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. 2014 Dec 31;6(7):4936–4952. doi: 10.18632/oncotarget.3210

FIGURE 2. Overexpression of galetin-3 in lung CSCs.

FIGURE 2

(A) The mRNA levels of galectins (Gal) in first and secondary passages of H1299 spheres were detected by RT-qPCR and normalized against mRNA levels of monolayers. (B) The mRNA levels of galectin-3 in NSCLC spheres compared to that in monolayers were detected by RT-qPCR. (C) Galectin-3 (Gal-3) was knocked down in H1299 cell line by lentiviral shGal-3 infection. Luciferase shRNA lentivirus (shLuc) was used as a control. The expression levels of galectin-3 were detected using RT-qPCR and Western blotting. (D) The mRNA levels of stemness-associated genes in monolayers (shLuc monolayer), spheres (shLuc sphere), or shGal-3-infected spheres (shGal-3 sphere) were detected by RT-qPCR. (E) shLacZ- or shGal-3-infected H1299 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 was conducted to investigate Oct4, Sox2, or Nanog promoter activity regulated by galectin-3. (F) The protein levels of Oct4, Sox2, and Nanog in shLuc- or shGal-3-infected H1299 spheres were detected by using flow cytometry. (G) H1299 cells from shLuc- or shGal-3-infected H1299 spheres were analyzed for CD44/CD133 double staining by flow cytometry. Control: isotype control. Data represents the mean ± SD of at least 3 independent experiments, *p < 0.05; **p < 0.01.