Figure 3

Effect of tumor necrosis factor-alpha (TNF-α) and mithramycin on Sp1 protein expression in fibroblast-like synoviocyte nuclei. Fibroblast-like synoviocytes were cultured to confluence in the presence or absence of 300 nM mithramycin for 30 minutes and then further incubated with or without TNF-α (1 ng/mL) for 24 hours. Nuclear extracts were processed for immunoblotting with an anti-Sp1 antibody. Anti-lamin C and anti-α-tubulin immunoblotting were included to assess the purities of nuclear and cytoplasmic fractions, respectively. Results are presented as mean ± standard error of the mean of five determinations. Statistical significance compared with controls was calculated by using the Mann-Whitney U test: compared with controls *P < 0.05; compared with samples with TNF-α alone ^†P < 0.01. MIT, mithramycin.