Dimethyl sulfoxide (dmso)

The THP-1 cells from the above-mentioned NF-κB assay were used to establish the viability of the monocytes after exposure to an increasing dose of zymosan or zymosan and TCP-25. In brief, 20 μl of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (5 mg/ml) were added to the cells and incubated for 90 min at 37°C. MTT is a yellow tetrazolium salt that is converted to insoluble purple formazan crystals by metabolically active cells, which are used as an indicator. At the end of incubation, the supernatant in the wells was carefully removed, and the formazan-formed crystals were dissolved in 100 μl of dimethyl sulfoxide (Duchefa Biochemie, Haarlem, Netherlands). The plate was then incubated at RT for 30 min on a shaker at low speed to allow for the solubilization of all crystals. The absorbance at 550 nm was then measured. A positive control was obtained by adding, to the untreated cells, a lysis buffer (Thermo Scientific, Rockford, IL, USA) and incubating at 37°C for 40 min before adding MTT. The absorbance value for untreated cells was considered to be 100%, and values for other treatments are shown in comparison to the untreated cells.

Cells were plated on flat‐bottom 96‐well culture plates (1 × 10⁴ cells per well) and incubated for 4 h with 0.5 mg·mL⁻¹ thiazolyl blue tetrazolium bromide (Sigma‐Aldrich). The resulting methylthiazole tetrazolium formazan crystals were treated with dimethyl sulfoxide (Duchefa Biochemie, Haarlem, The Netherlands). The absorbance of the sample at 540 nm was measured using a microplate reader (BioTek Instruments, Winooski, VT, USA).

Fetal bovine serum (FBS), minimum essential medium alpha medium (α-MEM), penicillin-streptomycin, and trypsin-EDTA were purchased from GIBCO (Grand Island, NY, USA). Dulbecco's Modified Eagle medium (DMEM) was obtained from Thermo Fisher Scientific (Pittsburgh, PA, USA). Cordycepin, alizarin red S, an Alkaline Phosphatase Assay Kit, and TRAP Staining Kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). RANKL was purchased from R&D systems (Minneapolis, MN, USA). 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from DUCHEFA Biochemie (Haarlem, The Netherlands). Mouse antibodies BMP-2, Runx-2, Osterix, Osteopontin, and sRANKL were purchased from Abcam (Cambridge, UK). Antibodies of mouse TRAF6, cFOS, phospho-cFOS, NFATc1, cathepsin K, p38 MAPK, phospho-p38 MAPK, ERK1/2, phospho-ERK1/2, JNK, and phosphor-JNK were provided by Cell Signaling Technology (Beverly, MA, USA). The anti-mouse HRP-conjugated secondary antibody and anti-rabbit HRP-conjugated secondary antibody were purchased from Enzo Biochem (Farmingdale, NY, USA). TOPreal™ qPCR 2X PreMIX and M-MLV cDNA Synthesis Kit were obtained from Enzynomics (Dajeon, KOR). PCR primers (OSCAR, Ctsk1, NFATc1, GADPH) were purchased from Qiagen (Hilden, DEU).

Cells (2 × 10⁴ cells/mL) were stabilized overnight on a 96-well plate and treated with WCSC (0, 0.25, 0.5, or 1%) for 24 h. The MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and the cells were incubated for 4 h at 37 °C. Then, formazan crystals were dissolved with dimethyl sulfoxide (Duchefa Biochemie, Haarlem, RV, Nederland), and the absorbance was measured at 540 nm using a multimode microplate reader (BioTek, Winooski, VT, USA).

Unless stated otherwise, materials were obtained from the following manufacturers: Duchefa Biochemie (Haarlem, The Netherlands): dimethylsulfoxide (>99.9 atom % D); Ecos-1 (Moscow, Russia): n-hexane (analytical grade), ethyl acetate (analytical grade), methylene chloride (analytical grade); Honeywell (Seelze, Germany): acetonitrile (>99.9%, LC-MS grade). All other chemicals were purchased from Merck KGaA (Darmstadt, Germany). Water was purified in house with a water conditioning and purification system, the GenPure Pro UV-TOC system (resistance 18 mΩ/cm, Thermo Fisher Scientific, Langenselbold, Germany).

TNBC cell lines, MDA-MB-231 (HTB-22), HCC1395 (CRL-2324), and HCC1937 (CRL-2336), with different BRCA1 statuses were purchased from the American Type Culture Collection (Rockville, MD, USA). Cell lines were grown in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) and Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 10,000 U/mL penicillin-streptomycin (Thermo Fisher Scientific). The cell lines were cultured at 37 °C in a 5% carbon dioxide (CO₂) incubator. Sub-culturing was performed when the cell confluence reached approximately 85–90% in a 100 mm dish (Corning, New York, NY, USA). Cells were cryopreserved in cell freezing medium containing 90% FBS + 10% dimethyl sulfoxide (Duchefa Biochemie, Amsterdam, The Netherlands).