Figure 6.

 mRNA stability after mithramycin treatment. The effects of mithramycin on COL1A1 mRNA stability were examined as described in "Materials and methods". (A) Confluent cultures of systemic sclerosis dermal fibroblasts were either kept in culture medium containing 10% FBS and ascorbic acid (50 µg/ml) (lanes 1–7) or were incubated for 4 hours with mithramycin (50 nmol/l; lanes 8–14) before addition of α-amanitin (1 µg/ml). Total RNA was prepared from cells at various intervals after α-amanitin addition and examined by northern analysis. Northern analysis was performed on samples prepared at the time of α-amanitin addition (time point 0): lanes 1, 2, 8, 9; at 6 hours after α-amanitin administration: lanes 3, 4, 10, 11; at 18 hours after α-amanitin treatment: lanes 5, 6, 12, 13; and at 24 hours after α-amanitin: lanes 7 and 14. (B) COL1A1 mRNA stability after mithramycin treatment assessed by densitometric analysis. Values shown were corrected to GAPDH and represent the average of duplicate cultures of different cell lines. The 24 hour value was not included in the graph because it was not performed in duplicate.