Fig 1: WNT16 treatment inhibited mineralization and promoted cell senescence of AS-osteoprogenitor cells during osteoblast differentiation. Both control and AS-osteoprogenitor cells were differentiated with osteogenic medium in the presence of vehicle or WNT16. Analysis of A ALP staining, B ARS staining, and VON staining, C HA staining, D intercellular ALP activity at indicated day, E quantitation of ARS and VON at day 21, and HA staining at day 28 (n=4 per each group). F SaOS2 cells were co-transfected with 3 μg ALP, BSP, OSE, or OCN promoter plasmid along with β-gal plasmid for 48 h followed by and WNT16 treatment for 24 h and then analyzed by luciferase assay (n=6 per each group). G SA-β-gal staining was performed at 28 days of osteoblast differentiation of control and AS-osteoprogenitor cells. Counting results of Fig. 3G (right panel) (n=4 per each group). AS-osteoprogenitor cells were differentiated into mature osteoblasts in the presence of vehicle and WNT16 (50ng/ml). Analysis of H immunoblotting for protein level and I RT-qPCR for mRNA level (n=3 per each group). Scale bar = 200 μm. Data are presented as the median and interquartile range. *p<0.05
Fig 2: WNT16 treatment facilitated cell senescence of AS-osteoprogenitor cells under H2O2-stimulation. A Experimental design for cell senescence treated with WNT16. AS-osteoprogenitor cells were exposed to H2O2 for 2 h and then incubated with vehicle or WNT16 treatment for 70 h (n=5 per each group). Analysis of B SA-β-gal staining, C counting results of SA-β-gal-positive cells, D assessment of osteoprogenitor cell lysates using hydrogen peroxidase assay (n=5 per each group), E immunoblotting for protein level, F protein quantification of Fig. 3E, and G RT-qPCR for mRNA level (n=4 per each group). H SaOS2 cells were transfected with TOP or FOP-flash in the presence β-galactosidase for 24 h, incubated with WNT16 treatment for 24 h, and then analyzed by luciferase assays (n=4 per each group). I Experimental design for cell senescence treated with siRNA against WNT16. AS-osteoprogenitor cells were transfected with 100 nM WNT16 siRNA #2 for 70 h, exposed to H2O2 for 2 h, and followed by analysis of H immunoblotting for WNT16 protein level and I qPCR for mRNA level (n=5 per each group). Data are presented as the median and interquartile range. *p<0.05, **p<0.01. Representative images are shown. Scale bar = 200 μm
Fig 3: WNT16 is highly expressed in facet joint of AS. A Heatmap diagram of differentially expressed probes mapped to Wnt genes in AS and control osteoprogenitor cells. In AS osteoprogenitor cells, WNT16 showed significant upregulated expression with a p value < 0.05 and fold change > 2 compared to the control (n=3 per each group). B Verification of mRNA level using qPCR of A for the Wnt family (n=6 per each group). C Immunoblotting for WNT16 protein level in control and AS-osteoprogenitor cells (n=5 per each group). D Immunohistochemistry (IHC) staining of WNT16 in spinal facet joint tissue from control or AS patients. Black arrows indicate bone-lining cells, and blue arrows indicate periosteum in facet joints tissues. Three representative images are shown (n=7 per each group). Data are presented as the median and interquartile range. *p<0.05, **p<0.01
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