Figure 5.

ACLY-mediated NF-κB full activation upregulated the SLC25A1 and ACLY genes. Human PBMC-derived macrophages were triggered by LPS in the presence or absence of SB, and (a,b) were used to carry out chromatin immunoprecipitation (ChIP) analysis with an antibody against subunit p65 of NF-κB. Then, specific primers were used in real-time PCR experiments to analyze the SLC25A1 and ACLY gene promoters. (c,d) Human PBMC-derived macrophages treated as reported above were used to carry out ReChIP experiments with a first antibody against subunit p50 of NF-κB and then with the anti-NF-κB p65 (acetyl K310) antibody. Specific primers were used in real-time PCR experiments to analyze the SLC25A1 and ACLY gene promoters. (e) CIC and ACLY protein levels were detected by western blotting in PBMC-derived macrophages treated as above. (f,g) iBMDM cells transiently cotransfected with the NF-κB (p65) wild-type (NF-κB wt) overexpression vector together with ACLY (p3000) (f) or SLC25A1 (p2000) (g) promoter vectors and treated with LPS in the presence or absence of SB (f,g, left panels) or in the presence of ACLY gene silencing (siACLY) (f,g, right panels), which were used for luciferase luminescence detection. (h) iBMDM cells transiently cotransfected with the NF-κB (p65) wild type (NF-κBwt) or mutated (NF-κBmut) and treated with LPS were used to quantify both endogenous ACLY and CIC proteins. Western blotting data presented are representative of at least 3 independent experiments, and the mean of the protein levels quantified against β-actin and normalized versus the mean of the proteins in the untreated cells (C) are reported under the image. In (a–d,f,g), data are representative of 3 independent experiments and are presented as means ± SD (error bars). Different letters above the bars indicate significant differences between treatments at p < 0.05, according to Tukey’s post hoc test performed after one-way ANOVA.