Il-1β secretion, Mitochondrial NADH cytochrome c reductase activity and ROS levels in stable CFTR knock down cells.

<p>Caco-2/pRSctrl cells (transfected with pRS control) and Caco-2/pRS26 cells (transfected with the shRNA pRS26) were preincubated 24 h in serum free media. A: Immune-dot blotting quantification of the IL-1β present in culture media. The results were expressed as pg/ml. B: Cells were treated for additional 24 h, as indicated. IL-1β: IL-1β (5 ng/ml); Ab-IL-1β: anti-IL-1β blocking antibody (30 ng/ml); Ab-Ctrl: anti-Histone H1 monoclonal antibody as negative control (30 ng/ml); p38 Inh: p38 inhibitor SB203580 (5 µM); IKK Inh: IKK inhibitor III (2 µM); JNK Inh: JNK inhibitor SP600125 (5 µM); MEK1/2 Inh: MAPK/AP-1 pathway inhibitor U0126 (5 µM). Spectrophotometric measurements of the mitochondrial NADH-cytochrome c reductase activity, expressed as percentage (%) relative to Caco-2/pRS26 control values. C: Mitochondrial ROS levels in the same experiments of panel B; the figure shows the MitoSOX fluorescence (5 µM for 10 min in Hank’s buffer). Results were expressed as percentage (%) relative to Caco-2/pRSctrl control values. D: Cellular ROS levels in the same experiments of panel B; the figure shows the DCF fluorescence (DCFH-DA 10 µM for 40 min in Hank’s buffer). Results were expressed as percentage (%) relative to Caco-2/pRSctrl control values. Measurements were performed in triplicate and data are expressed as mean ± SE of two independent experiments (n = 2). *indicates p<0.05 compared to Caco-2/pRS26 cells.