Paraquat

SH-SY5Y (ECACC, Salisbury, Wiltshire, United Kingdom), HCT-15, HCT116, HEK293, HEK293T and MCF-7 cells were cultured in DMEM/F-12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum. To obtain normal human matured neuronal cells in a culture system, ReproNeuro, a neuron progenitor derived from human iPS cells, was purchased from ReproCELL (Yokohama, Japan) and maintained in ReproNeuro maturation medium for 14 days according to the manufacturer’s instructions. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 6-hydroxydopamine hydrobromide (6-OHDA), cadmium chloride (CdCl₂), rotenone, tert-Butylhydroquinone (tBHQ), sulforaphane and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paraquat, caffeine and curcumin were purchased from Wako Chemicals (Osaka, Japan). The antibodies used were as follows: an antibody against PINK1 (rabbit monoclonal, 6946), an antibody against NRF2 (rabbit monoclonal, 12721), horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies, Danvers, MA, USA), and an antibody against Tubulin (mouse monoclonal, T5168; Sigma-Aldrich).

NIH-3T3 or MEF cells were seeded in 96-well plates (5 × 10³ cells/well for NIH-3T3 cells or 1 × 10⁴ cells/well for MEF cells) and incubated at 37°C in 5% CO₂. The next day, cells were treated with paraquat (0.5 mM) (Wako) with or without lactic acid bacteria (1, 10, 100 μg/mL) for 24 h. After treatment, the cells were washed with PBS and the cell proliferation rate was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions.