Nystatin

The following reagents were used. Cys-specific chemical crosslinkers: BMH and TMEA (Thermo Scientific), o-PDM and p-PDM (Sigma) and Bis-MTS (methanethiosulfonate) reagents (M1M through M17M) (Toronto Research Chemicals). Detergents: Brij L23, Brij 97 and Brij 99 (Sigma), Triton-X-100, NP-40 and Tween-20 (MP Biochemical) and Digitonin (Sigma and Calbiochem). o-phenanthroline (Wako). Polyene antibiotics: nystatin (Wako) and natamycin (Fluka). Other chemicals were purchased from Sigma, Wako Pure Chemical, Nacalai Tesque and BD.

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.