Figure 2

MWCNT induce pyroptosis in HBE cells. A) A dose response of MWCNT (1.5-24 μg/mL equivalents 0.37-6.0 μg/cm²) induced cell death after 24 hours exposure. Cells were analyzed by flow cytometry after labeling with PI. CB (24 μg/mL) and Min-U-Sil (100 μg/mL) were used as negative and positive particle controls respectively. H₂O₂ (1 mM) was used as positive control for oxidative stress induced cells death. B) Caspase-1 activation in HBE cells after 24 hours MWCNT exposure, analyzed by flow cytometry. CB (24 μg/mL) was used as negative control while Min-U-Sil (24 μg/mL) and LPS (1 μg/mL) were used as positive controls. C) Protection of cell death using specific inhibitors for cathepsin B (CA-074ME, 10 μM) and caspase-1 (Z-WEHD-FMK, 50 μM). Cells were pre-treated (for 1 hour) with these inhibitors before treatment with MWCNT (24 μg/mL) in the presence of inhibitor for further 24 hours and analyzed by flow cytometry. D) Modulation of cell death by specific siRNA against NLRP3 inflammasome. Transfection of HBE cells was performed using either specific siRNA against NLRP3 inflammasome or a nonspecific negative control siRNA. Cells were treated with MWCNT (24 μg/mL) for 24 hours. Cell death was measured by flow cytometry after labeling with PI. Data were analyzed by analysis of variance (ANOVA) followed by Tukey’s post hoc test. Graphs show average ± SEM of three independent experiments with triplicate of each conditions, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (between media-treated control and treatments).