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. 2018 Jul 13;293(36):13874–13888. doi: 10.1074/jbc.RA118.003443

Figure 4.

Figure 4.

Loss of the anti-inflammatory effect of nicotine in LPS-stimulated RAW264.7 macrophages due to dupα7 overexpression. The anti-inflammatory activity of nicotine acting on α7-nAChRs of macrophages exposed to LPS was analyzed by measuring the drug effect on nuclear translocation of NF-κB or TNFα production assayed by immunostaining/confocal microscopy or ELISA, respectively. A, representative confocal images of the cellular distribution of NF-κB (red) after staining with primary anti-NF-κB–p65 antibody and Alexa Fluor 555-conjugated secondary antibody in non-nucleofected cells or in cells nucleofected with dupα7.pcDNA3.1/Myc-His or with the corresponding empty vector. Successful expression of the dupα7-Myc subunit or empty vector in nucleofected cells was ensured by staining of the Myc epitope (green) with the primary anti-Myc antibody and the Alexa Fluor 488-conjugated secondary antibody. DAPI was used for nuclear staining (blue). In non-nucleofected cells, NF-κB was distributed throughout the cytosol in the absence of stimulation and was translocated to the nucleus in response to LPS (100 ng/ml; 1 h). Incubation of cells with nicotine prevents the NF-κB activation induced by endotoxin. This anti-inflammatory effect of nicotine was preserved in cells nucleofected with the empty vector while it is markedly reduced in cells overexpressing dupα7 subunits. B, percentage of cells with respect to total cells visualized in individual microscope fields from three independent experiments in which the NF-κB–p65 translocation to the nucleus has occurred (positive cells) in response to each experimental condition described above. The horizontal bars show the mean ± S.D. for the group; ***, p < 0.001 compared with non-nucleofected cells exposed to LPS. †††, p < 0.001 compared with non-nucleofected cells exposed to LPS and nicotine. C, histogram representing the effect of nicotine on TNFα production induced by LPS (100 ng/ml; 4 h) in cells nucleofected with dupα7.pcDNA3.1/Myc-His or with the empty vector. Once again, the anti-inflammatory effect of nicotine in cells nucleofected with the empty vector practically disappears after dupα7 overexpression. The bars show the mean ± S.E. from three independent experiments. ***, p < 0.001 after comparing the indicated bars. ns is nonsignificant.