Figure 8. Gd@C₈₂(OH)₂₂ impaired EMT by inhibition of TGF-β and HIF-1α expression under hypoxia.

MDA-MB-231 cells were transfected with hif-1α expressing plasmid and/or treated with 20 ng ml⁻¹ TGF-β with further culture in the presence of Gd@C₈₂(OH)₂₂ or PBS under hypoxia for 10 days. (a; A-E) F-ACTIN cytoskeleton of cells (scale bar, 12.5 μm). Immunofluorescence staining (a, F-Y) and western blot analysis (b) of E-CADHERIN (E-CAD), γ-CATENIN (γ-CAT), VIMENTIN (VIM) and FIBRONECTIN-1 (FN-1). Scale bar, 25 μm. (c) The migratory and invasive cells were examined using trans-well cell culture chambers and Matrigel-coated ones (mean±s.e.m., n=3 each). *P<0.05 and **P<0.01 (two-way ANOVA, Bonferroni’s post-hoc test). mRNA levels of EMT markers (d), hif-1α, hif-1β, tgf-β, snail, zeb1, twist1, e47 (e), il-6, il-8, mmp-2, mmp-9 and vegf (f) were analysed by qPCR (mean±s.e.m., n=3 each). ELISA analysis for expression of IL-6 (g) and IL-8 (h) in the MDA-MB-231 cell culture medium was determined. All the data are represented as mean±s.e.m. (n=3 each) with *P<0.05 and **P<0.01 (two-way ANOVA, Bonferroni’s post-hoc test).