Fig. 8.
Fig. 8. GM-CSF mRNA expression in BMSCs from triple-KO mice. / (A) BMSCs were stimulated with LPS (1 μg/mL) for up to 5 hours; cells were then harvested; RNA was extracted; and 14 μg of total cellular RNA from each time point was separated on a 1.5% agarose gel and used for Northern blotting with a mouse GM-CSF cDNA probe. The blots were exposed to autoradiographic film for 7 days. 2KO indicates mice deficient in both TNF-α receptors, but WT for TTP; 3KO indicates mice deficient in TTP and both TNF-α receptors. The arrows indicate the positions of the 2 GM-CSF mRNA species. Note the greater levels of total GM-CSF mRNA in the 3KO samples, compared with the 2KO, as well as the virtually complete absence of the smaller species of GM-CSF mRNA in the 3KO samples. Note also that the GM-CSF mRNA was almost undetectable by 5 hours in the 2KO cells, but was still present in readily detectable amounts in the 3KO cells. The same blots were hybridized with a rat GAPDH cDNA probe as a loading control. Exposure of the blots to autoradiographic film for GAPDH mRNA was 2 hours. (B) Relative amounts of GM-CSF mRNA after normalization to GAPDH mRNA. Solid diamonds, 2KO; open diamonds, 3KO cells. (C) BMSCs were stimulated with LPS (1 μg/mL) for 2 hours; the culture medium was then removed and replaced by fresh medium containing 5 μg/mL actinomycin D. Cells were harvested and RNA was extracted at 20-minute intervals for 160 minutes. Then, 15 μg of total cellular RNA from each time point was separated on a 1.5% agarose gel and used for Northern blotting with a mouse GM-CSF cDNA probe. The blot was exposed to film for 8 days. The arrows indicate the positions of the 2 GM-CSF mRNA species. Note the rapid disappearance of both species of GM-CSF mRNA in the samples from the 2KO cells, whereas GM-CSF mRNA levels remained essentially unchanged in the 3KO cells. Note also the virtually complete absence of the lower species of GM-CSF mRNA in the samples from the 3KO cells. The same blots were hybridized with a rat GAPDH cDNA probe as a loading control. Exposure of the blots to autoradiographic film for GAPDH mRNA was 4 hours. (D) 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 GM-CSF mRNA decay. With the use of these regressions, the estimated half-lives for GM-CSF mRNA were found to be 49 minutes in the 2KO cells but were impossible to determine in the 3KO cells.

GM-CSF mRNA expression in BMSCs from triple-KO mice.

(A) BMSCs were stimulated with LPS (1 μg/mL) for up to 5 hours; cells were then harvested; RNA was extracted; and 14 μg of total cellular RNA from each time point was separated on a 1.5% agarose gel and used for Northern blotting with a mouse GM-CSF cDNA probe. The blots were exposed to autoradiographic film for 7 days. 2KO indicates mice deficient in both TNF-α receptors, but WT for TTP; 3KO indicates mice deficient in TTP and both TNF-α receptors. The arrows indicate the positions of the 2 GM-CSF mRNA species. Note the greater levels of total GM-CSF mRNA in the 3KO samples, compared with the 2KO, as well as the virtually complete absence of the smaller species of GM-CSF mRNA in the 3KO samples. Note also that the GM-CSF mRNA was almost undetectable by 5 hours in the 2KO cells, but was still present in readily detectable amounts in the 3KO cells. The same blots were hybridized with a rat GAPDH cDNA probe as a loading control. Exposure of the blots to autoradiographic film for GAPDH mRNA was 2 hours. (B) Relative amounts of GM-CSF mRNA after normalization to GAPDH mRNA. Solid diamonds, 2KO; open diamonds, 3KO cells. (C) BMSCs were stimulated with LPS (1 μg/mL) for 2 hours; the culture medium was then removed and replaced by fresh medium containing 5 μg/mL actinomycin D. Cells were harvested and RNA was extracted at 20-minute intervals for 160 minutes. Then, 15 μg of total cellular RNA from each time point was separated on a 1.5% agarose gel and used for Northern blotting with a mouse GM-CSF cDNA probe. The blot was exposed to film for 8 days. The arrows indicate the positions of the 2 GM-CSF mRNA species. Note the rapid disappearance of both species of GM-CSF mRNA in the samples from the 2KO cells, whereas GM-CSF mRNA levels remained essentially unchanged in the 3KO cells. Note also the virtually complete absence of the lower species of GM-CSF mRNA in the samples from the 3KO cells. The same blots were hybridized with a rat GAPDH cDNA probe as a loading control. Exposure of the blots to autoradiographic film for GAPDH mRNA was 4 hours. (D) 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 GM-CSF mRNA decay. With the use of these regressions, the estimated half-lives for GM-CSF mRNA were found to be 49 minutes in the 2KO cells but were impossible to determine in the 3KO cells.

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