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. 2016 Aug 8;11(8):e0160431. doi: 10.1371/journal.pone.0160431

Fig 4. SIRT-2 inhibition reverses endotoxin tolerance in RAW cells.

Fig 4

A: SIRT-2 inhibitor AK-7 enhanced LPS induced cytokine response in sensitive cells. RAW cells were pre-treated with vehicle (DMSO) or AK-7 (25uM) for 1h before the stimulation with normal saline or LPS (100ng/ml) for 4h and TNF-α mRNA expression levels were studied in different groups. There was significant increase in TNF-α mRNA in AK-7 +LPS vs. Vehicle +LPS group. * p<0.05 vs. Vehicle +LPS using Tukey‘s post-hoc analysis; error bars: s.e.m. B SIRT-2 inhibitor AK-7 reverses endotoxin tolerance in RAW cells: Endotoxin tolerance was induced with LPS (100ng/ml) treatment for up to 24h. AK-7 (25uM) was added to cell culture at 4h and 6h after the beginning of LPS stimulation, followed by further stimulation with Vehicle or LPS (100ng/ml) for 4h and TNF-α, IL-6 and IL-1β mRNA expression in different groups were studied. While Vehicle treated groups remained endotoxin tolerant, there was a significant increase in all three cytokine mRNA expression in AK-7 +LPS vs. Vehicle +LPS treated groups. * p<0.05 vs. Vehicle +LPS using Tukey‘s post-hoc analysis; error bars: s.e.m. C: SIRT-2 deficiency increases NFkB p65 acetylation: We studied the effect of SIRT-2 deficiency on NFkB p65 acetylation using RAW cells. We treated RAW cells with SIRT-2 siRNA (si-SIRT2) or scramble control (si-Ctrl), and co-transfected with p65 and CBP plasmids (to increase baseline p65 acetylation) and blotted for antibodies against Ac-p65, total p65, SIRT-2 and GAPDH. NFkB p65 acetylation (Ac-p65) increased in cells with transfection with p65 + CBP. Cells treated with p65+CBP+si-SIRT-2 showed further increase in AC-p65 vs. p65+CBP+si-Ctrl, indicating SIRT-2 directly deacetylates NFkB p65.