FIGURE 5. Overexpression of galectin-3 in A549 cells promoted sphere-forming capacity and in vitro tumorigenicity.

(A) The morphology of galectin-3 (Gal-3)- or empty vector (ev)-transduced spheres. The expression levels of galectin-3 in Gal-3- or ev-transduced monolayers or spheres were detected by using RT-qPCR and Western blot. (B) The mRNA levels of stemness-related genes in Gal-3- or ev-transduced A549 monolayers or spheres were detected by RT-qPCR. (C) Gal-3- or ev-transduced A549 monolayers or spheres were cotransfected with pGL4-Oct4-luc, pGL4-Sox2-luc or pGL4-Nanog-luc, and pRL-SV40. After 48 h, luciferase reporter assays were conducted to investigate Oct4, Sox2, or Nanog promoter activity regulated by galectin-3. (D) The protein levels of Oct4, Sox2, or Nanog in Gal-3- or ev-transduced A549 monolayers or spheres were analyzed by flow cytometry. (E) Gal-3- or ev-transduced A549 monolayers or spheres (2 × 10⁴ cells) were seeded onto upper side of Matrigel-coated transwell inserts and incubated for 24 h to determine the invasive ability. (F) Gal-3- or ev-transduced A549 monolayers or spheres (1 × 10³ cells per 6-well plate) were seeded in soft agar for evaluating anchorage-independent tumor growth. (G) Gal-3- or ev-transduced A549 were seeded onto 96-well plates in serum-free tumor sphere media and cultured for 30 days. The primary spheres were dissociated to single cells and re-seeded to yield the second generation. Data represents the mean ± SD of at least 3 independent experiments, *p < 0.05; **p < 0.01.