Chlorpromazine

Overnight culture of L. monocytogenes strains were deposited onto TG cells at a multiplicity of infection (MOI) of 10 by centrifugation at 150 × g for 10 min at room temperature. To measure the efficiency of bacterial uptake, the infected cells were incubated at 37°C for 30 min, washed once with TS medium, and then incubated in TS medium containing gentamicin (50 µg/ml) for 30 min. The cells were washed thrice with phosphate-buffered saline (PBS) and lysed with cold distilled water. Colony forming units (CFUs) were determined by serial dilution on BHI agar plates. Wortmannin (5 μM, Sigma), Akt 1/2 kinase inhibitor (20 μM, Sigma), nystatin (20 μg/ml, Wako, Osaka, Japan), β-cyclodextrin (5 μg/ml, Wako), chlorpromazine (15 μg/ml, Wako), or amiloride (indicated concentrations, Sigma) was added to the RPMI 1640 medium 1 h before infection. Recombinant IFN-γ (3,000 units/ml, Cedarlane Laboratories, Ontario, Canada) was added to the TS medium 24 h before infection.

N2a cells were seeded at 1.0 × 10⁵ cells/ml in poly-D-lysine-coated 35 mm glass-bottomed dishes (MatTek Corp., Ashford, MA, USA). The medium was replaced with the differentiation medium containing 1 μM FITC-CysC or 300 nM rapamycin at 6 h after the transfection of pcDNA3.3-SOD1 or pAcGFP-N1-SOD1. The cells were further incubated for 24 h. To determine the CysC endocytotic pathway, the cells were treated with 25 μM chlorpromazine (Wako), 5 mg/ml filipin III (Enzo Life Sciences Inc., Farmingdale, NY, USA) or 25 μM 5-(N-Ethyl-N-isopropyl) amiloride (Enzo) for 1 h. The medium was replaced with the differentiation one with or without 1 μM FITC-CysC and incubated for another 1 h. The cells were stained with Lysotracker-Red (Life Technologies) according to the manufacturer's instructions to visualize the lysosomal acidic components. The cells were washed with PBS twice and observed by confocal laser scanning microscopy.