Fig. 6

Effect of CFTR knockdown on IL-8 mRNA and secretion levels in HT-29 cells. HT-29 cells were infected with a lentiviral vector carrying either a scrambled sequence or shRNAi against CFTR and analyzed for gene (a) and protein (b) expression of CFTR by Q-PCR and Western blotting. Results represent the means ± SEM of three independent experiments and are illustrated as % of controls after calculating the data as densitometric ratios of CFTR to the housekeeping gene RPL27 for gene expression (n = 3) or CFTR to β-actin for protein expression (n = 3). CFTR knockdown or control (Scrambled) cells were incubated 1 and 8 h in the absence or presence of 10 ng/mL of either TNF (c) or IL1-β (d). Total RNA was collected and subjected to Q-PCR using intron-spanning primers for the IL-8 gene. (e) CFTR knockdown or control (Scrambled) cells were incubated 24 h in the absence or presence of 10 ng/mL of either TNF or IL1-β and supernatants were collected to quantify the secretion of IL-8 by ELISA. Results represent the means ± SEM of n = 3 independent experiments and are illustrated as pg/mL normalized to total protein concentration. *p <0.05, **p <0.01 and #p <0.0001 vs. scrambled-infected cells