100 mm dishes

Murine RAW264.7 macrophages were subcultured to 70–80% confluence every 2–3 days in 100-mm dishes (Falcon, Bedford, MA, USA) in DMEM supplemented with 10% FBS, and were incubated in a humidified atmosphere containing 5% CO₂ and 95% air at 37 °C. For the experiments, the cells were seeded in 96-well plates for cell cytotoxicity, reactive oxygen species (ROS), and nitric oxide (NO) determination in DMEM containing 10% FBS for 24 h. The day before treatments, all cells were starved in DMEM containing 1% FBS overnight, and then treated with β-CD, tCIN, or CIs with or without 1 µg/mL LPS for further experiments.

PC12 cells derived from pheochromocytoma of the rat adrenal medulla were obtained from the Korean Cell line Bank (Seoul, Korea). PC12 cells were seeded at a density of 2×10⁶ / dish in 100mm dishes (Falcon; Becton-Dickinson, Oxnard, CA) in DMEM (Life Technologies-Invitrogen) with 10% heat-inactivated (56°C for 0.5 h) fetal bovine serum (FBS) and antibiotics at 37°C in a humidified atmosphere of 5% CO2. Cells were sub-cultured twice a week, and only those in the exponential growth phase were used in experiments. PC12 cells were subsequently incubated with 1 μM retinoic acid (Sigma Aldrich, USA) in 10% FBS containing DMEM for 24 hours.

GSCs were seeded in 100 mm dishes (Falcon) and incubated for 24 hours prior to treatment. After treatment, GSCs were collected and lysed in RIPA buffer, and centrifuged at 12 000 × g for 15 min. Supernatants were collected, and the total protein concentration was quantified using the bicinchoninic acid (BCA) assay kit. Equal amounts of proteins (30 μg) were separated by SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature for 2 h, the membranes were incubated with primary antibodies against rabbit anti-active caspase-3, rabbit anti-BAX, rabbit anti-Bcl-2, rabbit antiphospho-AMPK, rabbit anti-AMPK, rabbit antiphospho-acetyl CoA carboxylase (ACC), rabbit anti-ACC, rabbit antiphospho-AKT, rabbit anti-AKT, rabbit antiphospho-mTOR, rabbit anti-mTOR, rabbit antiphospho-4EBP1, rabbit anti-4EBP1, rabbit antiphospho-p70S6K, rabbit anti-p70S6K (All of the above antibodies were procured from Cell Signaling Technology), equal lane loading was confirmed using a monoclonal antibody against β-actin (Sigma-Aldrich). The membranes were washed three times with PBS-T (0.1% (v/v) Triton-X100) buffer for 0.5 hours and incubated with HRP-conjugated secondary antibody for 2 hours. After washing with the PBS-T buffer, the membranes were scanned with the Odyssey Infrared Imaging System (LI-COR).

NRK-52E cells, which are immortalized rat renal tubular epithelial cell lines, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Briefly, the cells were passaged every 3–4 days in 100 mm dishes (Falcon, Bedford, MA, USA) using Dulbecco’s Modified Eagle’s Medium-F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Life Technologies Inc., Gaithersburg, MD, USA), insulin–transferrin–sodium selenite media supplement (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). For experimental use, these cells were incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C for 24 h and sub-cultured at 70%–80% confluence. After being washed extensively with phosphate-buffered saline (PBS), the cells were treated with either 0.1 µg/mL linagliptin (Santa Cruz Biotechnology Inc. Dallas, TX, USA. Cat. # sc-364721, Lot # D1921), 10 μg/mL LPS (lipopolysaccharide from Escherichia coli 0111:B4, Sigma-Aldrich, St. Louis, MO, USA, product #L2630, Lot #095M4163V), or LPS + linagliptin for 2 h. The cells were harvested at the end of treatment for molecular analysis. All cellular experiments were repeated three times for semi-quantitative real-time polymerase chain reaction (RT-PCR) and Western blot analysis.

HUDEP-2 cells and HiDEP-1 cells were grown in Stemline II Hematopoietic Stem Cell Expansion Medium (Sigma) supplemented with 50 ng/mL recombinant stem cell factor (SCF; R&D systems), 3 IU/mL recombinant erythropoietin (EPO; Kyowa Kirin), 1 μM dexamethasone (Dex; Sigma), and 1 μg/mL doxycycline (Sigma). A total of 1 × 10⁵ human CD34⁺ progenitor cells derived from bone marrow or peripheral blood (Lonza) were cultured on 35-mm dishes (BD Falcon) in StemSpan SFEMII medium with 50 ng/mL SCF, 5 IU/mL EPO, and 10 ng /mL interleukin 3 (IL-3; R&D systems). The medium was changed on day 4 to SCF, EPO, and 1 μM Dex, and the cells were transferred to 60-mm dishes (BD Falcon). Seven days after the onset of differentiation, cells were counted and 1 × 10⁶ cells were re-plated on 100-mm dishes (BD Falcon) with SCF, EPO, and Dex. The rat myoblast cell line H9c2, HEK293T, and PLAT-E cells were maintained in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin. All cells were cultured at 37 °C under 5% CO₂.