Effect of TNF-α and DRM destabilization on AA release.

<p>Calu-3 cells were incubated overnight with either ³H-labelled AA, treated with 100 U/mL TNF-α for 10 min, 10 mM mβCD for 1 h, with or without preincubation with 15 µM pyrrolidine for 45 min, or with a combination of the different treatments. For combined treatments, TNF-α was added for the last 10 min of incubation. After incubation, supernatants were collected and radioactivity measured by a scintillation counter. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are expressed as percent increment with respect to control. Asterisks denote p<0.05 with respect to control unless indicated otherwise, n = 3.</p

Effect of TNF-α and DRM destabilization on eicosanoid production.

<p>Calu-3 cells were treated with either 100 U/mL TNF-α for 10 min, 10 mM mβCD for 1 h, with or without preincubation with 15 µM pyrrolidine for 45 min, or with a combination of the different treatments. For combined treatments, TNF-α was added for the last 10 min of incubation. After incubation the supernatant was collected, either immediately or after 3 h of incubation in fresh medium, and subjected to ELISA for LTB4 and PGE2 determination. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are expressed as percent of control values. Asterisks denote p<0.05 with respect to control, n≥3.</p

Effect of CFTR inhibition on eicosanoid, AA and IL-8 release.

<p>A: Iodide efflux (CFTR activity) measurements on Calu-3 cells. Effect of 10 min incubation with 10 µM Inh172 in Calu-3 cells (left). Effect of forskolin activation (right). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are representative of three experiments. B: Calu-3 cells were treated with either 20 µM Inh172 or Gly-101 for 20 min. In a separate experiment, HeLa cells were treated with 20 µM Inh172 for 20 min. The supernatant was collected right after treatment and subjected to ELISA for LTB4 and PGE2 determination. C: Calu-3 cells were incubated with or without 20 µM Inh172 for 20 min. After incubation, supernatants were removed and fresh DMEM medium containing fetal calf serum was added. After 3 h supernatants were harvested for IL-8 determination. All results are expressed as percent of control values.</p

Impact of TNF-α and DRM destabilization on IL-8 release.

<p>IL-8 production by Calu-3 cells after proinflammatory stimulation, functional inhibition of CFTR and DRM disruption. Calu-3 cells were incubated with either 100 U/mL TNF-α for 10 min, 10 mM mβCD for 1 h, or with a combination of both treatments. After incubation, supernatants were removed and fresh DMEM medium containing fetal calf serum was added. After 3 h of incubation, supernatants were harvested for IL-8 determination. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are expressed as percent of control values. Asterisks denote p<0.05 with respect to control, n≥3.</p