly determine the role of XT-I in cartilage GAG anabolism, we overexpressed XT-I in human chondrocytes and cartilage explants. We observed high expression of recombinant XT-I protein in chondrocytes at 48 h after transfection (Fig. 5A). Figure 4. Analysis of the effect of XT-I knockdown on GAG synthesis in chondrocytes and cartilage explants. Cells were transfected with XT-I shRNA, or control shRNA or left untreated and then examined for mRNA expression (A) and GAG synthesis (B). Total RNA was isolated from cells of each group at 72 h posttransfection and XT-I expression level was determined by qPCR analysis. XT-I mRNA levels are represented as a ratio relative to those in negative controls or untreated controls. GAG synthesis was studied by 35S-sulfate incorpo- ration. (C) Cartilage explants from normal region of human femoral condyle of three patients were transfected with XT-I shRNA or control shRNA and then GAG synthesis was analyzed by 35S-sulfate incorpo- ration. Measurements were normalized to control shRNA. Data represent the mean 6 SD of 3 independent observations for each group. **,*Significantly (P,0.01, P,0.05, respectively) lower than control shRNA. doi:10.1371/journal.pone.0034020.g004 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e34020