FIG. 9.

Termination of ERK1,2 signaling rescues cyclin D1 expression in a subset of cells. (A) Three hours after stimulation with medium containing 10% FBS with or without SIN-1 (1 mM), the culture medium was removed and replaced with medium containing 10% FBS and the indicated concentration of U0126 or an equal volume of DMSO as a vehicle control. After an additional 3 h, cell lysates were prepared and examined for levels of phospho-ERK and cyclin D1 by immunoblotting. CDK4 was used as a loading control. Immunoblotting showed that inhibition of ERK signaling partially restored cyclin D1 expression; CDK4 was used as a loading control. (B) Serum-stimulated C10 cells were treated with either SIN-1 or GO (15 mU/ml) for 3 h, the medium was removed, and cells were incubated in medium containing either 10% FBS (−) or FBS with 5 μM U0126 (+) for an additional 3 h. Total-cell lysates were examined for the levels of c-Fos and phospho-ERK1,2. (C) Cells were transfected with empty vector (pA3), CD1-963AP1mut, or CD1-963 luciferase reporter plasmids; replated; and then made quiescent by serum starvation. Lysates were prepared prior to serum stimulation (Synch) or after stimulation with medium containing 10% FBS with or without catalase (2,000 U/ml), SIN-1 (1 mM), or GO (15 mU/ml). (D) Cells were transfected with the CD1-963 reporter plasmid, serum starved, and treated with medium with 10% FBS with or without SIN-1 or GO for 3 h. The medium was then removed and replaced with medium containing 5 μM U0126, 10 μM SB 203580, or an equal volume of DMSO and incubated for an additional 6 h before lysates were prepared. All samples were assayed for luciferase activity and protein concentration. The results are expressed as relative luciferase units (RLU) per microgram of protein.