Figure 6

Analysis of mesenchymal marker expression in LECs after intervention with NF-κB signalling. LECs were pretreated with 10 μ M of the I-κBα phosphorylation inhibitor Bay11-7082 for 0.5 h and then stimulated with 1 μ M 12(S)-HETE for 0.2 h. Cells were harvested and analysed by western blotting using (A) anti-ZEB1 and anti-VE-cadherin antibodies. MCF-7 cells were used as negative control. (B) Blots were analysed with anti-S100A4 antibody. Equal sample loading was controlled by Ponceau S staining and β-actin analysis. Confocal immunofluorescence images of LECs at the rim of CCID (C) induced by an MCF-7 spheroid; (D) and from a similar position after treatment with 10 μ M Bay11-7082. LECs were grown on coverslips until confluence when MCF-7 spheroids were transferred on top of LECs and co-incubated for 4 h at 37°C to allow CCID formation. LECs were stained with anti-S400A4 antibody (green), anti-VE-cadherin antibody (red), and DAPI (blue). (C) S100A4 is well expressed and VE-cadherin interactions are disrupted. (D) Upon Bay11-7082 treatment, VE-cadherin structures again appear well developed (although unconnected to VE-cadherin structures of neighbouring cells), whereas S100A4 expression is decreased. Scale bar: 15 μ M.