Recombinant mouse ifn γ

Human primary mesangial cells (HMCs, ScienCell Research Laboratories Inc., Basel, Switzerland) were cultured in Mesangial Cell Medium (ScienCell Research Laboratories Inc.). Mouse mesangial cells (MMCs; ATCC, Manassas, VA, USA) were derived from glomerular explants of SV40 transgenic mice on the C57BL/6 background [14 (link)] and were cultured in Dulbecco's modified Eagle's medium/Ham's F12 medium (3 : 1 mixture) (ATCC) with 5% fetal bovine serum (FBS) (HyClone Laboratories Inc., South Logan, UT, USA). JAWSII immature mouse DCs (originating from the C57BL/6 mouse strain) (ATCC) were cultured in Alpha Minimum Essential Medium with 20% FBS and 5 ng/ml murine GM-CSF (PeproTech, Rocky Hill, NJ, USA) [15 ]. All the cells were grown in a humidified atmosphere (5% CO₂) at 37°C. The HMCs were stimulated with 50 ng/ml human IFN-γ (Sigma-Aldrich, St. Louis, MO, USA) for 48 h [16 (link)]. The MMCs were stimulated with 50 ng/ml recombinant mouse IFN-γ (Sigma-Aldrich) for 48 h, and the JAWSII cells were stimulated with 2 μg/ml lipopolysaccharide (LPS) (from Escherichia coli O111:B4, Sigma-Aldrich) for 48 h.

Conditionally immortalized mouse podocytes were cultured on BD BioCoat Collagen I plates (BD Biosciences, NJ, USA) at 33 °C in the presence of 20 U ml⁻¹ mouse recombinant IFN-γ (Sigma, St Louis, MO, USA) to enhance expression of a thermosensitive T antigen27 (link). Mouse podocyte cell lines are routinely characterized in the lab based on morphology and gene expression patterns. Cells for all experiments reported herein were between passage 4–12. Cells were confirmed to be free of mycoplasma contamination. To induce differentiation, podocytes were maintained at 37 °C without IFN-γ for 10–12 days. Podocytes prepared for experiments involving high glucose (HG, 25 mM) conditions were serum deprived for 24 h before addition of HG. Likewise, control cells were serum deprived and cultured with normal glucose, NG, (5 mM). Cell culture experiments were repeated at least three independent times.

Conditionally immortalized mouse podocytes were a kind gift from Jochen Reiser (Rush University, Chicago, IL). In brief, cells were cultured at 33°C in RPMI (Corning) containing 10% FBS (GenDepot), antibiotic antimycotic solution (Corning), and 20 U/ml mouse recombinant IFN-γ (Sigma) to enhance expression of a thermosensitive T antigen. The cells were differentiated at 37°C in DMEM (Corning) supplemented with 5% FBS and antibiotic antimycotic solution without IFN-γ on collagen type I (Gibco) coated dishes for 7–12 days. For imaging, differentiated cells were trypsinized, dissociated, and plated onto collagen type I coated coverslips. Podocytes prepared for experiments involving high glucose conditions (HG, 25mM) were serum deprived for 24 hours prior to addition of HG. Control cells were cultured with normal glucose (NG, 5.5mM). For primary cultured podocytes, isolated podocytes from WT and Ndufs4^podTg mice were cultured with DMEM containing NG, 5% FBS, and antibiotic antimycotic solution, while those from Ins2^Akita/+ and Ins2^Akita/+;Ndufs4^podTg mice were cultured with DMEM containing HG, 5% FBS, and antibiotic antimycotic solution just before each experiment.