Figure 4. Gd@C₈₂(OH)₂₂ nanoparticles impeded EMT by the abrogation of TGF-β expression under normoxia.

MDA-MB-231 cells were cultured with 20 ng ml⁻¹ TGF-β supplement for 24 h after treatment with PBS or Gd@C₈₂(OH)₂₂ for 21 days. (a) Fluorescent visualization of F-ACTIN cytoskeleton was observed (A–D). Scale bar, 12.5 μm. E-T is the immunofluorescence staining of E-CADHERIN (E-CAD), γ-CATENIN (γ-CAT), VIMENTIN (VIM) and FIBRONECTIN-1 (FN-1). Scale bar, 25 μm. (b) mRNA levels of E-cadherin, γ-catenin, Vimentin and Fibronectin-1 were analysed by qPCR (mean±s.e.m., n=3 each). *P<0.05 (two-way ANOVA, Bonferroni’s post-hoc test). (c) The protein expression of E-CADHERIN (E-CAD), γ-CATENIN (γ-CAT), VIMENTIN (VIM) and FIBRONECTIN-1 (FN-1) were detected by western blot. (d) Cell migration and invasion were examined using trans-well cell culture chambers and Matrigel-coated ones (mean±s.e.m., n=6 each). *P<0.05, **P<0.01 (two-way ANOVA, Bonferroni’s post-hoc test). (e) mRNA levels of tgf-β, hif-1α, snail, zeb1, twist1 and e47 were analysed by qPCR (mean±s.e.m., n=3 each). (f) mRNA levels of il-6, il-8, mmp-2, mmp-9 and vegf were analysed by qPCR (mean±s.e.m., n=3 each). (g,h) ELISA analysis of IL-6 (g) and IL-8 (h) to determine their concentrations in the cell culture medium. 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).