Hmgb1

Ovaries were collected from 40-day-old CD1 mice, frozen and stored at −20 °C. Total protein lysate was extracted with a pestle and motor using cell lysis buffer (Cell Signaling Technology, 9803S) in the presence of a protease inhibitor cocktail (Life Technologies, 78440). Denaturing SDS–PAGE was performed with 25 μg of the total protein lysate using standard western blotting techniques on a 4–15% polyacrylamide gel (Bio-Rad, 456–1084). The polyvinylidene fluoride membrane was blocked following transfer using 3% Amersham ECL Prime Blocking Reagent (GE Healthcare, RPN418) and incubated in primary antibodies as follows: 1:1,000 HMGB1 (Abcam, ab18256) and 1:1,000 GAPDH (Cell Signaling Technologies, 5174S) in TBS+0.1% Tween-20. As a control for expression, the HMGB1 blocking peptide (Abcam, ab18650) was also used. A ratio of 1:5 HMGB1 antibody to HMGB1 peptide was incubated overnight at 4 °C prior to application. Membranes were then incubated in horseradish peroxidase conjugated anti-rabbit secondary antibody (GE Healthcare, NA931-1ML), and protein was visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232).

The protein levels of IL-6 (Abcam, UK), IL-8 (Abcam, UK), IL-1β (Cyagen, China) and HMGB1 (BIOVISION, USA) in pleural effusion samples were analyzed using ELISA kits. The assay was completed strictly following the manufacturer’s instructions. The OD value at 450 nm was monitored with a Perlong DNM-9602 Microplate reader (Beijing, China).

Prior to being sacrificed, the mice were not allowed access to any food for 8 h. Under anesthesia with isoflurane, blood was withdrawn from the left femoral artery. WBCs, lymphocytes and neutrophils were each measured using routine complete blood counts. Plasma levels of insulin (#630-07289, Shibayagi, Gunma, Japan), TGF-β1 (DY1679-05, R&D Systems, Minneapolis, MN, USA), leptin (MOB00B, R&D Systems, Minneapolis, MN, USA), soluble P-selectin (DY737, R&D Systems, Minneapolis, MN, USA), glutamine (ab197011, Abcam, Cambridge, MA, USA) and HMGB1 (E4864, BioVision, Mountain View, CA, USA) were all measured using Enzyme Immunosorbent Assay (ELISA) kits, following the procedures provided by the respective manufacturers. Plasma levels of circulating citrullinated histone H3 were measured using self-prepared assay plates which had been coated with an antibody against citrullinated histone H3 (NB100-57135, Novus Biologicals, Centennial, CO, USA). The contents of immunocomplexes were quantified using a spectrophotometer at 450 nm. The level of fasting glucose was measured using a hand-held Accucheck glucometer (Roche Diagnostics, Indianapolis, IN, USA). The HOMA-IR index was calculated as [fasting insulin (μU/mL) × fasting glucose (mmol/L)]/22.5, and used as the reported method [32 (link)].

OA was obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium carboxymethylcellulose (CMC-Na) was provided by Sinopharm (Shanghai, China). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) microplate test kits were obtained from Nanjing Jiancheng Bioengineering Institute (Jiancheng Biotech, China). TNF-α enzyme-linked immunosorbent assay (ELISA) kits were acquired from eBioscience (San Diego, CA, USA). The RNA polymerase chain reaction (PCR) kit was purchased from Takara Biotechnology (Dalian, China). The antibodies used in this study included those against HMGB1, TLR4 (Epitomics, Burlingame, CA, USA), TNF-α, caspase-3, caspase-9, Bcl-2, Bax, Beclin 1, LC3, c-Jun NH2-terminal kinase (JNK), phospho-JNK (p-JNK), and β-actin (Proteintech, Chicago, IL, USA).

Ethyl pyruvate and Con A were purchased from Sigma-Aldrich (USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-2, IL-6, TNF-α and IL-1β were purchased from R&D systems (Minneapolis, MN, USA). HMGB1 ELISA kit was purchased from Shino-Test Corporation (Oonodai, Kanagawa, Japan). Antibodies used in this study include HMGB1 (Epitomics, CA), IL-2 (Biolegend, CA), IL-6 (Proteintech, CA), TNF-α (Santa Cruz, CA), NF-κB (Proteintech, CA), IL-1β (Biolegend, CA), IκB α (Cell signal technology, CA) and IκB β (Cell signal technology, CA).

Liver tissue was homogenized as previously described [4 (link),34 (link)]: 50 μg proteins were separated on 6%–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were blotted into polyvinylidene fluoride (PVDF) membranes (Biorad, Madrid, Spain) and immunoblotted overnight at 4 °C with antibodies directed against total and phospho-AKT (Ser473), cleaved caspase 3, cleaved caspase 9, total and phospho-GSK3β (ser 9), cytochrome C, and phospho-VDAC (Cell Signaling, Beverly, MA, USA); GRP78, CHOP, ATF4 and total and phospho-PERK (Santa Cruz Biotechnology, Santa Cruz, California, USA), HMGB-1(Abcam, Milton, Cambridge, UK), and β actin (Sigma Chemical, St. Louis, MO, USA). After washing, membranes were incubated with appropriate peroxidase-conjugated secondary antibody for 1 h. Immuno-labeled bands were detected using Western-Bright ECL-HRP Substrate (Advansta, Menlo Park, CA, USA) and quantified using the Quantity One software for image analysis. Results were expressed as densitometry ratios between the protein of interest and the loading control (β-actin).

For proteins extraction from supernatant (conditioned media), trichloroacetic acid (TCA) precipitation method was used. Briefly, cell culture medium was centrifuged to remove cell debris before incubation with 2% sodium deoxycholate (1/1,000 v/v) for 30 min on ice. TCA was then added to a final concentration of 7.5% and incubated on ice for 30 min. Proteins were recovered by high speed centrifugation (15,000 g for 20 min at 4°C) before two washing steps with ice-cold acetone and resuspension of the protein pellet in RIPA buffer. Immunoblotting was performed as previously described (20 (link)). Bip (Cell Signaling Tech., ref. 3177), Cleaved-PARP (Cell Signaling Tech., ref. 5625), Hsp90 (BD Biosciences, ref. 610419), Annexin A1 (Zymed, ref. 71–3,400), and HMGB1 (Abcam ref. ab18256) antibodies were diluted at 1/1,000 (v/v) and β-actin antibodies (Sigma, ref. A5441) at 1/2,500 (v/v) in a TBST (Tris-buffered saline, 0.1% Tween 20) solution with 1% w/v non-fat dry milk.