Heat inactivated fetal bovine serum

RAW264.7 and BV2 cells, obtained from ATCC, were cultured in RPMI 1640 and Dulbecco’s Modified Eagle Medium (Nakalai Tesque, Kyoto, Japan), respectively, containing 10% heat-inactivated fetal bovine serum (Sigma, Burlington, MA, USA) and 1% penicillin–streptomycin solution (Nakalai Tesque) in 5% CO₂ at 37 °C. ASK1 KO RAW264.7 cells were established in our previous study^{11 (link)}. HEK293A cells obtained from Thermo Fisher Scientific (Waltham, MA, USA) were cultured in Dulbecco’s Modified Eagle Medium (Nakalai Tesque) containing 5% heat-inactivated fetal bovine serum (Sigma, Burlington, MA, USA) and 1% penicillin–streptomycin solution (Nakalai Tesque) in 5% CO₂ at 37 °C.

Human foreskin fibroblasts (hFSF) were acquired from ATCC (SCRC-1041^TM) Manassas, Virginia, USA. The cells were cultured in DMEM (Thermo Fisher, Waltham, MA, USA) with 15% heat-inactivated fetal bovine serum (Merck, Darmstadt, Germany), 1% non-essential amino acids (Merck, Darmstadt, Germany), 1% L- glutamine (Merck), 1% sodium pyruvate (Merck, Darmstadt, Germany) and 0.5% Pen/Strep (Merck, Darmstadt, Germany). Dox and MT were acquired from Merck (Darmstadt, Germany). FuB was acquired from Cayman chemical (Ann Arbor, MI, USA).
Solvent control (solvent co) for Dox and MT was H₂O. Solvent co for FuB was DMSO. Results were normalized to solvent co (H₂O) or otherwise indicated. Transfection results were normalized to FLAG-TC, siRNA control, or otherwise indicated.

HK-2 and mTEC, kindly provided by Prof. Huiyao Lan, were cultured in DMEM/F-12 (HyClone, Logan, UT, USA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (Merck Millipore, Darmstadt, Germany) at 37 °C in a humidified incubator under 5% CO₂. Cells were treated with cisplatin (5 μmol/l, 24h) with/without 0.1 μg/ml of BMP-7 for 24 h and then harvested for RNA isolation or used to determine apoptosis. To determine CP regulation of BMP-7 expression, cells were treated with vehicle or specific inhibitor for 0.1 μmol/l of TSA or 1 mmol/l of VPA for 24 h. Cells were harvested and RNA was used for HDAC2 expression studies by real-time PCR. To determine the effect of BMP-7 knockdown on TSA-induced suppression of cisplatin-mediated epithelial cell apoptosis, siRNA specific to BMP-7 was transfected (50 nmol/l). Twenty-four hours after transfection, cells were treated with/without cisplatin and TSA or VPA for 24 h, and then cells were harvested to quantify apoptosis by flow cytometry. At 80% confluency, cells were treated with cisplatin with/without 0.1 μg/ml of BMP-7 or 0.1 μmol/l of TSA or 1 mmol/l of VPA for 24 h. Cells and supernatants were harvested and subjected to cytokine and gene expression analysis.