Fig. 5

Glycolytic inhibitors suppress LPS-induced transcriptional activity of NF-κB via inhibition of NAD+/SIRT1-mediated p65 acetylation. a BV-2 microglial cells were pretreated with 2-DG (1000 μM) or 3-BPA (100 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 1 h. The expression of p65 subunit of NF-κB p65 and p-p65 subunit of NF-κB in the total, cytosolic, and nucleus lysates was determined by Western blotting (left), and the relative protein levels were quantified by densitometric analysis (right). b BV-2 microglial cells were pretreated with 2-DG (1000 μM) for 30 min followed by LPS (200 ng/mL) treatment for 1 h. The extracts were subjected to immunoprecipitation (IP) with anti-p65 antibody or anti-IgG followed by immunoblotting analysis with anti-acetylated-p65 antibody and anti-p65 antibody. The expression of ac-p65 was determined by Western blotting (upper), and the relative protein levels were quantified by densitometric analysis (lower). c HEK293T cells were cotransfected with p300 and p65 plasmids for 24 h, followed by 2-DG (1000 μM) treatment for 30 min and then treated with TNF-α (10 ng/mL) for 1 h. The extracts were subjected to immunoprecipitation (IP) with anti-p65 or anti-IgG followed by immunoblotting analysis with anti-acetylated-p65 antibody and anti-p65 antibody. The expression of ac-p65 was determined by western blotting (upper) and the relative protein levels were quantified by densitometric analysis (lower). d BV-2 microglial cells were pretreated with 2-DG (600–1000 μM) for 30 min, followed by LPS (200 ng/mL) treatment for 16 h. The NAD⁺/NADH ratio was measured as described in the methods and materials section. e–g BV-2 microglial cells were transfected with si-SIRT1 (40 nM) or si-Scramble for 48 h, followed by LPS (200 ng/mL) treatment for 6 h. The mRNA expression of SIRT1, TNF-α, and IL-1β was measured by qPCR. Data are presented as mean ± S.D. (n=3) and are representative of results obtained from three independent experiments. *p<0.05, **p <0.01 compared to the LPS, si-Scramble or si-Scramble + LPS group, respectively