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. Author manuscript; available in PMC: 2014 Oct 17.
Published in final edited form as: Oncogene. 2013 May 13;33(16):2110–2122. doi: 10.1038/onc.2013.160

Figure 2. YKL-40 expression is associated with interaction of N-cadherin/β-catenin/SMa and cell-cell adhesion in GSDCs.

Figure 2

A. GSDC control and YKL-40 shRNA cell lysates were probed for N-cadherin (N-cad), β-catenin (β-cate), YKL-40, SMa, VEGF, and actin expression through western blotting. VEGF mRNA levels were also tested by RT-PCR with GAPDH as a loading control. B. (Left) Immunoprecipitation of N-cad from control and YKL-40 shRNA cell lysates was tested for its association with β-cate by western blotting against β-cate. (Right) Immunoprecipitation of β-cate from control and YKL-40 shRNA cell lysates was tested for its association with SMa by western blotting against SMa. IgG protein levels were used to ensure equal levels of antibody pull down. C. GSDC control and YKL-40 shRNA cells were treated with or without a neutralizing anti-VEGF antibody (100 ng/ml) overnight. Immunocytochemistry of N-cad (green) and β-cate (red) to determine overlapping staining (yellow) indicated by arrows. Cell nuclei were stained by DAPI (blue). A bar: 10 μm. Quantification of co-staining between N-cad and β-cate by normalization of overlapping images to cell number. N=3, *P≤0.05 compared with control. D. Representative pictures of the cell aggregation assay. Cells were subjected to serum-free media with either IgG or an N-cad neutralizing antibody (Ab, 50 μg/ml) for one hour. Cell aggregates of 10 or more cells were counted for each condition and quantified below. A bar: 100 μm. N=3, * P≤0.05 compared with control.