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Tissue samples and cells were homogenized with RIPA lysis buffer (Beyotime Biotech, Shanghai, China). Protein concentrations were determined by using a BCA protein assay kit. The proteins were separated on a 12% SDS‐polyacrylamide gel and transferred to a PVDF membrane. After blocking with 5% bovine serum albumin (BSA) in TBS, the membrane was incubated with primary antibody at 4°C overnight. The secondary antibody was conjugated to horseradish peroxidase and incubated at room temperature for 1 hour. Immunoreactivity was detected with an enhanced chemiluminescence kit (Beyotime Biotech) by a ChemiDoc™ MP System (Bio‐Rad). The following antibodies were used: HMGB1 (1:5000, Abcam), RAGE (1:1000, Abcam), RAGE (1:200, Santa Cruz), TLR4 (1:1000, Protein Tech), TLR4 (1:200, Santa Cruz) and β‐actin (1:1000, Abcam); pNF‐κB/NF‐κB, pJNK/JNK and pERK/ERK were all purchased from Cell Signaling Technology and incubated at a dilution of 1:1000.

Licochalcone A (Lico A), purity up to 98%, was provided by the Chengdu Herbpurify CO., LTD (Chengdu, China). Dimethyl sulfoxide (DMSO), d-Galactosamine, LPS (Escherichia coli 055:B5), and inhibitors of autophagy (3-methyladenine, 3-MA) were offered by Sigma-Aldrich (St. Louis, MO, USA). Antibodies against P-AMPK/AMPK, Nrf2, HO-1, and Lamin B were offered by Abcam (Cambridge, MA, USA); and Atg7, Atg16, Beclin1, Atg12, Atg5, Atg3, LC3, NLRP3, ASC, Casapase-1, IL-1β, P-ERK/ERK, P-P38/P38, P-P65/P65, P-IκBα/IκBα, P-JNK/JNK, TFEB, P-P62, and β-actin were obtained from Cell Signaling. Additionally, ALT, AST, MDA, ROS, GSH, and SOD test kits were obtain Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), if not otherwise indicated.

Immunoblotting analysis was conducted according to procedures previously described.^{53 (link)} Antibodies against LC3B, caspase-3, PARP, IRE1-α, p-ERK/ERK, p-Akt/Akt, and p-PKA/PKA were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin antibody was purchased from Developmental Studies Hybridoma Bank (DSHB, Iowa City, IA, USA). Binding of antibodies was detected using HRP-coupled anti-rabbit or anti-mouse immunoglobulin (Cell Signaling Technology), and visualized using Pierce ECL Western Blotting Substrate (ThermoFisher, Waltham, MA, USA). Density of bands was quantified, and then normalized with β-actin as loading control.

The tibia was thoroughly flushed bone marrow cells with saline. The flushed tibia was homogenized using a handy sonicator and then centrifuged at 12,000 × g for 30 minutes. The supernatant was subsequently assayed for the protein concentration using the DC Protein Assay Kit (BioRad, Hercules, CA). Equal amounts (20 μg) of total protein were subjected to 10% SDS-PAGE and electroblotted onto a PVDF membrane. The membranes were blocked in 2% blocking reagent (ECL Advance Blocking Agent, GE Healthcare) and probed with antibodies against Cx43 (1:1,000; Cell signaling), RANKL (1:1,000; abcam), SOD2 (1:2,000; SOD-110; StressGen), Sclerostin (1:500; abcam), p-ERK, ERK (1:1,000; Cell signaling), GAPDH (1:1,000; Cell signaling) and ACTIN (1:2,000; Cell signaling). The signals were detected using the ECL system (ECL plus, GE Healthcare) and a luminoimage analyzer LAS-3000 mini (Fuji Film, Tokyo, Japan).

Phosphate-buffered saline (PBS) was purchased from Bio Basic Inc. (Markham, Ontario, Canada). 2,3,5-Triphenyl-tetrazolium chloride (TTC), cresyl violet, Evans blue (EB), and propidium iodide (PI) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). The optimal cutting temperature compound cryostat embedding medium was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies against B-cell lymphoma 2 (Bcl-2), bcl-2-like protein 4 (Bax), phospho-phosphoinositide 3-kinase (p-PI3K), protein kinase B (PKB, AKT), phospho-extracellular signal-regulated kinase (p-ERK), ERK, phospho-c-Jun N-terminal kinase (p-JNK), JNK, phospho-p38 mitogen-activated protein kinase (p-p38), p38, and manganese superoxide dismutase (MnSOD) were purchased from Cell Signalling Technology (Danvers, MA, USA). PI3K, p-AKT, phospho-mammalian target of rapamycin (p-mTOR), sirtuin 1 (SIRT1), and β-actin were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Aquaporin 4 (AQP4) was purchased from Abcam Inc. (Milton, Cambridge, UK). The bovine serum albumin (BSA) standard and enhanced chemiluminescence (ECL) western blotting chemiluminescent substrate were purchased from Thermo Fisher Scientific.

Human embryonic kidney 293E (HEK293E) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with antibiotics and 5% fetal bovine serum (FBS, Invitrogen) in the presence of M-CSF. RAW264.7 cell, a murine macrophage cell line, was cultured in DMEM supplemented with antibiotics and 10% FBS in the absence of M-CSF. Antibodies used in this study were as follows: His antibody from Roche; NFATc1, c-Fos, ATP6V0D2, actin, p-Akt, and Akt antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); M-CSF antibody from Abcam; and p-ERK, ERK, p-JNK, JNK, p-p38, and p38 antibodies from Cell Signaling Technology (Boston, MA, USA).

29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) from human plasma, U73122 (a phosphoinositide-specific phospholipase C (PLC)-γ-specific inhibitor), SP600125 (c-Jun N-terminal kinase (JNK) inhibitor), PD98059 (extracellular signal-regulated kinase (ERK) inhibitor), SB203580 (p38 inhibitor), Bay 11-7082 (nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor), 2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (BAPTA, a permeable Ca²⁺ chelator), thapsigargin (TG, an inhibitor of ER Ca²⁺-ATPase), and an antibody against β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against TonEBP and lamin-B1 were obtained from Abcam (Cambridge, UK), and antibodies against p-nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (Iκ-Bα)/Iκ-Bα, p-p38/p38, p-ERK/ERK, p-JNK/JNK, and p-protein kinase C (PKC)/PKC were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Small interfering RNAs (siRNAs) against TonEBP and TLR-2 were purchased from Bioneer (Daejeon, South Korea). Antibodies against MMPs 1, 3, and 13 were obtained from R&D Systems (Minneapolis, MN, USA).