The plates were maintained at 22°C for 5 days. KA count were realized after incubation of 300 μL of KA with or without 15 μL of MFN1032, MFN1030 or V1 (ratio 10%) in SM at 22°C for 5 days. Serial dilutions were plated on Hektoen enteric agar (bioMerieux) at 37°C to select KA. For some assay, 150 μL of MFN1032, MFN1030, V1 (0.5 OD580nm) or 300 μL of KA (1 OD580nm) were plated in SM-agar plates and 2 μL of serial dilution of D. discoideum culture (respectively Capmatinib research buy 1000,100, 10 or 1 D. discoideum per μL)

were spotted on the bacterial layer. The plates were maintained at 22°C for 2 days. Cell culture and infection conditions Macrophage cell line J774A.1 was grown in Dulbecco’s modified Eagle Minimal Essential Medium (DMEM) (Lonza) containing 10% foetal calf serum (FCS) supplemented with 2 mM L-glutamine, 100

μg.mL-1 penicillin, 100 μg.mL-1 streptomycin and 2 mM pyruvic acid. The cells were seeded 20 h before infection in 24-well culture plates at 3 × 105 cells per well. Bacterial strains were grown overnight in LB (NaCl 5 g/l), diluted to 0.08 OD580nm and grown for approximately 4 h more for P. fluorescens and 2 h more for P. aeruginosa to an OD580nm between 1.0 and 1.5. For the cytotoxicity assay, one day before infection, the macrophages were GDC-0941 manufacturer antibiotic starved. The macrophages were infected with bacteria resuspended in 1 ml of DMEM in order to give an MOI (multiplicity of infection) of 5 (15 × 105 bacteria.mL-1). Carnitine palmitoyltransferase II selleck screening library After 4 hours of incubation under controlled atmosphere (37°C, 5% CO2), lactate dehydrogenase (LDH) present in the supernatant was measured in each well using cytotox 96® enzymatic assay (Promega). LDH is a stable cytosolic

enzyme released by eukaryotic cells and is an overall indicator of necrosis. J774A.1 cells exposed to Triton X100 (0.9%) were used as a control of total release (100% LDH release). The background level (0% LDH release) was determined with serum free culture medium. The percentage (%) of total lysis was calculated as follows: , where B (baseline) is a negative control and T (total lysis) is a positive control. X is the OD490nm value of the analysed sample. For in vitro microscopy, macrophages were infected with MFN1032 strain expressing Green Fluorescent Protein (pSMCP2.1 carrying gfp gene), resuspended in 1 ml of DMEM, in order to give an MOI of 10 and incubated for 10 min at 37°C, 5% CO2[37]. The medium was supplemented with 500 ng.mL-1 EtBr, which enters only into dead cells. Infection was followed using an inverted Zeiss (LSM 710) confocal laser-scanning microscope with an oil immersion 63X/1.40 plan-apochromatic objective. Plates were excited with a wavelength of 488 nm for GFP (emission: 493-539 nm) and 514 nm for EtBr (emission 589-797). 3D modelisation and orthographic representation were processed using Zen® 2009 (Zeiss) software and a Kernel of 3×3 (x, y) was applied.