FIGURE 4.

The effect of cordycepin is gene specific and unrelated to mRNA stability. (A) ASM cells were pretreated with cordycepin (50 μM) or vehicle for 60 min, then TNF or vehicle (Ctrl) was added. Fifteen minutes after TNF or vehicle addition, 500 μM thiouridine was added. Cells were lysed 45 min after addition of thiouridine, RNA was isolated, and thiouridine-labeled RNA was purified as described. RT-qPCR for the indicated mRNAs was performed and normalized to RT-qPCR values for ribosomal 18S RNA. Values shown are relative to the average value for untreated. Error bars represent standard deviations over thee biological replicates. (B) ASM cells were treated with TNF in the presence or absence of cordycepin as described in A, but thiouridine was left out in one plate of cells (TNF no 4sU). Values shown are relative to the TNF induced signal (TNF) in each replicate. Error bars represent standard deviations over two biological replicates. (C) Airway smooth muscle cells were treated with TNF for 1 h, when 5 μg/mL of the transcription inhibitor actinomycin D was added with or without cordycepin at the indicated concentrations. RNA was isolated at the indicated times and RT-qPCR performed as described. Error bars are standard deviations over three biological replicates.