Figure 5

Mithramycin suppression of tumor necrosis factor-alpha (TNF-α)-induced gliostatin (GLS) expression in fibroblast-like synoviocytes (FLSs). Confluent FLSs were incubated in the presence or absence of 10 to 300 nM mithramycin for 30 minutes and then with TNF-α for 24 hours. GLS gene expression levels are represented relative to β-actin (A). Immunoreactive GLS was determined by enzyme immunoassay (B). Results are presented as mean ± standard error of the mean (SEM) of four determinations. Confluent cells were incubated in the presence (closed circle) or absence (closed square) of 300 nM mithramycin for 30 minutes and then with TNF-α as indicated. Control FLSs were cultured without TNF-α (closed triangle). GLS mRNA (C) and protein (D) expression levels are presented as mean ± SEM of four determinations. Statistical significance was calculated by using the Mann-Whitney U test: compared with controls *P < 0.05, **P < 0.01; compared with samples with TNF-α alone ^†P < 0.05, ^††P < 0.01. MIT, mithramycin.