1. Phenotype-related differential α-2,6- or α-2,3-sialylation of glycoprotein N-glycans in human chondrocytes 
S. Toegel, M. Pabst, S.Q. Wu, J. Grass, M.B. Goldring, C. Chiari, A. Kolb, F. Altmann, H. Viernstein, F.M. Unger 
Osteoarthritis and Cartilage 
Volume 18, Issue 2, Pages (February 2010)
DOI: /j.joca
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Analysis of sialylated and free H5N4F1 glycans in human chondrocytes. (A) Scheme showing all the diantennary N-glycans considered in this study with the abbreviated composition and the full structural description according to the proglycan system29,32. S stands for sialic acid, H for hexose, N for N-acetylglucosamine (GlcNAc) and F for fucose. (B) Proportional distribution of free and sialylated H5N4F1 glycans in human chondrocytes. PCs cultured in ML or alginate beads (3D) are compared to SW1353 chondrosarcoma cells and immortalized C-28/I2 chondrocytes. PCs from three donors were analyzed in triplicate and respective mean values are presented. The standard deviations were between 1 and 10% of the mean values for all measurements.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Phenotypic changes of primary human chondrocytes under exposure to inflammatory cytokines. (A) Morphological change of chondrocytes under exposure to TNF-α for 24h. (B) Altered cell number (white bars) and proliferation rate (black bars) of chondrocytes treated with IL-1β and TNF-α. Data are presented as mean values±SD. (C) Reduced sGAG content in chondrocytes treated with TNF-α measured in cells (white bars) and culture medium (black bars). Data are presented as mean values±SD. (D) Fold-changes of AGC, COL2 and COL1 transcription in human chondrocytes isolated from three donors (PC1, PC2, PC3) and exposed to IL-1β and TNF-α with respect to untreated cells (control).
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
mRNA levels of N-glycan related sialyltransferases in human chondrocytes exposed to pro-inflammatory cytokines. PCs were treated in duplicate with 10ng/ml IL-1β (A) or 40ng/ml TNF-α (B) for 24h and SiaT expression was analyzed in duplicate using qPCR. Expression levels were log transformed (log base of 2) and alterations with respect to the control group were displayed as log-fold changes. Mean values and SD of six donors are presented. Asterisks indicate significant differences with respect to untreated controls (P<0.05). (C) Proportional distribution of SiaT mRNA levels in untreated control cells and under exposure to IL-1β or TNF-α.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Regulation of H5N4F1 glycan sialylation under IL-1β and TNF-α exposure. PCs of three donors were cultured separately for 5 days in the presence of 10ng/ml IL-1β (grey bars) or 40ng/ml TNF-α (black bars). N-glycan sialylation was analyzed in triplicate using LC–ESI-MS. Values are means±SD relative to that of untreated cells (grey bars) set at 1.0.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

6. Supplementary Fig. 1
Melting curves were generated by heating amplified qPCR products stepwise from 55°C to 95°C while continuously monitoring the fluorescence. Melting curves confirmed the absence of primer dimers and verified the specific amplification of single qPCR products of ST6Gal1, ST6Gal2, ST3Gal3, ST3Gal4, and ST3Gal6.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions