Fig. 5.

RGS4-UTR-dependent mRNA instability involves new protein synthesis. A: translation inhibitor cycloheximide (CHX) alone induced a significant increase in mRNA expression of endogenous RGS4. Cultured colonic smooth muscle cells after serum starvation were treated with CHX (12.5 μg/ml) for indicated time periods, and RGS4 mRNA level was determined by real-time RT-PCR. The fold change was relative to the level before CHX treatment. B: transcription inhibitor actinomycin D (AD) prevented CHX-induced upregulation of RGS4 mRNA, while CHX prevented IL-1β-induced upregulation of RGS4 mRNA. Cells were pretreated with AD (10 μM) 1 h and CHX 30 min before IL-1β treatment for indicated time periods, and RGS4 mRNA level was determined by absolute quantitative real-time RT-PCR using rabbit RGS4 plasmid as standard. C: RGS4 3′-UTR-dependent instability of luciferase was alleviated by pretreatment with CHX. Cells were transfected with pcDNA3-rLuc-RGS4-UTR(fl) for 24 h, serum starved for 24 h, and treated with vehicle control, CHX, and IL-1β 10 ng/ml for 24 h. Renilla luciferase was measured. Data represent means ± SE of 4 separate experiments. *P < 0.05 and **P < 0.01, statistically significant increase by ANOVA compared with corresponding control.