Dmem f12 medium

Peripheral blood cells from two patients with the MAPT R406W mutation were obtained (Ikeuchi et al., 2011 ), which were maintained in T cell medium, consisting of KBM502 medium (Kohjin Bio, Saitama, Japan) supplemented with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific, Waltham, MA, USA).
iPSCs were maintained on irradiated mouse embryonic fibroblasts or SNL 76/7 feeder cells in iPSC medium, consisting of DMEM/F12 medium (Wako, Osaka, Japan) supplemented with 0.08 mM MEM-Non Essential Amino Acid solution (Sigma-Aldrich, St. Louis, MO, USA), 2 mM L-glutamine, 20% (v/v) Knockout Serum Replacement (Thermo Fisher Scientific), 80 U/mL penicillin, 80 μg/μL streptomycin, 0.1 mM 2-mercaptoethanol, and 10 ng/mL basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA). Feeder-free iPSCs were maintained on culture dishes coated with 0.25–0.5 μg/μL iMatrix-511 (Laminin-511E8) (Wako) in StemFit AK02N medium (Ajinomoto, Tokyo, Japan). Medium was changed every day for iPSCs on feeder and every other day for feeder-free iPSCs.

We described the detailed information and history how the tissues of Boninflying fox we obtained in the section of Ethics Statement. We collected the tissues of 5^th finger of the right-wing and skin and transferred into the National Institute of Environmental Studies in Tsukuba. We maintained the primary cells in the DMEM/F12 medium (Wako Chemical, Osaka, Japan) containing 10% fetal bovine serum (FBS) and 1 X antibiotics mixture (Nakarai Tesque, Kyoto, Japan). We preserved the expanded cells in liquid nitrogen tank with 10% dimethyl sulfoxide (DMSO) medium until use. We carried out the surgical procedure and handling based on the Act on Welfare and Management of Animals (Act No. 105 of October 1, 1973).

SV40 MES13, a mouse mesangial cell line, was obtained from American Type Culture Collection (Manassas, VA) and was cultured in DMEM/F12 medium (Wako, Tokyo, Japan) with 14 mM HEPES (Nacalai tesque, Kyoto, Japan), 5% fetal bovine serum (Life Technologies, Grand Island, NY), 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich, Munich, Germany). Cells were incubated in a humidified incubator with 5% CO₂ at 37 °C. Cells were serum-starved for 24 h before IS stimulation experiments. We used 50 mM Tris-HCl buffer as a vehicle control because of the IS solvent.
(P)RR siRNA (L-063641-01-0005, Thermo Scientific, Waltham, MA) or non-targeting control siRNA (D-001810-10-20, Thermo Scientific) were transfected into SV40 MES13 cells (final concentration, 10 nM) by Lipofectamin2000 (Thermo Scientific) according to the manufacturer’s protocol.
To assess the effects of IS or siRNA on mesangial cell growth and viability, the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (10009365; Cayman Chemical Co., Ann Arbor, MI) was performed according to the manufacturer’s protocol.

HK-2 cells, a human-derived PT cell line, were purchased from ATCC (Manassas, VA, #CRL-2190). The cells were cultured in D-MEM/F12 medium (Wako, Osaka, Japan) containing 10% FBS and penicillin/streptomycin. To stimulate gluconeogenesis, confluent HK-2 cells were cultured in conditions of increased oxygen supply by shaking at 60 rpm. The cells were treated with Na BHB in serum-free HBSS (+) for 3 h. Total RNA was extracted using RNeasy Mini kit (Qiagen, MD) and cDNA was synthesized for real-time qRT-PCR analysis. siRNA against human CEBPB was generated by Ambion (Thermo Fischer Inc. MA). Scramble or CEBPB siRNA was transfected using Lipofectamine RNAiMAX reagent (Thermo Fischer Inc. MA) following the manufacture's protocol. Seventy-two hours after transfection, total RNA was extracted for qRT-PCR analysis. For measurement of glucose production, cells were cultured with Krebs-Ringer-buffer (KRB) (135 mM NaCl, 3.6 mM KCl, 2 mM NaHCO₃, 0.5 mM NaH₂PO₄, 0.5 mM MgCl₂, 1.5 mM CaCl₂, and 10 mM Hepes, pH7.4). After Na BHB treatment, the supernatant was collected for measurement of glucose, and the cells were subjected to NaOH lysis for protein extraction. The glucose concentration in medium was measured by Glucose-Glo assay (Promega Corp., WI). The values were normalized by the protein concentration.

SH-SY5Y cells (American Type Culture Collection, CRL-2266) were maintained in DMEM/F-12 medium (Wako Pure Chemical Industries) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO₂. HL-60 cells were grown in RPMI 1640 medium (Wako Pure Chemical Industries) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO₂. Cells were plated onto 35 mm glass-bottom culture dishes (MatTek) before transfection. Transient transfection was carried out using FuGENE HD (Promega). All experiments were performed 2 to 3 days after transfection.

Epithelial barrier function was evaluated by TER analyses. HCE-T cells were seeded at a density of 5,000 cells per well into Transwell plates containing a 0.4 μm pore polyester membrane insert (Corning Inc., Cat# 3470). After 24 h, the medium was replaced with fresh DMEM/F-12 medium (FUJIFILM-Wako, Osaka, Japan) containing 1% FBS with or without either 5% CSE or 5% CSE plus 1 mM NAC. The TER values were measured every 24 h until harvest using an epithelial volt-ohm meter (Millicell ERS-2; Merck Millipore) and a cup-shaped electrode (Endohm-6; World Precision Instruments, Sarasota, FL, USA).