Time course of GM-CSF mRNA accumulation in BMSCs stimulated with LPS.

(A) 60 mm dishes containing confluent BMSCs derived from wild-type (WT) or TTP-deficient (KO) mice were prepared as described in “Materials and Methods.” Cells were then stimulated with LPS (1 μg/mL), and RNA was extracted at different time points for up to 8 hours. Then, 16 μg of total cellular RNA was separated on a 1.5% agarose gel, and Northern blotting and hybridization with a mouse GM-CSF cDNA probe were performed as described in “Materials and Methods.” Blots were exposed to autoradiographic film in the same cassette for 3 days. The arrows indicate the position of the 2 species of GM-CSF mRNA, of approximately 1.0 and 0.8 kb. Note the clear presence of the 2 species in the WT cells, whereas the 0.8-kb band was decreased in the KO cells. Note also that GM-CSF mRNA was already detectable after 1 hour in the KO cells, but was not detectable until 2 hours in the WT cells. The same blots were then hybridized with a rat GAPDH cDNA probe as a loading control, as indicated. Exposure of the blots to film for GAPDH mRNA was 2 hours. (B) Relative amounts of GM-CSF mRNA after normalization to GAPDH mRNA. Solid symbols, WT; open symbols, TTP-deficient cells.