Collagen coated dishes

Mouse primary myoblasts were isolated from 2 – 4 day old C57/Bl6 mice and cultured in proliferation media as previously described [23 (link)]. To induce differentiation, the proliferation media was removed, the cells were washed twice with PBS, and the media was replaced with 10ml of differentiation media, as previously described [23 (link), 24 (link)]. All cells were grown on collagen-coated dishes (Becton Dickinson Labware, Bedford, MA), were passage-matched, were not used past passage nine, and were not allowed to grow past approximately 80% confluency.
Mouse primary myoblasts were stably transduced as previously described [23 (link), 37 (link)] with the MSCV-IRES-puromycin empty vector or vector containing FLAG epitope-tagged PAX3-FOXO1 (FLAG-PAX3-FOXO1) [21 (link), 22 (link)]. Three days post-transduction, cells were selected using puromycin, as previously described [22 (link)]. The stably transduced cells were harvested and pooled from three independent transductions to create a single population that express each construct.

Primary human hepatocytes (PHH) (provided by Prof. M. Rivoire) were prepared from HBV, HCV and HIV negative adult patients undergoing lobectomy or segmental liver resection for medically required purposes unrelated to this research program. PHH were prepared and cultured as described elsewhere [45 (link)]. Briefly, the cells are plated overnight in collagen-coated dishes (BD Biosciences) at 2×10⁵ cells/cm² in William’s medium (Life Technologies) supplemented with 10% FetalClone II (Thermo Scientific), 1% penicillin/streptomicine and 1% glutamine (Life Technologies), as well as 5 μg/ml Insulin and 5×10^-7 M hydrocortisone hemisuccinate (Sigma Aldrich). After 24 h PHH are extensively washed in serum-free medium and kept in serum-free medium for one more day to counter select the growth of contaminating fibroblast and endothelial cells, and infected 48 hours after plating with HBV virus (i.e. inoculum) produced in HepG2.2.15 cells [46 (link)].

The kidney cortex was dissected and the minced specimens were digested with Hank’s balanced salt solution with 3 mg/mL collagenase (Sigma‒Aldrich, St. Louis, MO) and were incubated at 37 °C for 1 h. The cells were washed with phosphate buffered saline (PBS) through a serial sieving, and centrifuged at 500 × g for 5 min. The cells were incubated in DMEM/F12 (Lonza, Walkersville, MD) for 4–6 h and floating tubular cells in the media were collected and cultured on collagen-coated dishes (BD Biosciences, San Jose, CA) until epithelial cell colonies were established. To identify tubular epithelial cells, a fluorescence-activated Cell Sorting Calibur instrument (BD Biosciences) with FITC-labeled anti-AQP1 staining (Abcam, Cambridge, MA) was used at 4 °C for 30 min^{12 (link),13 (link)}.

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.