TNF- mRNA stability in BMSCs after the addition of actinomycin D.

(A) Confluent BMSCs were stimulated for 2 hours with LPS (1 μg/mL). The culture medium was then removed and replaced with fresh medium containing 5 μg/mL actinomycin D. Cells were harvested, and total cellular RNA was extracted at 15-minute intervals for 60 minutes. Then, 20 μg of total cellular RNA from each time point was separated on a 1.5% agarose gel, and Northern blotting and hybridization with a mouse TNF-α cDNA probe were performed. The blots were exposed to autoradiographic film in the same cassette for 2 days. The arrow indicates the position of TNF-α mRNA. The same blot was then hybridized with a rat GAPDH cDNA probe as a loading control; the exposure of this blot for GAPDH was 12 hours. (B) Relative amounts of TNF-α mRNA after normalization to GAPDH mRNA. Solid diamonds, WT; open diamonds, TTP-deficient cells. The dotted lines represent the linear regression of TNF-α mRNA decay. With the use of these regressions, the estimated half-lives for TNF-α mRNA were found to be 35 minutes in the WT cells and 90 minutes in the TTP-deficient cells.