Insulin transferrin sodium selenite supplement

Primary C57BL/6 renal tubular epithelial cells were isolated as described previously^{28 (link)}. Kidneys were digested using multi-tissue dissociation kit and GentleMacs (Miltenyi, Bergisch Gladbach, Germany), incubated with CD326 (EpCAM) microbeads (Miltenyi) and passed through LS columns. The positive cell fraction was suspended in defined K1 medium: DMEM/F12 medium supplemented with 25 ng/ml epidermal growth factor (Sigma Aldrich, St Louis, MO), 1 ng/ml prostaglandin E₁ (Cayman Chemicals, Ann Arbor, MI), 5 × 10⁻¹¹ M triiodothyronine (Sigma Aldrich), 5 × 10⁻⁸ M hydrocortisone (Sigma-Aldrich), insulin–transferrin–sodium selenite supplement (Sigma Aldrich), 1% penicillin/streptomycin (Thermofisher Scientific, Waltham, MA), 25 mM HEPES (Thermofisher Scientific), and 5% FCS (Thermofisher Scientific) and cultured on collagen-coated dishes (BD Biosciences, Franklin Lakes, NJ).
Cell passages 2–4 were used for experimental work. Primary RTEC cultures were treated for 24 h in the following groups: untreated, LPS only (1 μg/mL, InvivoGen; San Diego, USA), LPS + colchicine (1 μM), LPS + metformin (1 mM). In further experiments, RTEC were subjected to normoxia (FiO₂ 21%) or hypoxia (FiO₂ 1%) for 24 h. All drug treatments were administered 2 h prior to LPS stimulation or hypoxia. Cell lysates were collected for RNA and protein.

Collagenase I, dexamethasone, ascorbate-2-phosphate, pantothenic acid, and insulin-transferrin-sodium selenite supplement were purchased from Sigma Aldrich (St. Quentin Fallavier, France). Trypsin, Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, and phosphate buffered saline (PBS) were provided by Hyclone (Cergy-Pontoise, France). Fetal bovine serum (FBS) for ADSCs was purchased from Gibico (Paris, France). LY294002 was purchased from Cell Signaling Technology (CST, Beverly, MA, USA).

Human kidney cells (HK2) were maintained in DMEM medium (GIBCO, Invitrogen, CA, USA) with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin (PS). HK-2 cell line was originally purchased from ATCC. Primary tubular epithelial cells (pTECs), isolated from the kidney of wild-type or Mlkl-deficient mice kindly provided by James Murphy, Parkville, Australia^{33 (link)}, were maintained in DMEM/F12 medium with 10% FCS, 1% PS, 125 ng/ml prostaglandin E1 (Calbiochem, Germany), 25 ng/ml EGF, 1.8 mg/ml l-thyroxine, 3.38 ng/ml hydrocortisone and 2.5 mg/ml of insulin–transferrin—sodium selenite supplement (all from Sigma-Aldrich, Germany)^{8 (link),34 (link)}. All cells were stimulated with crystals of CaP 1 mg/ml (0.2–1 µm size; rhomboid and prism shape; Santa Cruz Biotechnology, Germany), silica 1 mg/ml (1–1.5 µM size; sphere shape; Alfa aesar, Germany), TiO₂ 0.5 mg/ml (80 nm size; sphere shape; Io-li-Tec, Germany), cholesterol 3 mg/ml (0.2–1 µm size; rhomboid shape; Sigma, Germany), CaOx 1 mg/ml (1–2 µm size; rhomboid and prism shape; Alfa aesar, Germany) and MSU 0.5 mg/ml (needle like shape, 1–2 µm size; Invivogen, Germany). Cells were pretreated with cytochalasin D (10 µM, Sigma, Germany), NSA (5 or 10 µM, Millipore, Germany), dabrafenib (10 µM, Selleckchem, Germany) or Nec-1s (100 µM, Bio-vision, Milpitas, CA, USA) 30 min before exposing to crystals.