GM-CSF mRNA stability in BMSCs after 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 20-minute intervals for 160 minutes. Then, 15 μg of total cellular RNA from each time point were separated on a 1.5% agarose gel, and Northern blotting and hybridization with a mouse GM-CSF cDNA probe were performed. The blots were exposed to autoradiographic film in the same cassette for 8 days. Arrows indicate the positions of the 2 species of GM-CSF mRNA. Note that the smaller band is essentially absent in the KO cells; note also that the GM-CSF mRNA dramatically decreased after 1 hour in the WT cells, whereas its level remained largely unaffected in the KO cells. The same blot was then hybridized with a rat GAPDH cDNA probe as a loading control; the exposure of this blot for GAPDH was 4 hours. (B) Relative amounts of GM-CSF mRNA after normalization to GAPDH mRNA. Solid diamonds, WT; open diamonds, TTP-deficient cells. The dotted lines represent the linear regression of the GM-CSF mRNA decay values. With the use of these regressions, the estimated half-lives for GM-CSF mRNA were found to be 99 minutes in the WT cells but were impossible to determine in the TTP-deficient cells.