Fig. 3: NF-κB regulates IL-8 production under basal and stimulated conditions.

A A549 cells were treated with media with or without glucose (25–0 mM). Cells were collected at indicated time points to analyze p65 and IĸBɑ by western blot. Results are shown compared to 24 h Glc + and are expressed as Mean of fold change ± SEM (N > 4). B Representative western blot of A is shown. C A549 cells were transfected with a NF-κB reporter plasmid based on luciferase and treated with media containing either TNFα (10 ng/ml + Glc 25 mM, called ‘T’), glucose (25 mM, called ‘+’) or without glucose (0 mM, called ‘−’) for indicated time points, in 1% dFBS. Cells were lysed and luminescence measured for each sample. Results are shown compared to Glc + samples of each time point. Mean of fold change ± SEM (N = 3). D A549 cells were treated for 6 h with media containing either TNFα (10 ng/ml + Glc 25 mM, indicated as ‘T’), glucose (25 mM, ‘+’) or deprived of glucose (0 mM, ‘−’). Cells were collected and nuclear proteins extracted. p65 binding to NF-ĸB site in CXCL8 promoter was measured by qDPI-ELISA. Results are shown compared to CTRL. Mean of fold change ± SEM (N = 4). E A549 and HeLa cells were treated with media containing either TNFα (10 ng/ml + Glc 25 mM, called ‘T’), glucose (25 mM, called ‘+’) or without glucose (0 mM, called ‘−’) for 24 h. Cells were lysed and luminescence measured for each sample. Results are shown compared to HeLa cells treated with glucose media, mean of fold change ± SEM (N = 2). Statistical analysis performed using Ratio paired t-test. p-value: *p < 0.05, **p < 0.01, ***p < 0.001.