Figure 4: Lactate promotes HMGB1 acetylation and release.

(a) RAW 264.7 cells were transfected with indicated shRNA for 48 h and then stimulated with LPS (100 ng ml⁻¹) for 6–24 h. The lactate level in the culture medium was assayed (n=3, means±s.e.m., *P<0.05 versus control shRNA group by analysis of variance (ANOVA) LSD test). (b,c) RAW 264.7 cells were transfected with GRP81 shRNA and control shRNA for 48 h and then stimulated with lactate (5 mM) for 6 h. GRP81 expression (b), cell viability (c) and HMGB1 release (c) were assayed (n=3, means±s.e.m., *P<0.05 by ANOVA LSD test). (d) RAW 264.7 cells were stimulated with 3-phosphoglycerate (3PG, 2 mM), 2-phosphoglycerate (2PG, 2 mM) and PEP (2 mM) for 6 h and levels of lactate and HMGB1 release were assayed (n=3, means±s.e.m., *P<0.05 versus untreated group by ANOVA LSD test). (e) RAW 264.7 cells were stimulated with lactate (5 mM) for 6 h and levels of acetylated HMGB1 were assayed. (f) RAW 264.7 cells were stimulated with LPS (100 ng ml⁻¹) for 24 h and the levels of acetylated HMGB1 were assayed. (g) RAW 264.7 cells were stimulated with LPS (100 ng ml⁻¹), lactate (5 mM) and PEP (2 mM) for 24 h. HDAC activity was assayed (n=3, means±s.e.m., *P<0.05 versus untreated group by ANOVA LSD test). (h–j) RAW 264.7 cells were stimulated with Trichostatin A (TSA) (2 μM) and/or LPS (100 ng ml⁻¹) for 24 h. HDAC activity (h) and acetylated HMGB1 (i) and HMGB1 release (j) were assayed (n=3, means±s.e.m., *P<0.05 by ANOVA LSD test). (k) RAW 264.7 cells were stimulated with lactate (5 mM, 6 h) with or without anti-HMGB1-neutralizing antibody (2 μg ml⁻¹) and levels of TNF-α release were assayed (n=3, means±s.e.m., *P<0.05 by ANOVA LSD test).