A: RAW cells undergo endotoxin tolerance as early as 4h after LPS stimulation: To study endotoxin tolerance in RAW cells, we stimulated cells with LPS (100ng/ml) and re-stimulated with another LPS challenge (100ng/ml) at 0, 4, 6, 8, and 24 hour time points for four hour. We studied TNF-α mRNA expression. The cells increased TNF-α mRNA expression in response to LPS re-stimulation only at 0h time point. RAW cells were unable to increase TNF-α mRNA further to LPS re-stimulation at 4, 6, 8 and 24h time points, indicating endotoxin tolerance. * p<0.05 vs. respective NS group Tukey‘s post-hoc analysis; error bars: s.e.m. B: SIRT-2 protein expression increased during endotoxin tolerant phase in RAW cells: RAW cells were treated with LPS (100ng/ml) for 0, 1, 4, 8, 18, and 24 h. Whole cell lysates were collected for western blotting of proteins SIRT-2 and CPA (housekeeping gene). Representative image out of three experiments shows that was increased in SIRT-2 expression in 18 and 24h after LPS stimulation. C SIRT-2 deacetylates NFkB p65: We studied the effect of SIRT-2 expression on NFkB p65 acetylation using HEK293 cells. SIRT-2 plasmid was co-transfected with p65 or/and CBP plasmids into HEK293 cells (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 while it decreased in cells transfected with p65+CBP+SIRT-2, indicating SIRT-2 directly deacetylates NFkB p65.

https://doi.org/10.1371/journal.pone.0162560.g001