Lps e coli 026 b6

EFLS (n = 3) cells were cultured with and without 10µg/ml LPS (E. Coli 026:B6, Sigma–Aldrich, Poole, UK) for 6 or 16 h. Culture media was collected, the cells washed using PBS, and lysed in 1× SDS sample‐loading buffer (62.5 mM Tris–HCl, pH 6.8 at 25°C, 2% w/v SDS, 10% glycerol, 50 mM DDT, 0.01% w/v bromophenol blue, or phenol red). Cell lysate and culture media protein concentrations were measured using the Pierce™ 660 Protein Assay (Life Technologies; Carlsbad, CA). Protein (40 µg) was extracted from each culture media sample using StrataClean™ Resin according to manufacturer protocol and eluted by boiling in 15 µl 1× SDS sample buffer. Cell lysate and culture media protein samples were run on SDS‐PAGE gels and blotted onto nitrocellulose membranes, which were then probed using a sheep anti‐mouse ADAMTS5 antibody (a kind gift from Amanda Fosang, Melbourne Australia)14 at a dilution of 1:500 followed by an anti‐goat/sheep IgG‐peroxidase secondary antibody at a dilution of 1:1000 (A9452; Sigma–Aldrich). Antibody localization was visualized using Western Lightening‐Plus chemiluminescence reagent (Perkin Elmer, Beaconsfield, UK), and imaged on a UVP ChemiDoc‐it imaging system. Intensity of bands was quantified using imageJ software.

LPS (E. coli 026:B6) and 1826-CpG DNA (5′-TsCsCsAsTsgsAsCsgsTsTsCsCsTsgsAsCsgsTsT-3′) were purchased from Sigma (St. Louis, MO) and TIB Molbiol (Berlin, Germany), respectively. YB-1 antibodies were obtained from Cell Signaling Technology (Danvers, MA) and Santa Cruz Biotechnology (Santa Cruz, CA). Actinomycin D (Act. D) and Trichloroacetic acid (TCA) were purchased from Sigma.

PUN (purity ≥98%) was obtained from Sigma-Aldrich (St. Louis, USA) and dissolved in phosphate-buffered saline (PBS). PBS was used as the vehicle control. Recombinant RANKL was purchased from R&D Systems (Minneapolis, USA). Minimal essential medium (DMEM/high glucose) and fetal bovine serum (FBS) were purchased from HyClone (Logan, USA). LPS (E. coli 026: B6) was obtained from Sigma-Aldrich. Primary antibodies to phospho-NF-κB p65 (#3033), phospho-IκBα (#2859), NF-κB p65 (#8242), IκBα (#4814), phospho-ERK (#4370), phospho-p38 (#4511), phospho-JNK (#4668), ERK (#4695), p38 (#8690), and JNK (#9252) were purchased from Cell Signaling Technology (Boston, USA). Anti-rabbit and anti-mouse HRP-conjugated secondary antibodies were obtained from Multi Sciences (Shanghai, China). A CCK-8 assay kit was purchased from ApexBio (Boston, USA).

The LPS (E. coli 026:B6) and Griess reagent were obtained from Sigma (St. Louis, MO, USA). Glycyrrhizic acid, liquiritigenin, berberine, baicalin, baicalein, forsythiaside-A and poncirin were also purchased from Sigma. Enzyme-linked immunosorbent assay (ELISA) kits for tumour necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-1β were acquired from Pierce Endogen (Thermo Scientific, Waltham, MA, USA). The PGE₂ ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA). The primary antibodies for the phospho (p)-inhibitor of NF-κB α (I-κBα), p-ERK, p-jun NH₂-terminal kinase (JNK), p-p38, ERK, JNK and p38 antibodies were purchased from Cell Signaling (Danvers, MA, USA). COX-2 and iNOS antibodies were obtained from BD Biosciences (San Jose, CA, USA), while the NF-κB, IκBα, β-actin and lamin A/C antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Fresh bone marrow cells flushed from femurs and tibias were passed through 70-µm cell-strainers. After erythrocytes lysis, BM cells were incubated with supernatants from YTS169, YTS191 (anti-CD4; Therapeutic Immunology Group, Oxford, UK), M5/114, and RA3-3A1, followed by sheep anti-rat Dynabeads^®. After magnetic separation, cells were transferred to 24-well plates in the presence of 20 ng/ml mouse recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF; R&D Systems). On days 2 and 4, medium containing small non-adherent cells was removed and replaced with fresh GM-CSF-containing medium. For maturation, 1 µg/ml LPS (E. coli 026:B6, Sigma) was added on day 6 for the final 12 h.

LPS and IFN-γ treatment: Cells were treated with 100 ng/ml LPS (E. coli 026:B6, Sigma-Aldrich, St. Louis, MO, USA) and 0.1 ng/ml IFN-γ (Sigma-Aldrich) for 6 h or 24 h. Stock solutions of 1 mg/mL LPS in a serum-free culture medium and 10 μg/mL IFN-γ in a serum-containing culture medium, were prepared and stored at −20°C.
MPP+ and rotenone treatment: Cells were treated with 10, 25, 50 and 100 μM MPP+ or 20, 40, 100 and 150 nM rotenone (both from Sigma-Aldrich) for 6 h or 24 h, in the absence or presence of LPS/IFN-γ. Stock solutions of 50 mM MPP+ in milliQ H₂O and 10 mM rotenone in DMSO were freshly prepared on the day of treatment. DMSO in the cell cultures was always below 1/1,000.
Treatments were added directly to the culture medium.