P ikkα β

After treatment, HPDLCs were collected and lysed in RIPA buffer for protein extraction, as per the product’s instructions. Following sodium dodecyl sulfate/polyacrylamide gel electrophoretic separation, the extracted proteins were transferred on to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and then cultured with the primary antibodies against caspase-3 (Abcam), cleaved caspase-3 (Abcam), TLR4 (Abcam), p-TAK1 (Abcam), p-IKK-α/β (Abcam), p-IκBα (Abcam), NF-κB p65 (Abcam) and β-actin (Abcam) at 4°C overnight. Afterward, the membranes were immunoblotted with a secondary antibody conjugated to horseradish peroxidase (Abcam) for 1.5 h at room temperature. The immunoblots were detected by enhanced chemiluminescence from Pierce (Rockford, IL, U.S.A.) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.).

hDPCs were lysed using RIPA lysis buffer. Total protein was measured using a BCA Protein Assay Kit (Beyotime, Haimen, China). Thirty micrograms of protein was separated by electrophoresis on 8% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) with transfer buffer containing 10% methanol. After blocking with TBST containing 5% nonfat milk at room temperature for 1 h, the membranes were probed overnight at 4°C with the primary antibodies (1:2000) including IKKα/β, p-IKKα/β, IκBα, p-IκBα, p65, p-p65, p38, p-p38, ERK1/2, p-ERK1/2, JNK, p-JNK (CST, USA) and GAPDH (Abcam, UK). Subsequently, the membranes were incubated for 1 h with secondary antibodies at a dilution of 1:2000 (CST, USA) at room temperature. After the membranes were thoroughly washed with TBST buffer, bands with target proteins were visualized with enhanced chemiluminescence reagents (Millipore, USA) and observed using an ImageQuant LAS 4000 mini system (GE Healthcare Life Sciences, USA). The blots were quantified and normalized using the ImageJ 1.47 software program (National Institutes of Health, MD, USA).

For immunohistochemistry analysis, liver sections were dewaxed and placed in 1x citrate buffer (ThermoFisher Scientific) for high‐pressure antigen repair over 5 minutes. The sections were then allowed to cool naturally, before moving on to blocking procedures. Subsequently, the sections were incubated with a p‐NF‐κBp65 (1:100; Abcam) or an p‐IKKα/β (1:100, Abcam) antibody. The next day, after incubation with respective secondary antibodies, the sections were coloured with the 3, 3'diaminobenzidine (DAB) reagent, red dyed with haematoxylin, dehydrated and dried with ethanol, made transparent with xylene and sealed with neutral gum. These sections were then observed and imaged under the BX51 optical light microscope (Olympus).

LPS (Escherichia coli 055:B5) was provided by Sigma (St. Louis, United States). ML385 was purchased from Med Chem Express (MCE, United States). TNF-α and IL-1β ELISA kits were obtained from Absin (Shanghai, China) and Cusabio (Wuhan, China), respectively. Myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) commercial kits were purchased from Jiancheng Corporation (Nanjing, China). NADPH oxidase activity commercial kit was supplied by Genmed Company (Shanghai, China). The ROS detection kit, BCA Protein Assay Kit, and 2′7′-dichloro-dihydro-fluorescein diacetate (DCFH-DA) were provided by the Beyotime Company (Shanghai, China). Antibodies against Poldip2, Nox4, Nrf2, Heme Oxygenase-1 (HO-1), phosphorylated IκB kinase α/β (p-IKKα/β), IKKα, phosphorylated IκBα (p-IκBα), IκB-α, p-P65, and P65 were obtained from Abcam (Cambridge, United States) or Novus (Littleton, United States). Antibody against β-actin was provided by Proteintech Corporation (Wuhan, China).

Analysis of the proteins extracted from ankle joints by western blotting was performed using standard methods. Equivalent amounts of protein from each sample were separated in 10% SDS-polyacrylamide gel and blotted onto a PVDF membrane. The membrane was then blocked with 5% milk (BD, Sparks, MD, USA); incubated with antibodies against OPG, RANKL, p-p65, p-IKKα/β, IκBα, and β-actin (Abcam, Cambridge, UK) overnight; and then hybridized with HRP-conjugated secondary antibody for 1 h. The immunoreactive bands were visualized using an ECL system (CLINX, Shanghai, China). The relative intensities of bands were quantified using Image J.

Total protein was extracted from kidney and mouse NRK-52E cells using the RIPA cell lysis buffer system (Cell Signaling Technology, USA), supplemented with phosphatase inhibitors, and was quantified by BCA-protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). An aliquot (20 μg) of the proteins was separated by 10% SDS-PAGE for protein electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). Following blocking in 5% nonfat dry milk for 1 h at room temperature, the membrane was incubated at 4°C overnight with primary antibodies. The following antibodies were used: mouse RIPK1, p-RIPK3/RIPK3, p-MLKL/MLKL, IL-1β, anti-active caspase-3, phosphor-inhibitor κB α (p-IκBα), phosphor-inhibitor of kappa B kinase α/β (p-IKKα/β), GAPDH (all antibodies dilution ratios are 1 : 1000; Abcam, Cambridge, UK), and rabbit p-RIPK1. After washing three times with phosphate-buffered saline with Tween-20 (PBST), the membrane was incubated with HRP-conjugated secondary antibody (1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Bands were quantified using ImageJ software and normalized to GAPDH.

DCZ0805 was synthesized by the Shanghai Institute of Materia Medica (Chinese Academy of Sciences, Shanghai, China). It was dissolved in dimethyl-sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 100 mM, stored at − 20 °C and diluted at desired concentrations into each well plate with cell suspension. The Cell Counting Kit-8 (CCK-8) was obtained from Yeasen (Shanghai, China), the Annexin-V/propidium iodide (PI) apoptosis detection kit was purchased from BD Pharmingen (Franklin Lakes, USA), and Z-VAD-FMK was obtained from Selleck Chemicals (Houston, USA). Recombinant human TNF-α was obtained from R&D Systems (Minneapolis, MN, USA). Antibodies against cleaved caspase-8, caspase-3, and β-actin were purchased from Cell Signaling Technology (Beverly, USA). Antibodies against CDK4, CDK6, cyclin D1, Bcl-2, Bcl-xL, Bax, caspase-9, IκBα, p-IκBα, p-IKKα/β, NF-κB-p65 and p-NF-κB-p65 were obtained from Abcam (Cambridge, MA, USA).