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Fig. 8 | Particle and Fibre Toxicology

Fig. 8

From: Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation

Fig. 8

Release of HMGB1 and dsDNA from MWCNT-treated cells and inhibitory effects of antibodies on 8-nitroG formation. a Release of HMGB1 from MWCNT-treated cells. A549 cells were treated with 1 μg/ml of MWCNT for indicated durations at 37 °C. Then the concentration of HMGB1 in the culture supernatant was measured by a commercial ELISA kit. b Release of dsDNA from MWCNT-treated cells. A549 cells were treated with 1 μg/ml of MWCNT at 37 °C, and the concentration of dsDNA in the culture supernatant was measured with a Quantus fluorometer. a, b Data represent means ± SD of 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the control; # p < 0.05, compared with CNT-S-treated cells. c Inhibitory effects of anti-HMGB1 and anti-RAGE antibodies on MWCNT-induced 8-nitroG formation. A549 cells were pretreated with 10 μg/ml anti-HMGB1 or anti-RAGE antibody (or corresponding control IgG) for 30 min and then treated with 1 μg/ml CNT-L for 8 h. Then fluorescent immunocytochemistry was performed as described in Methods. Hoechst, Hoechst 33258. Magnification, X200. d Quantitative image analysis for effects of the antibodies on 8-nitroG formation in MWCNT-treated cells. Staining intensity per cell was analyzed by an ImageJ software. Relative staining intensity of the control was set at 1. Data represent means ± SD of 8 (control and MWCNT alone) or 4 (MWCNT plus an antibody) independent experiments. ***p < 0.001, compared with the control; ## p < 0.01 and ### p < 0.001. e 8-NitroG formation induced by culture supernatant of MWCNT-exposed cells. A549 cells were treated with 1 μg/ml of MWCNT for 8 h, and the culture supernatant was given to fresh A549 cells, followed by incubation for 2 h at 37 °C. 8-NitroG formation was examined by fluorescent immunocytochemistry. f Quantitative image analysis for 8-nitroG formation induced by culture supernatant of MWCNT-treated cells. Staining intensity was analyzed as described in (d). Data represent means ± SD of 3-4 independent experiments. ***p < 0.001, compared with the non-treated control; ## p < 0.01 and ### p < 0.001, compared with the cells treated with control cell supernatant; $$ p < 0.01. Statistical analysis was performed by ANOVA followed by Tukey’s test

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