Figure 6.

Effect of Hsp90 knockdown on the EDP-regulated pro-MMP-2 and uPA secretion by HT-1080 cells. (A) Real-time PCR analysis of Hsp90α transcript 2 mRNA relative expression 48 h after treatment with Hsp90 siRNA vs negative control siRNA-treated cells (−). Results (mean±s.e.m.; n=3) were expressed as the ratio of Hsp90α transcript 1 mRNA to EEF1a1 mRNA. (B) Western blotting analysis and quantification of intracellular Hsp90 using a rabbit anti-Hsp90 monoclonal antibody and probing with anti-actin 72 h after treatment with Hsp90 siRNA vs negative control siRNA-treated cells (−) as described in Materials and Methods section. Results (mean±s.e.m.) were expressed as the percentage of negative control siRNA-treated cells (EDP-untreated cells). (C) Immunolocalisation of Hsp90 72 h after treatment with siRNA to Hsp90 mRNA vs negative control siRNA-treated cells (−). Cells were cultured on glass slides, fixed with paraformaldehyde and labelled with an anti-Hsp90 antibody (green). Scale bar: 20 μm. (D) Gelatin zymography, gelatin plasminogen zymography and western blotting analysis of conditioned media in the presence (+: 50 μg ml⁻¹) or in the absence (−) of EDPs 72 h after treatment with Hsp90 siRNA vs negative control siRNA-treated cells. Quantitative evaluation of protein secretions by HT-1080 cells on gelatin zymography, gelatin plasminogen zymography and by western blotting. Results (mean±s.e.m.) were expressed as the percentage of control (negative control siRNA/EDP-untreated cells).