FIGURE 4. Increased galecitn-3 associated with β-catenin in lung CSCs.

(A) Cignal Finder Reporter Array consist of 10 dual-luciferase reporter assays was performed. H1299 monolayers or spheres were dissociated and seeded onto 96-well plates (4 × 10³ cells). Cells were transfected with the reporter plasmids and incubated for 48 h. Firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay system. (B) The β-catenin promoter activity was examined by TOPFlash system. H1299 monolayers or spheres (4 × 10³ cells per 96-well plate) were cotransfected with TOP or FOP reporter construct and pRL-SV40. After 48 h, the firefly luciferase enzyme activity was measured using the Dual-Luciferase Reporter Assay system and normalized to Renilla luciferase activity. (C) shLuc- or shGal-3-infected monolayers or spheres were cultured for 3 days and treated with DSS (5 mM) 30 minutes before lysis. We performed immunoprecipitation (IP) in the presence of lactose (5 mM) using anti-galectin-3 (Gal-3) antibody (left) or anti-β-catenin (β-cat) antibody (right), and then immuno-blotted using anti-galectin-3 or anti-β-catenin antibody. IgG, negative control; WCL, whole cell lysate control. Data represents the mean ± SD of at least 3 independent experiments, *p < 0.05; **p < 0.01.