Fig. 5.

1 Day of butter-flavored e-cig aerosol exposure under sub-ohm conditions-induced oxidative stress-mediated lung cell injury is alleviated by n-acetyl cysteine (NAC) pre-treatment. H292 cells were pre-treated with or without NAC at a concentration of 5 mM for 12 h before exposure to butter-flavored e-cig aerosol for 1 day at the air–liquid interface (ALI). a Exposure to butter-flavored e-cig aerosol and NAC pre-treatment had no significant effect on cellular viability. b Levels of extracellular lactate dehydrogenase (LDH) were significantly increased by the butter-flavored e-cig aerosol compared to its respective air control; NAC pre-treatment prevented this elevation. c Levels of extracellular reactive oxygen species (ROS) were significantly increased by the butter-flavored e-cig aerosol compared to its respective air control; NAC pre-treatment allowed ROS to remain at control levels. d Exposure to butter-flavored e-cig aerosol and NAC pre-treatment had no significant effect on the extracellular protein concentration of 8-OHdG. This ELISA was analyzed for cell inserts (n = 3 per group). Each cell insert was further evaluated in duplicate. e Levels of extracellular nitric oxide (NO) were not significantly changed by the butter-flavored e-cig aerosol compared to its respective air control; however, the NAC pre-treatment significantly elevated the levels of NO compared to the e-cig exposed-cells that were not pre-treated with NAC. f The transepithelial electrical resistance (TEER) values were significantly decreased by the butter-flavored e-cig aerosol compared to its respective air control, and NAC pre-treatment significantly improved the TEER values. For assays (a, f), data are from one experiment representative of results from three independent experiments, each performed in triplicate (n = 3 per group). For each cell insert, bioassays were further evaluated in duplicate or triplicate. For all assays (b–f) data were normalized to cell count and are presented as mean ± SEM. Comparisons between groups were made with one way ANOVA followed by the Tukey's Multiple Comparison Test. *p < 0.05: significantly different from respective air control; ^ξp < 0.05: significantly different from e-cig aerosol exposed-cells without NAC pre-treatment. g Heatmap of dysregulated genes in H292 cells pre-treated with or without NAC followed by exposure to butter-flavored e-cig aerosol. Data are from H292 cells from distinct experiments. Data are presented as fold-change over the respective air-control group. Fold-changes > ± 1.5 were considered significant. Results from other independent experiments are represented in Additional file 1: Figures S2 and S3