Bay 11 7082

After reaching 80 % confluence, ADSCs were seeded in six-well plates (5 × 10⁵ cells/well). The cells were treated with the following reagents for 24 hours: (a) ADSC basal medium as a control; (b) stimulant CRP alone; (c) stimulant plus inhibitors or block antibodies, including ERK inhibitor (PD098059, 10 μM), PI3K inhibitor (LY294002, 5 μM), and nuclear factor-kappa beta (NF-kB) inhibitor (BAY-11-7082, 5 μM) (all from Cell Signaling Technology, Danvers, MA, USA), or anti-CD16 (2 μg/ml; R&D Systems, Inc., Madison, WI, USA), anti-CD16/32 (1 μg/ml; Abcam Inc., Cambridge, UK), and anti-CD64 (1:100, 3 μg/ml; R&D Systems Inc.); or (d) inhibitors and block antibodies alone. Doses of the inhibitors or block antibodies were determined according to previous laboratory characterization and published data. Supernatants and cell extractions were collected 24 hours after treatment.

C2C12 mouse myoblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Corning, NY, USA) supplemented with 10% fetal bovine serum and antibiotics at 37 °C in a humidified atmosphere containing 5% CO₂. To differentiate into myotubes, C2C12 cells were maintained in DMEM supplemented with 2% horse serum for 72 h, and the medium was replaced every other day. Recombinant mouse HMGB1 (BioLegend Inc., San Diego, CA, USA) was dissolved in phosphate-buffered saline (PBS). Specific inhibitors of p38 MAPK (SB203580), PI3K (LY294002), MEK (PD98059), JNK (SP600125), and NF-κB (BAY 11-7082) were purchased from Cell Signaling Technology (Danvers, MA, USA) and dissolved in the recommended solvents. An adenovirus expressing atrogin 1 (Ad-atrogin 1/FBXO32) was kindly provided by Prof. J.-S. Chun of the School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea^{40 (link)}.

The human monocytic cell line (purchased from CTCC, Wuhan University, Wuhan, China) was cultured as previously described (Xie et al., 2017 (link)). psiRNA-hTLR2, psiRNA-hTLR4, psiRNA-hTLR6, pZERO-hTLR2, pZERO-hTLR4, pZERO-hTLR6, pDeNy-hMyD88, pDeNy-hMal, and control plasmids (psiRNA-LucGL3, pZERO-mcs, and pDeNy-mcs) were obtained from InvivoGen (Carlsbad, United States). The inhibitor of NF-κB, BAY11-7082, was obtained from Cell Signaling Technology (Beverly, MA, United States). Anti-phosphorylated and anti-total IκBα and p65 mouse antibodies were obtained from Abcam (Cambridge, MA, United States). Anti-β-actin antibody was bought from Sigma-Aldrich (St, Louis, MO, United States). Cy3-conjugated goat anti-mouse IgG antibody was obtained from Invitrogen (Frederick, MD). Antibodies against TLR2, TLR4, TLR6, MyD88, and Mal were purchased from Abcam (Cambridge, MA, United States). Neutralizing antibodies against TLR2, TLR4, and TLR6 were obtained from Novus. Complete protease inhibitor cocktail was purchased from Roche Applied Science (Mannheim, Germany). Other reagents were purchased from Thermo Fisher Scientific (Waltham, United States).

BGC-823 gastric cancer cells were grown in RPMI 1640 (Gibco, USA) supplemented with 10% newborn bovine serum (Gibco) in a CO₂ incubator at 37°C containing 5% CO₂. The PI3K/Akt inhibitor LY294002, the nuclear factor-κB (NF-κB) inhibitor BAY11-7082, and the MAPK inhibitor UO126 were from Cell Signaling Technology (USA). The three signal pathway inhibitors were dissolved in dimethyl sulfoxide (DMSO, China) solution.

Immobilized Glutathione Column was purchased from Thermo scientific (Rockford, USA) and Ni²⁺-charged chromatographic column was provided by GE healthcare (Buckinghamshire, United Kingdom). Polymyxin B – agarose which is used for endotoxin removal was provided by Sigma Corp. (Santa Clara,CA). Rabbit monoclonal antibodies including anti-phospho-Akt, anti-Akt, anti-phospho-MAPKs, anti-MAPKs, and anti-phospho p65 were provided by Cell Signaling Technology Corporation (Beverly, MA). Mouse monoclonal antibody anti-actin was provided by Santa Cruz Biotechnology Corporation (Santa Cruz, CA). P38 MAPK inhibitor SB203580, extracellular signal-regulated kinase inhibitor U0126, JNK inhibitor SP600125, Janus kinase inhibitor AG490, phosphatidylinositol 3-OH kinase (PI3K) inhibitor LY294002, and IκB-α phosphorylation inhibitor BAY11–7082 were provided by Cell Signaling Technology. DMSO was used to dissolve AG490, BAY117082, PD98059, and SP600125, while water was used to dissolve LY294002 and SB203580. The final concentration of DMSO was 0.1% (volume/volume) in all the cell culture experiments.