Traf6

Human serum albumin (HSA) was obtained from Baxter (Deerfield, IL, United States). LPS, D-galactosamine, ALT kit, and AST kit were purchased from Sigma-Aldrich (St Louis, MO, United States). IL-6, IL-22, HMGB1 ELISA kits were purchased from BIKW Co., Ltd. (Beijing, China). Antibodies against Ubiquitin (Cat.3936), TRAF6 (Cat.8028), p38 (Cat.9212), p-p38 (Cat.9216), p65 (Cat.8242), p-p65 (Cat.3033), JNK (Cat.9252), p-JNK (Cat.4668), STAT1 (Cat.14995), and Actin (Cat.3700) were obtained from Cell Signaling Technology (United States). The magnetic RNA-Protein Pull-Down Kit was purchased from Thermo Fisher (United States). Real-time PCR kits were from Takara (Japan). NEAT1 lentivirus, sh-NEAT1 lentivirus, AAV8, and AAV8-NEAT1 were purchased from Genechem (China).

Human recombinant FAM19A5 was purchased from Biovendor (Brno, Czech Republic). Murine recombinant M-CSF and murine RANKL were purchased from Peprotech (Rocky Hill, NJ, USA). LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). WKYMVm and WRW4 were synthesized from Anygen (Gwangju, Korea). Polymyxin B, PTX, and PD98059 were purchased from Calbiochem (San Diego, CA, USA). LY294002 was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA, USA). MK-2206 was purchased from Selleck Chemicals (Houston, TX, USA). Phospho-Akt (Ser473), Akt, phospho-ERK, ERK, RANK, TRAF6, and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). c-fos and NFATc1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

After induction model, THP-1 cell was fixed with 4 % paraformaldehyde for 15 min and then incubated with using 0.25 % Triton X100 for 15 min at room temperature. THP-1 cell was incubated with Pellino1 (1:100, 31,474, Cell Signaling Technology) and TRAF6 (1:100, 8028, Cell Signaling Technology) at 4˚C overnight after blocking with 5 % BSA for 1 h. After washing with PBS for 15 min, THP-1 cell was incubated with goat anti-rabbit IgG-cFL 555 (1:100, sc-362,272, Santa Cruz Biotechnology) or anti-mouse IgG-cFL 488 antibody (1:100, sc-362,267, Santa Cruz Biotechnology) for 2 h at room temperature and stained with DAPI for 15 min and washed with PBS for 15 min. The images of THP-1 cell obtained using a Zeiss Axioplan 2 fluorescent microscope (carl Zeiss AG, Oberkochen, Germany).

Total proteins were isolated from treated human T/C-28a2 cells using the lysis buffer and were quantified using the bicinchoninic acid (BCA) kit (Vazyme, Nanjing, China), followed by being loaded onto the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After separation for 1 h, proteins were transferred to the polyvinylidene fluoride (PVDF) membrane (Bio-Rad, California, USA) and the membrane was incubated with the primary antibody against type 2 collagen (1:500, #ab34712, Abcam, USA), Aggrecan (1:500; #216965, Abcam, USA), TRAF-6 (1:2000; #8028, Cell Signaling Technologies, USA), IκBα (1:3000; #4814, Cell Signaling Technologies, USA), p-IκBα (1:1000, #5209, Cell Signaling Technologies, USA), NF-κB p65 (1:2000; #3036, Cell Signaling Technologies, USA), lamin B1 (1:5000, #13435, Cell Signaling Technologies, USA), and β-actin (1:10,000, #3700, Cell Signaling Technologies, USA). Then, the membrane was incubated with the Horseradish Peroxidase (HRP)- linked goat anti-mouse (1:3000, #7076, Cell Signaling Technologies, USA) or goat anti-rabbit (1: 2000, #7074, Cell Signaling Technologies, USA) secondary antibodies, followed by 3 washes and exposure to ECL solution (Vazyme, Nanjing, China). Lastly, the quantification was conducted on the bands using the Image J software and normalized to the internal control β-actin or lamin B1.

Prostate CAFs were seeded in 6-well plates at a density of 3 × 10⁵ cells/well and were treated with MPSSS at different concentrations (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/ml) for 24 h. Then the cells were lysed with RIPA buffer supplemented with 100 mΜ phenylmethylsulfonyl fluoride (PMSF), 25 μg/ml aprotinin, 1 mM sodium orthovanadate, and 50 nM NaF to obtain the total protein content. The total protein concentrations were determined by the BCA protein assay. Equal amounts of protein samples (30 μg/sample) were separated on 10% SDS polyacrylamide gels under denaturing conditions and were then electrotransferred onto nitrocellulose membranes (GE Healthcare, Milwaukee, WI, U.S.A.) for 70 min at 100 V. Then, the membranes were blocked with 3% BSA in PBS-T (0.1% Tween-20) for 1 h and were incubated overnight at 4°C with the primary antibodies. The primary antibodies used were against α-SMA, phospho-NF-ĸB p65, NF-ĸB p65, phospho-TAK, TAK, phospho-IKKα/β, TRAF6, and MyD88 (all from Cell Signaling Technology, U.S.A.) and were diluted at 1:1000; the primary antibody against GAPDH (Tianjin Sungene Biotech Co., Ltd.) was diluted at 1:2000 and was used as an internal standard.

Primary antibodies for iNOS, COX-2, β-actin, PARP, phospho-PI3K p85, PI3K, Akt, phospho-Akt, NF-κB, phospho-NF-κB, IκB-α, phospho-IκB-α, IKKβ, phospho-IKKα/β, phospho-p38, p38^MAPK, phospho-JNK, JNK, phospho-ERK1/2, ERK1/2, MKK4, MKK6, MyD88, IRAK1, TRAF6, phosphor-TAK1, TAK1, phospho-c-fos, phosphor-c-jun, phosphor-ATF2, IL-1β, TNFα, and horseradish peroxidase-conjugated secondary antibody were from Cell Signaling Technology (Danvers, MA, USA). SB203580, SP600125, and PD98059 were from Sigma-Aldrich (St. Louis, MO). Consensus oligonucleotides for NF-κB and AP-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). γ⁻³² P-Labeled ATP was purchased from ICN (Costa Mesa, CA). A prostaglandin E₂ EIA kit was from Enzo life Sciences (Ann Arbor, MI, USA). Easy Blue™ RNA extraction kit was from iNtRON, Korea. Millipore MILLIPLEX™ Mouse Cytokine/Chemokine enzyme-linked immunosorbent assay kit was from Millipore Corp. (St. Charles, MO). All materials, equipment, and biotinylated marker proteins for gel electrophoresis were from Bio-Rad. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Torilin (98%) was prepared as previously indicated in [26 (link)] and dissolved in dimethyl sulfoxide and freshly diluted in culture media for all experiments.