Figure 9.

VCAM-1 mRNA stability at 16 hours and 10 days after treatment of fibroblast-like synoviocytes with various combinations of cytokines. Cells were cultured with medium-only (untreated), TNF-α (10 ng/ml), IL-4 (10 ng/ml), or IL-13 (10 ng/ml) and, in addition, were treated with TNF-α (10 ng/ml) in combination with IL-4 or IL-13. Cytokines were re-added at days 5 and 10. At 16 hours (A) or 10 days (B) after the start of the culture, Actinomycin-D (5 μg/ml) was added and mRNA was isolated at 0, 2, 4, or 10 hours thereafter. To measure the amount of mRNA transcripts, semiquantitative RT-PCR was performed with VCAM-1-specific and GAPDH-specific primers. C: Normalized results of three separate PCR experiments. Normalized data are expressed as the ratio of each VCAM-1 band intensity to the respective GAPDH band intensity. Medium-only (open circles); TNF-α (closed circles); IL-4 (open squares); TNF-α and IL-4 (closed squares).