Fig. 7

Inhibitory effect of TLR9 siRNA on MWCNT-induced 8-nitroG formation. a Immunofluorescent images of reduced TLR9 expression and the absence of 8-nitroG formation in siRNA-transfected cells. A549 cells were transfected with negative control or TLR9 siRNA (siRNA-1 and siRNA-2). TLR9 expression and 8-nitroG formation were examined by fluorescent immunocytochemistry. Hoechst, Hoechst 33258. Magnification, X400. b Relative staining intensity of TLR9 in siRNA-transfected A549 cells. The staining intensity of TLR9 per cell was analyzed by an ImageJ software. Relative staining intensity of the control was set at 1. Data represent means ± SD of 4 independent experiments. ***p < 0.001, compared with the control; ^### p < 0.001, compared with negative control siRNA-transfected cells. c Reduction of TLR9 expression in siRNA-transfected cells. A549 cells transfected with TLR9 siRNA were lysed and treated, and then Western blotting was performed to detect TLR9 and GAPDH as described in Methods. d Relative intensity of TLR9 expression in siRNA-transfected A549 cells. The relative band intensity of TLR9 normalized by GAPDH was measured with an ImageJ software. Data represent means ± SD of 3 independent experiments. **p < 0.01 and ***p < 0.001, compared with the control; ^# p < 0.05 and ^## p < 0.01, compared with negative control siRNA-transfected cells. e Inhibitory effect of TLR9 siRNA on MWCNT-induced 8-nitroG formation. After siRNA transfection, A549 cells were treated with 1 μg/ml CNT-L for 8 h. 8-NitroG formation was analyzed by fluorescent immunocytochemistry. Magnification, X200. f Quantitative image analysis for effects of TLR9 siRNA on MWCNT-induced 8-nitroG formation. Staining intensity was analyzed as described in (b). Data represent means ± SD of 3-4 independent experiments. ***p < 0.001, compared with the control, ^### p < 0.001, compared with MWCNT alone, ^$$ p < 0.01 and ^$$$ p < 0.001, compared with MWCNT + negative control siRNA. g Effect of TLR9 siRNA on NO release from MWCNT-exposed cells. After siRNA transfection, A549 cells were treated with 1 μg/ml CNT-L for 4 h. Data represent means ± SD of 5-6 independent experiments. ***p < 0.001, compared with the control; ^# p < 0.05 and ^### p < 0.001, compared with CNT-L-treated cells. Statistical analysis was performed by ANOVA followed by Tukey’s test