Fig. 5.
Effect of cross-linking Siglec-15 on TGF-β secretion in monocytic cells. (A) Lung cancer cell lines T47D and HCC827 that show differential expression of Siglec-15 ligands were co-cultured with THP-1 mock and human Siglec-15-expressing monocytic cells for 24 h in serum free medium. (B) T47D cells were co-cultured with U937 mock and human Siglec-15-expressing cells. (C) H460 wildtype and sTn-expressing (ST6GalNAc-I) cells were co-cultured with THP-1 mock and Siglec-15-expressing cells. (D) Mock-transfected and Siglec-15-expressing THP-1 cells were incubated with monoclonal antibodies against human Siglec-15 and crosslinked with anti-mouse IgG F(ab’)2 secondary antibody. The cells were cultured in macrophage serum-free medium for 24 h. To determine secretion of TGF-β, supernatants were concentrated as described in the Materials and Methods, acidified to release active TGF-β, neutralized and analyzed using Human TGF-β1 Quantikine ELISA kit according to the manufacturer’s instructions. The results are mean concentrations ± SD for two independent experiments.