LUHMES cells were grown in a humidified incubator at 5% (v/v) CO2 and 37°C. Plastic cell culture vessels were pre-coated with 50 ng/mL poly-L-ornithine and 1 µg/mL fibronectin (Sigma Aldrich, UK) prepared in distilled water for 3 h. After removal of the coating solution, the culture flasks were washed 3 times with distilled water and air dried before use. Non-differentiated cells were maintained in proliferation medium, consisting of Advanced Dulbecco’s modified Eagle’s medium/F12 supplemented with 1× N-2 supplement (Gibco/Invitrogen, UK), 2 mM L-glutamine (Sigma, UK) and 40 ng/mL recombinant basic fibroblast growth factor (R&D Systems, UK). For differentiation, cells were cultured in proliferation medium for 24 h and were then switched to ‘+/+’ medium for 7 days. The ‘+/+’ medium consisted of Advanced Dulbecco’s modified Eagle’s medium/F12 supplemented with 1× N-2 supplement (Gibco/Invitrogen, UK), 2 mM L-glutamine (Sigma, UK), 1 mM dibutyryl-cAMP (Sigma Aldrich), 1 µg/mL tetracycline (Sigma Aldrich, UK) and 2 ng/mL recombinant human GDNF (R&D Systems, UK).
Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature and were then permeabilised and incubated for 1 h at room temperature in blocking solution (PBS, 1% BSA and 0.2% Triton X-100). Then, the cells were incubated overnight at 4°C with an anti-β3 tubulin antibody (Abcam, UK) in blocking solution. Next, the cells were washed and incubated with Alexa Fluor 594 anti-mouse (Invitrogen, UK; 1:1000 dilution) for 1 h at room temperature, followed by incubation with anti-rabbit Alexa-488 (1:1000) as a secondary antibody for 1 h. Nuclear staining was performed by incubation of the cells with 10 μg/ml Hoechst 33342 (Molecular Probes, UK) for 10 min.
Protein extraction and western blotting
Protein extracts were prepared by scraping the cells in lysis buffer (100 mM KCl, 20 mM HEPES, pH 7.5, 5% (v/v) glycerol, 10 mM EDTA, 0.1% (v/v) Triton X-100, 10 mM DTT, 1 μg/mL leupeptin, 1 μg/mL antipain, 1 μg/mL chymostatin, and 1 μg/mL pepstatin). The cell suspensions were cleared by centrifugation at 21,000 g for 10 min at 4°C, and the protein concentrations of the supernatants were determined by Bradford assay (Bio-Rad, UK). All supernatants were mixed with 4× LDS loading buffer (Invitrogen, UK). For SDS-PAGE, equivalent amounts of proteins were resolved on 4–12% NuPAGE precast gels (Invitrogen, UK), and were transferred onto to PVDF membranes (Millipore). The membranes were blocked with TBS (0.15 M NaCl and 10 mM Tris-HCl, pH 7.5) containing 5% (w/v) dried non-fat milk for 1 h at room temperature and were then probed with the indicated primary antibody, followed by incubation with the appropriate HRP-conjugated secondary antibody. Antibody complexes were visualised using Pierce’s enhanced chemiluminescence system (ECL). The primary antibodies employed for western analysis were anti-αTubulin (Cell Signalling, cat no. 2144), anti-tyrosine hydroxylase (Abcam, cat. no. ab112) and anti-HSP60 (Cell Signalling, cat. no. 4870).
Cell viability assay
Cells were plated in proliferation medium at a density of 50,000 cells per well in 96 well plates and were grown to approximately 70% confluency. The medium was then replaced with proliferation or ‘+/+’ medium, and the cells were further grown for 7 days. Next, they were incubated in the medium in the absence or presence of PQ at several concentrations (1, 10, 100 and 500 µM) for an additional 24 h. Cell viability was analysed by Alamar Blue assay (Invitrogen, UK) according to the manufacturer’s instructions.
Global metabolic profiles were obtained from cells grown in plastic flasks (T175) and harvested with trypsin (5 mL ATV-trypsin, consisting of 138 mM NaCl, 5.4 mM KCl, 6.9 mM NaHCO3, 5.6 mM D-glucose, 0.54 mM EDTA, and 0.5 g/L trypsin from bovine pancreas type-II-S; Sigma, UK). The cells were collected in 50 mL Falcon tubes by adding 40 mL Advanced DMEM/F12 medium and were then centrifuged at 300× g for 5 min at room temperature. Next, the supernatants were removed carefully, and the cell pellets were flash frozen on dry ice and stored at 80°C. Metabolic profiles were obtained using Metabolon Platform (Metabolon Inc., USA) as previously described. Essentially, 5 replicates were analysed for each condition (n = 5).