MitoTEMPO

Immortalized mouse podocyte cell line was a kind gift from Professor Peter Mundel (Harvard Medical School, Charlestown, MA, USA). These cells were grown on collagen I (Catalog # A10483-01, Life Technologies Corporation, Grand Island, NY, USA) coated dishes with low glucose (5.5 mM) Dulbecco's modified Eagles medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin at two different temperatures.^{49 (link), 50 (link)} At 33 °C, cells are allowed to proliferate (permissive condition) in the presence of 20 U/ml mouse recombinant IFN-γ (R & D Systems, Minneapolis, MN, USA). For the induction of differentiation (non-permissive condition), podocytes were thermo-shifted to 37 °C in the absence of IFN-γ for 14 days. Expression of synaptopodin, a specific marker for podocyte differentiation, was gradually increased during culture in non-permissive conditions (37 °C), whereas it was not detected in podocytes maintained under permissive conditions (33 °C with interferon-γ).
Palmitate and oleate were prepared by conjugation with fatty-acid-free BSA. First, sodium palmitate or sodium oleate (100 mM) was dissolved in autoclaved distilled water (DW) at 70 °C for 30 min. BSA (10%) was also dissolved in autoclaved DW at 55 °C for 30 min and filtered. Dissolved palmitate or oleate solution was added dropwise into the BSA solution and stored as a stock (10 mM) at −80 °C until use. KRB solution contained (in mM) 135 NaCl, 3.6 KCl, 2 NaHCO₃, 0.5 NaH₂PO₄, 0.5 MgSO₄, 1.5 CaCl₂, 10 HEPES, 5.5 glucose (pH 7.4, 318 mOsm/kg H₂O). 2′,7′-Dichlorofluorescein diacetate DCF-DA, mitoSox, JC-1 and TMRM were purchased from Molecular Probes (Invitrogen, Grand Island, NY, USA). SSO was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and mitoTEMPO was purchased from Enzo Life Sciences (Enzo Life Sciences, Inc., Farmingdale, NY, USA). Sodium palmitate, sodium oleate, fatty-acid-free BSA, MTT, 4′,6′-diamidino-2-phenylindole (DAPI), edelfosine, DKI R59022, GF109203X and other drugs were purchased from Sigma (St. Louis, MO, USA).

Intracellular infection assays were performed as described in (14 (link)) with some modifications. Cells were seeded at a density of 10⁶ cells per well or 8 × 10⁵ cells per well in a 6-well or 96-well tissue culture plate (Nunc; Thermo Fisher Scientific), respectively, and allowed to attach overnight at 37°C in a 5% CO₂ humidified atmosphere. Cells were infected at a multiplicity of infection (MOI) of 10 for up to 3 hours. Following infection, the medium was aspirated, and the cells were washed three times in PBS and incubated with gentamicin (150 μg/ml) (Sigma-Aldrich) and penicillin G (50 μg/ml) (Sigma-Aldrich) to kill extracellular bacteria and MTX (0.515 μg/ml), or varying concentrations of vancomycin (0.06 to 75 μg/ml) (Sigma-Aldrich) and MTX (0.515 μg/ml), in complete DMEM for 18 to 24 hours to selectively kill extracellular bacteria. The antibiotic-containing medium was then removed, and the cells were washed three times in PBS before addition of 2% Triton X-100 (Sigma-Aldrich) PBS solution to lyse the cells for enumeration of the intracellular bacteria. Variations of this assay included pretreatment of mammalian cells, before bacterial infection, with MTX (0.515 μg/ml) followed by antibiotic treatment only or cotreatment of cells at the time of infection with either MitoTEMPO (80 μM) (Sigma-Aldrich) or pepstatin A (10 μg/ml) (Sigma-Aldrich).

Bacterial growth assays were carried out in complete DMEM as described previously (43 (link)). Two microliters of overnight cultures grown in DMEM was added to 200 μl of medium in a 96-well plate with the indicated concentrations of MTX and/or vancomycin. In some assays, MitoTEMPO was also added to a final concentration of 80 μM. The OD₆₀₀ at the zero time point was established. Bacteria were grown statically in 96-well plates at 37°C for up to 24 hours. Final OD₆₀₀ measurements were acquired using a Tecan M200 microplate reader.