Recombinant mouse interferon γ

Conditionally immortalized mouse podocytes, stably expressing functional human AngII type 1 receptor (AT1R+), and control cells transfected with an empty vector (AT1R-) were a generous gift of Dr. Hsiang-Hao Hsu, Department of Medicine D, University Hospital Muenster, Germany (24). The cells were cultured in RPMI 1640 at 33°C, in the presence of 10% fetal bovine serum (FBS), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10 U/ml mouse recombinant γ-interferon (Sigma-Aldrich, Poland). Differentiation was induced by shifting the cells to 37°C. Cell culture was continued for an additional 10–14 days without γ-interferon and with the concentration of FBS reduced to 5%. To maintain stable cell transfection, all culture media contained 75 μg/ml geneticin (G418, Sigma-Aldrich, Poland). All experiments were performed with differentiated podocytes that were cultured for 5 days after initiation by switching the cells to experimental media containing D-glucose at 5.6 mmol/L (normal glucose, NG) or 30 mmol/L (high glucose, HG). The media were prepared using RPMI 1640 without glucose (Sigma-Aldrich, Poland) with osmolarity adjusted by the addition of mannitol. NG and HG media were supplemented with 5% FBS, G418, and antibiotics, as indicated above. Cell viability was assessed by measuring lactate dehydrogenase (LDH) levels as described previously (25 (link)), which was not <87%.

Conditionally immortalized mouse podocytes, originally from Dr. Peter Mundel (Division of Nephrology, Massachusetts General Hospital, Harvard University), were kindly provided by Prof. Niansong Wang (Shanghai Sixth People’s Hospital, China). The podocytes were cultured as previously described (Mundel et al., 1997 (link)). Briefly, the podocytes were cultured at 33°C in RPMI1640 medium (Gibco, United States) with 10% fetal bovine serum (Gibco, United States), 100 U/ml penicillin-streptomycin (Gibco, United States), and 30 U/ml recombinant mouse interferon-γ (Sigma, United States). To induce podocyte differentiation, the cells were cultured at 37°C in medium lacking interferon-γ for 10–14 days. The methods of verifying phenotype after differentiation were provided in Supplementary Material.

A conditionally immortalized murine podocytes were kindly provided by Dr. Peter Mundel (Mount Sinai School of Medicine, New York). Podocytes were maintained in RPMI 1640 medium (HyClone, USA) containing 10% heat-inactivated fetal calf serum (Gibco, USA), 100 U/mL penicillin G, and 100 μg/mL streptomycin in an incubator with 5% CO₂. During podocyte proliferation, the medium was mixed with 10 U/mL recombinant mouse interferon-γ (Sigma, USA), and the cells were maintained at 33°C. Then podocytes were cultured at 37°C to induce differentiation without interferon-γ for 10–14 days. 15–25 passages of podocytes were used in the following experiments.
Cells were cultured in serum-free RPMI 1640 for at least 8 h and pretreated with 5 μmol/L FTY720 (Cayman Chemicals, Ann Arbor, MI, USA) for 30 min followed by treatment with 2 μmol/L S1P (Sigma Chemical Co.) for 24 h.

The mouse podocyte cell line MPC5 used in our study was first established by Professor Peter Mundel et al. [26 (link)] in 1997, and it is widely used in many podocyte studies [26 (link), 27 (link)]. The culture method for MPC5 podocytes was similar to the originally described method. Briefly, podocyte proliferation occurred in a 33°C incubator using RPMI 1640 proliferation medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin-streptomycin (Gibco, USA) and 10 U/ml recombinant mouse interferon-γ (Sigma, USA). When the podocytes reached 60%-80% confluence, they were transferred to a 37°C incubator for differentiation for 10–14 days with RPMI 1640 medium in the absence of interferon-γ. The calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO (ALLN) (Santa Cruz Biotechnology, USA) was dissolved in dimethyl sulfoxide (DMSO) (GeneChem, China), and the appropriate amount of DMSO was added to each control sample. PAN (Sigma, USA) was dissolved in water. Podocytes were treated with different concentrations of PAN (25, 50, 75, 100, or 125 μg/ml) for 24 h, and 75 μg/ml was selected as the appropriate concentration for subsequent studies, including migration assays, F-actin staining, and calpain inhibition experiments.

The conditionally immortalized mouse podocyte cell line MPC5 used in our study was a kind gift from Professor Peter Mundel. MPC5 was firstly established in 1997 by Peter Mundel et al. Culture of MPC5 was performed as original described [9] (link). Briefly, the podocytes were cultured at 33°C in RPMI1640 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin-streptomycin (Gibco, USA) and 10 U/ml recombinant mouse interferon-γ (Sigma, USA). To induce podocyte differentiation, the cells were cultured at 37°C in the absence of interferon-γ for 10–14 days before experimental use. Rapamycin (Santa Cruz, USA) and ku0063794 (LC Laboratories, USA) were dissolved in dimethyl sulfoxide (DMSO) (Genechem, China), and the appropriate amount of DMSO was added to each control sample.

Conditionally immortalized murine podocytes were kindly provided by Dr. Peter Mundel (Mount Sinai School of Medicine, New York, NY, USA) and were grown at 33 °C in RPMI-1640 medium (HyClone, USA) containing 10% heat-inactivated fetal calf serum (Gibco, USA), 100 U/ml penicillin G, 100 μg/ml streptomycin, and 10 U/ml recombinant mouse interferon-γ (Sigma, USA) in the presence of 5% CO₂. Then, podocytes were cultured at 37 °C in interferon-γ-free medium for 7–14 days to induce differentiation and some cell groups were pretreated with STI571 (10 μM, 30 min, Enzo Life Sciences, Switzerland), which suppresses the phosphorylation of c-Abl.
COS7 cells were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured in a 37 °C incubator in Dulbecco's modified Eagle's medium (HyClone, USA) containing 10% fetal calf serum (Gibco). When the cells reached approximately 60% confluence, 20 μg/ml Cyt D (Enzo Life Sciences) was added to the medium for 30 min to depolymerize the actin cytoskeleton.