Fig. 1: Lung cancer cell lines with different genetic backgrounds secrete IL-8 in nutrient-poor and nutrient-rich conditions.

A A549 cells were plated and incubated in media containing glucose 25 mM supplemented with 10% dialyzed FBS for the times shown. IL-8 protein concentration was measured by ELISA. Results are expressed as Mean ± SEM of at least 4 experiments. B, C H2126 and SW900 cells were plated and incubated for the indicated time points. IL-8 was measured by ELISA. Results are expressed as Mean ± SEM (N = 3). D Lewis Lung Carcinoma cells were seeded and the supernatant was collected at indicated time points to analyze the IL-8 orthologue KC by ELISA. Results are expressed as Mean ± SEM (N = 3). E Table representing significant mutations of the lung cell lines and the amount of IL-8 they secrete 24 h after medium replacement. F A549 cells were plated and incubated in media with different concentrations of dialyzed FBS (dFBS), with TNFα (10 ng/ml) or with media deprived of glucose (0 mM), as indicated for 24 h. IL-8 was measured by ELISA. Results are expressed as Mean ± SEM (N = 4). G A549 cells were plated for 24 h at normal conditions. Cells were then incubated at 0.1 or 21% of O₂ in the hypoxic chamber for 24 hours. Supernatant were collected and secreted IL-8 measured. Results are expressed as Mean of fold change of pg of IL-8 / mg of proteins from cells in culture ± SEM (N = 3). H A549 cells were pretreated with 80 nM Actinomycin D (ActD) for 1 h; then cells were cultured in glucose-rich media in presence or absence of ActD (80 nM) supplemented with 10% dFBS for the indicated time points (0.5–6 h). Secreted IL-8 was measured by ELISA. Results are expressed as Mean ± SEM (N = 3). I A549 cells were pretreated with either TNFα (10 ng/ml + Glc 25 mM), glucose (25 mM) or without glucose (0 mM) for 3 h. Then media was replaced with fresh treatment media also containing ActD (80 nM) for times shown (0.25–4 h). CXCL8 mRNA decay was measured for each condition. Results are expressed as Mean fold change ± SEM (N = 3, except for TNFα that is equal to 2). Statistical analysis was performed for A–D using a paired t-test comparing to the shortest time point, for G compared to 21% O₂ G+, for H each time point ActD versus control sample. p-value: *p < 0.05, **p < 0.01, ***p < 0.001.