AS/S and IB3-1/S9 cells were pretreated with parthenolide or vehicle (placebo) for 1 h and then stimulated with IL-1β and/or TNFα for the indicated time. Subsequently, media and total RNA were prepared and analyzed for protein production and mRNA expression. The mRNA contents were normalized with for GAPDH and the results are given in relative units. (A) Parthenolide pretreatment significantly increased IL-8 mRNA expression in AS cells at 3 and 6 h (p = 0.001 and p = 0.00009, respectively) compared to S cells (inset). (B) Parthenolide pretreatment significantly inhibits IL-8 mRNA accumulation in IB3-1 cells at 3 h (p = 0.0004) compared to S9 cells (inset). (C) HTE cells were treated with 20 μM CFTRinh172 for 3 weeks (media changed every other day). When cells were ready, the inhibitor was added to basal and apical side and the media was replenished every day for 3 days. On the fourth day parthenolide or DMSO were added, one hour later cells were stimulated with or without TNFα for 3 h. Parthenolide significantly decreased IL-8 secretion in HTEinh172 at 3 h (p = 0.004), whereas, parthenolide pretreatment did not affect IL-8 mRNA accumulation (C, inset). (D) AS cells were pretreated with parthenolide or vehicle (placebo) for 1 h and then stimulated with TNFα alone or TNFα/IL-1β for 3 h. Subsequently, media and total RNA were prepared and analyzed for mRNA accumulation. The mRNA content was normalized with GAPDH and the results are given in relative units. Parthenolide pretreatment had no significant changes on AS-stimulated with TNFα; however it significantly increased IL-8 mRNA accumulation in AS-stimulated with TNFα/IL-1β (p = 0.0009). Results are shown as means ± SEM (n = 4-6 for each time point and condition).