Fig. 1. Degradation kinetics of V5-αENaC mRNA with a Tet-Off system in the presence and absence of Actinomycin D. Alveolar epithelial cells were transiently cotransfected with the pTet-Off plasmid and the pTRE-tight plasmid coding for αENaC cDNA bearing a V5 epitope upstream of its open reading frame and complete 3'UTR sequences. The cells pretreated or not with Actinomycin D (5.0 ug/mL) for 30 min were incubated thereafter with doxycycline (1.0 ug/mL) from 15 min to 6 h. Expression of V5-αENaC mRNA was measured by quantitative RT-PCR and presented as percentage ± SEM of V5-αENaC mRNA expression of untreated cells (t=0) after normalization with tTA-Ad. V5-αENaC mRNA half-life was estimated by one-phase decay nonlinear regression for each cell preparation. Multiple regression analysis revealed a statistically significant difference in V5-αENaC mRNA stability in cells treated with Actinomycin D compared to untreated cells (P<0.0001). Cells from at least four different rats (n≥4) were used for each experimental condition.