Fig 2: LPS-induced primary response gene expression varies significantly by lobe at 5 h of exposure. LPS-induced fold change of primary and secondary response genes relative to left lateral lobe. Expression following 5 h of exposure of the primary response genes (a) Tnf, (b) Cxcl1, (c) Nfkbia, and (d) Tnfaip3 and secondary response genes (e) Nos2 and (f) Il6. Values are means ± SE (n = 9/time point). *P < 0.05 vs. left medial lobe; †P < 0.05 vs. caudate process; $P < 0.05 vs. papillary process; #P < 0.05 vs. right lateral lobe; %P < 0.05 vs. right medial lobe; by Kruskal-Wallis test.

Fig 3: TSA blocked the inflammatory induction of the TNF-a/TNFAIP3 axis. RAW246.7 cells were pre-treated or not for 30 min with 100 ng/mL TSA (A,B) or as indicated (C) and then stimulated with 1 µg/mL LPS or left untreated (-) prior to mRNA extraction. (A–C) The regulation of different inflammatory mediators was investigated at the mRNA level using RT-PCR. Data are normalized to the housekeeping RNA (18S) and expressed as the mean ± SEM (n = 3) of 2-??Ct relative to (-). * p < 0.05 versus respective LPS stimulation using an unpaired t-test. (B) Cell viability was assessed using a live–dead assay. The results are expressed as the mean ± SEM (n = 3) of the percentage of live cells. Each dot represents one independent experiment. The lack of statistically significant differences compared with (-) was evaluated using one-way ANOVA with Dunnett’s post hoc test.

Fig 4: HDAC3 knockdown partially recapitulated the effects of TSA. (A) RAW246.7 cells were transfected with the indicated siRNAs: target gene expression was evaluated using qPCR (upper panels) and expression at the protein level using Western blot analysis (lower panels). The results depict the percentage of target gene expression (mean ± SEM, n = 3) (upper panels) and a representative Western blot experiment; scr: scramble. Dots represent results of individual experiments. (B) siRNA-transfected RAW246.7 cells were treated with 1 µg/mL LPS for 60 min prior to mRNA extraction. The expression of TNF-a, IL-6, TNFAIP3 and CCL5 was investigated at the mRNA level using RT-PCR. Data were normalized to the housekeeping RNA (18S) and expressed as the mean ± SEM (n = 3) of the percentage of production in scr cells stimulated with LPS alone. As a comparison, cells transfected with scramble siRNA were also pre-treated with 100 ng/mL TSA. * p < 0.05 versus scr using an unpaired t-test; # p < 0.05 versus scr TSA using one-way ANOVA with Dunnett’s post hoc test. Dots represent results of individual experiments. (C) RAW246.7 cells transfected with scr or HDAC3 siRNA were treated with 1 µg/mL LPS for 60, 120 and 240 min with or without a 100 ng/mL TSA pre-treatment prior to mRNA extraction. The expression of TNF-a and TNFAIP3 was investigated at the mRNA level using RT-PCR. Data are normalized to the housekeeping RNA (18S) and expressed as the mean ± SEM (n = 3) of 2-??Ct relative to (-). * p < 0.05 versus respective scr or # p < 0.05 versus respective HDAC3 siRNA using an unpaired t-test.

Fig 5: NF-?B signaling pathway plays critical role in the induction of A20MF-THP-1 cells A., BMDM B. and MF-THP-1 cells expressing gC1qR-specific shRNA or luciferase-specific shRNA C. were treated with core protein (10µg/mL) in different times, cell lysates were analyzed by immunoblotting with indicated antibodies. D. MF-THP-1 cells were treated with C1q (70µg/mL) for 30 minutes, cell lysates were analyzed by immunoblotting with indicated antibodies. E. MF-THP-1 cells were pretreated with IMD 0354 (3µM) for 30 minutes, then incubated with core protein for 3 and 6 hours, cell lysates were analyzed by immunoblotting to examine the A20 expression.