Figure 2. Inflammatory stimuli and other stresses interfere with ARE mRNA degradation.

A) HEK-293T cells were transfected with CMV-ARE (ARE) and cultured for 40h. One hour before total RNA isolation, cells were treated with PBS, 50 ng/ml IFN-α, 50 ng/ml Il-1β, or with 50 ng/ml TNF-α. Quantitative one-step RT-PCR assays were used to measure ARE mRNA levels. ARE mRNA levels were normalized against GAPDH mRNA levels and increases were calculated using PBS-treated sample controls. Results are expressed as the mean of 3 independent experiments, with the error bar representing the standard deviation. B) HEK-293T cells were transfected with either CMV-ARE (ARE) or CMV-ΔARE (ΔARE) and cultured for 40 additional hours. Prior to RNA isolation, cells were treated with 50ng/ml TNF-α for 0.25, 0.5, 1, 2, and 4 hours. Quantitative one-step RT-PCR assays were performed and ARE and ΔARE mRNA increases were calculated using the time 0 point as 1. C) HeLa Tet-off cells were transfected with Tet-ARE and cultured for 40 additional hours. Then cells were treated with either PBS or 50 ng/ml TNF-α for 15 min. before doxycycline was added to block transcription. Samples were analyzed as in B. ARE mRNA levels were expressed as a percentage of the amount existing at time 0. D) As C, but the presence of ARE mRNA in the cells was analyzed by Northern-blotting. β-actin was used to ensure equal loading. E) As C, but HeLa Tet-off cells were pretreated with PBS (PBS), hydrogen peroxide (H₂O₂), MNNG, or anisomycin (Aniso) 30 min. before addition of doxycycline. Samples were analyzed as in B.