FIGURE 3. Galectin-3 knockdown in H1299 resulted in reduction of CSC formation, chemoresistance, tumorigenicity, and tumor initiation in vivo.

(A) shLuc- or shGal-3-infected H1299 cells were seeded onto 96-well plates in serum-free tumor sphere media and cultured for 12 days. The primary spheres (1° sphere) were dissociated to single cells and re-seeded to yield the second generation (2° sphere) and third generation of lung spheres (3° sphere). (B) shLuc- or shGal-3-infected parental or sphere cells (500 cells per 6-well plate) were seeded in soft agar for evaluating anchorage-independent tumor growth. (C) To determine the invasive ability, shLuc- or shGal-3-infected spheres (2 × 10⁴ cells) were seeded onto upper side of Matrigel-coated transwell inserts and incubated for 24 h. (D, E) shLuc- or shGal-3-infected monolayers or spheres were cultured in 96-well plates and treated with cisplatin (D) or paclitaxel (E) for 48 h. Cell viability was examined by Celltiter Glo Luminescent Cell Viability Assay. (F) shLuc- or shGal-3-infected spheres were dissociated and inoculated subcutaneously into NOD/SCID mice (10⁶/100 μl cells). Tumor volume was measured every week. Experiments were performed in quadruplicate and the tumor formation was detected 10 weeks after injection. Data represents the mean ± SD of at least 3 independent experiments, *p < 0.05; **p < 0.01.