Fig. 6

TNF-α-induced MMP9 expression in M13SV1-Cre cells was blocked by minocycline. M13SV1-Cre and MDA-MB-435-pFDR1 cells were treated for 3 days with 100 ng/ml TNF-α, 10 μg/ml minocycline and a combination of both. The expression levels of a) CLDN1, b) ICAM1 and c) MMP9 were determined by qPCR, Western blot analysis and zymography (only MMP9). Significantly decreased MMP9 mRNA levels were detected in TNF-α and minocycline-treated M13SV1-Cre cells as compared with TNF-α-treated cells and were validated by zymography. d) Co-immunoprecipitation data show that MMP9 binds to ICAM1. ICAM1 expression and co-immunoprecipitated MMP9 were markedly decreased in the lysates of TNF-α + minocycline-treated M13SV1-Cre cells compared with the lysates from TNF-α-stimulated cells. e) No differences in sICAM1 levels were detected in M13SV1-Cre and MDA-MB-435-pFDR1 cells that were treated with TNF-α, minocycline and a combination of both. Shown are the mean ± SD or representative Western blot data for at least three independent experiments. Statistical analysis: ANOVA F-test and Scheffé post-hoc test: * = p < 0.05; ** = p < 0.01; *** = p < 0.001