Rat osteoblast-like UMR106 cells (CRL-1661; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 25 mM glucose and 1 mM pyruvate (Gibco, Life Technologies, Thermo Scientific, Darmstadt, Germany), supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Life Technologies) at 5% CO2 and 37 °C. Serum depletion was accomplished for 24 h or 48 h by incubating the cells in culture medium with 1% or 0% FBS and additional 10 nM 1,25(OH)2D3 (Tocris, Bioscience, Bristol, UK) to enhance Fgf23 expression [40 (link)]. Cells were seeded into 6-well plates (Greiner Bio-One, Frickenhausen, Germany) for 24 h. Subsequently, cisplatin, PAC-1 or doxorubicin (all from Tocris Bioscience) were added at the indicated concentrations for 24 or 48 h or the FBS concentration was reduced as described above. Il-6 signaling was blocked through gp130 inhibitor SC144 (1 µM, Tocris Bioscience). NFκB inhibitors withaferin A (Tocris Bioscience) and wogonin (Merck, Darmstadt, Germany) were used at concentration of 500 nM and 100 µM, respectively, where indicated.
To study cell proliferation, cells were trypsinized after 24 h or 48 h, respectively, and counted on a Neubauer hemocytometer.
To study cell proliferation, cells were trypsinized after 24 h or 48 h, respectively, and counted on a Neubauer hemocytometer.
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