Donkey anti-Goat IgG-AF647 Antibody

Acetate attenuates inflammasome activation in vitro. a–d ELISA of IL-1β (a, b) and IL-18 (c, d) in BMDMs treated with LPS+nigericin or LPS+ATP at different doses (20, 30, 35, 40, and 60mM) (n=3); e ELISA of IL-1β after sodium acetate treatment (n=3); f ELISA of IL-1β at different time points after acetate treatment in LPS-primed ATP-treated BMDMs (n=3); n=biological replicates; Data are presented as the mean±SEM. *P<0.05, **P<0.01, and ***P<0.001, Student’s t-test compared with the control group

Acetate suppresses NLRP3 inflammasome activation at different time points in vitro. a–d ELISA of IL-1β (a), IL-18 (b), TNF-α (c), and IL-6 (d) production after acetate treatment in BMDMs. Acetate (10, 20, and 30mM) was administered 30min before LPS priming, 30min before nigericin stimulation, simultaneously with nigericin or 30min after nigericin stimulation. All treatments showed suppressive effects in a dose-dependent manner (n=3); *P<0.05, **P<0.01, and ***P<0.001, Student’s t-test compared with the LPS+Nig group; P<0.05, P<0.01, and P<0.001, oneway ANOVA for comparison. e Western blot of pro-caspase-1, caspase-1-p20, and IL-1β-p17 in supernatant and NLRP3, pro-caspase-1, caspase-1-p20 and β-actin in cell lysate after indicated treatment; f–h ELISA of IL-1β after treatment with poly(dA/dT), MDP and flagellin (n=3). n=biological replicates. Data are presented as the mean±SEM

Acetate suppresses inflammasome-mediated peritoneal inflammation in vivo. a–h Acetate (20mM) suppressed MSU- (a–d) and alum(e–g)-induced peritoneal inflammation. Flow cytometry revealed that neutrophils (CD11b+ GR-1+)( a, e), macrophages (F4/80+)( b, f), peritoneal exudate cells (PECs) (c, g) and IL-1β (ELISA) in the peritoneal lavage fluid (PLF) (d, h) were decreased in the acetate pretreatment group; i–l acetate suppressed LPS-induced peritonitis (n=3). Serum levels of IL-6 (i), TNF-α (j), IL-1β (l), and IL-1β in PLF (k), as measured by ELISA, decreased in the acetate treatment group (n=3); *P<0.05, Student’s t-test compared with the model group (MSU, Alum or LPS). n=biological replicates. Data are presented as the mean±SEM

Acetate-induced suppression of the NLRP3 inflammasome occurs via the sAC-PKA axis and promotes polyubiquitination of NLRP3. a, b ELISA of IL-1β indicated that acetate (20mM) suppression in BMDMs was dependent on sAC. Inhibition of sAC (KH7) (a) or siRNA interference for two types of sAC (b) reversed the suppressive effects of acetate (n=6). c ELISA of IL-1β indicated that the inhibition of PKA (H89) reversed the suppressive effects of acetate in BMDMs (n=3). d Immunoprecipitation of NLRP3 polyubiquitination. The K48 and K63 ubiquitin chains were enhanced after acetate treatment (n=3). e ELISA of IL-1β indicated that siRNA interference of MARCH7, an E3 ligase involved in NLRP3 ubiquitination, reversed the suppressive effects of acetate in BMDMs (n=6). n=biological replicates. Data are presented as the mean±SEM. *P<0.05 and **P<0.01, Student’s t-test compared with the control group

Acetate promotes NLRP3 degradation through autophagy. a–c ELISA of IL-1β indicated that acetate (25, 30, and 35mM) mediated suppression of the NLRP3 inflammasome in a manner dependent on autophagy but not the proteasome. Inhibition of autophagy (3-MA and bafilomycin A1) reversed the suppression of IL-1β (a, b), whereas inhibition of the proteasome induced few alterations (c) in BMDMs (n=3). c, d Western blot of NLRP3, ASC, p62, and LC3B. The degradation of NLRP3 was accompanied by increased NLRP3 expression and decreased p62 expression in BMDMs (n=3). n=biological replicates. Data are presented as the mean±SEM. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001, Student’s t-test compared with the control group (a–c) or one-way ANOVA for comparisons of differences (d)

Acetate enhances autophagy in a dose-dependent manner. a, b Western blot (a) and quantification (b) of LC3B after treatment with bafilomycin A1 and acetate (mM) (n=3) in BMDMs. n=biological replicates. Data are presented as the mean±SEM. **P< 0.01 and ***P<0.001, one-way ANOVA for comparisons of differences