Glutamine

Oligodendrocytes were prepared as previously described [25 (link)]. Briefly, primary cultures of glial cells were prepared from the brains of newborn Wistar rats and oligodendrocytes were mechanically removed by shaking after 10–14 days in culture. Oligodendrocyte precursor cells were re-plated (1.2x10⁶ cells/6 cm dish) on poly-L-lysine (PLL)-coated culture dishes (1.2x10⁶ cells/6 cm dish) supplemented with glass cover slips (Fisher Scientific, Schwerte, Germany). Cells were grown in serum-free DMEM (Gibco/BRL, Grand Island, NY, USA), supplemented with 2 mM glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml sodium selenite (Roche Diagnostics, Mannheim, Germany) at 10% CO₂. Two hours after seeding, when cells were attached to the culture dishes, medium was replaced and recombinant human α-Syn (10 μg/ml, prepared as previously described, [29 (link)]) was added. Cells were incubated for 3–6 days as indicated.

RCC cells were grown in serum-free medium supplemented with 0.1% BSA (Roche) and 2 mM glutamine (MilliporeSigma) for 24 hours prior to treatment with either TGF-β (1 ng/mL, Calbiochem) or 10 µM TGF-β inhibitors (SB431542, Sigma-Aldrich; LY2109761 and LY364947, Selleckchem) for the indicated times. HK2 cells were cultured with 3% FBS prior to exposure with serum-free medium supplemented with 0.1% BSA (Roche) and 2 mM glutamine for 24 hours, followed by treatment with TGF-β (1 ng/mL).