Figure 3. Inflammatory stimuli and other stresses interfere with si20-mediated mRNA degradation.

A) HEK-293T cells were transfected with pSuper-si20 and CMV-ΔARE and cultured for 40 h. One hour before total RNA isolation, cells were treated with PBS, 50ng/ml IFN-α, 50ng/ml Il-1β or 50ng/ml TNF-α. Samples were analyzed as in 2A B) HEK-293T cells were transfected and cultured as in A. 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 run to measure ΔARE mRNA levels in HEK-293T cells. ΔARE mRNA levels were normalized against GAPDH mRNA levels. ΔARE mRNA increases were calculated by using the time 0 point as 1. C) HeLa Tet-off cells were transfected with Tet-ΔARE and pSuper-si20 and cultured for 40h. Then cells were treated PBS or 50 ng/ml of TNF-α 15 min. prior to doxycycline treatment. Samples were analyzed as in B. ΔARE mRNA levels were expressed as a percentage of the amount existing at time 0. D) As C, but the presence of ΔARE mRNA in the cells was analyzed by Northern-blotting. Re-probing 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 the addition of doxycycline. Samples were analyzed as in B. ΔARE mRNA levels were normalized against GAPDH mRNA levels. ΔARE mRNA levels were expressed as a percentage of the amount existing at time 0.