Pac 1

Madin-Darby Canine Kidney cells (MDCK; CCL-34, ATCC, Manassas, VA, USA) were cultured at 37°C and 5% CO₂ in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12; (Gibco, Life Technologies, Darmstadt, Germany) plus 5% fetal bovine serum (FBS; Gibco), 1% glutamine, and 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco). NRK-52E (CRL-1571, ATCC) cells were cultured in DMEM (Gibco) with 5% newborn calf serum (NBCS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C and 5% CO₂. Human HK-2 cells (CRL-2190, ATCC) were cultured in DMEM with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO₂. For the experiments, cells were first seeded into 6-well plates (Greiner Bio-One, Frickenhausen, Germany) for 24 h. Subsequently, cisplatin, PAC-1, doxorubicin (all from Tocris Bioscience, Bristol, UK), or paclitaxel (MP Biomedicals, Eschwege, Germany) were added for 24 h as indicated. For serum starvation, culture medium was replaced by serum free medium. After 24 h, cells were either trypsinated and counted with a Neubauer hemocytometer or analyzed for RNA isolation. Selective PPARγ inhibitor SR-202 (Biomol, Hamburg, Germany) was added to the culture medium along with cisplatin at 200 μM. Cell culture supernatants were collected and frozen for further use.

Doxycycline hyclate, MG-132, and TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine) were purchased from Sigma-Aldrich (U.S.A.) and PAC-1 was purchased from Tocris Bioscience (U.K.). Antibodies against GFP (B-2), β actin (8H10D10), Fas (CH11), TRPM7 (N74/25), and poly(ADP-ribose) polymerase-1 (PARP-1, SC-7150) were purchased from Santa Cruz Biotechnology (U.S.A.), Cell Signalling Technology (U.S.A.), Millipore (Germany), NeuroMab (U.S.A.), and Santa Cruz Biotechnology (U.S.A.), respectively. DNA oligomers were purchased from Cosmogenetech (ROK). K79 peptide (RVDDAMTPRMWHGLIF) was synthesized by Anygen (ROK).

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.