Effects of FGF2 on SDF-1 mRNA stability. (A) Analysis of SDF-1, Flt3-L, and GAPDH mRNA stability. S-17 cells (80%-90% confluent) were cultured in medium alone or in medium supplemented with 5 μg/mL α-amanitin for 8, 16, 24, 32, 40, or 48 hours at 37°C. Levels of specific mRNAs were measured by semiquantitative RT-PCR. Amplified products were separated on agarose gels. (B) To evaluate the effect of FGF2 on SDF-1 mRNA decay, S-17 cells were preincubated in medium alone or with FGF2 (50 ng/mL) for 6 hours, and then α-amanitin (5 μg/mL) was added to cultures. FGF2-pretreated cells were also tested without α-amanitin addition. After incubation for 1.5, 3, 4.5, 6, 7.5, 10, and 24 hours, SDF-1 and GAPDH mRNAs were measured by semiquantitative PCR. The amount of cDNA used for each amplification reaction was based on the results of PCR for GAPDH showing equivalent amounts of product amplified from all samples. PCR products of SDF-1 and GAPDH were separated on a 3% and 2% agarose gel, respectively, and visualized under a UV light (left panel; representative experiment of 3). Relative SDF-1 mRNA level (SDF-1/GAPDH) was calculated by densitometric scanning using NIH image software. Percent relative SDF-1 expression is defined as percent of the initial ratio of relative SDF-1 expression (right panel). The results reflect the means (± SD) of 3 independent experiments. The asterisk denotes statistical significance (amanitin alone vs FGF2 plus amanitin, P < .05).