Fig. 3

Effect of CFTR knockdown on IL-8 steady-state mRNA levels and IL-8 mRNA stability. CFTR knockdown or control (Scrambled) cells were incubated 1 and 8 h in the absence (a) or presence (b) of 25 ng/mL of either TNF or IL1-β. Total RNA was collected and subjected to Q-PCR using intron-spanning primers for the IL-8 gene. For mRNA stability, cells were incubated with 25 ng/mL of TNF (c) or IL1-β (d) for 1 h and then subjected to actinomycin D (5 μg/mL) to block transcription. Cells were incubated in the presence of actinomycin D for 2 and 8 h after which RNA was collected and analysed by Q-PCR for IL-8 mRNA expression. IL-8 mRNA was normalized to that of the housekeeping gene RPL27. Data represent the means ± SEM of n = 3-5 independent experiments. Steady state mRNA levels are reported as fold induction over basal levels. For mRNA stability, values represent percentages of remaining mRNA versus mRNA levels before the addition of actinomycin (time 0). *p <0.05 and #p <0.0001 vs. scrambled-infected cells