, From day 3, water flasks were supplemented with ampicillin 1 g/liter and with an antibiotic concoction consisting of vancomycin 5 mg/ml, neomycin 10 mg/ml, metronidazole 10 mg/ml, and amphotericin B 0.1 mg/ml, administered by antibiotic gavage daily for 7 days. A gavage volume of 10 ml/kg of body weight was delivered with a stainless steel tube without prior sedation of the mice. From the day following the end of the microbiota-depleting treatment, Briefly, antibiotic treatment started with 3 days of amphotericin B 0.1 mg/ml, administered by gavage daily
13% energy from fat) or HFD (235HF; SAFE, France) (45% energy from fat; cholesterol, 0.17 mg/g), both sterilized by gamma irradiation. The experiment was carried out twice. Mice were weighed weekly, and fresh stool samples were taken up. Mice were killed by cervical dislocation after 1 h of fasting, and blood, liver, and small intestine were recovered and immediately used for measurement of serum parameters (see the supplemental material), histological procedures (jejunum), RNA extraction ,
The m-ICcl2 murine intestinal epithelial cell line was cultured as previously described (28) and seeded on Costar transwell plates with a 0.4-m-pore-size filter (Thermo Fisher) at a density of 3.10 5 /cm 2 . The medium was changed every 2 to 3 days until complete differentiation occurred ,
, Bacterial strains. L. paracasei ATCC 334 (formerly referred to as Lactobacillus casei ATCC 334) and a nonpathogenic murine E. coli isolate (13) were grown at 37°C in aerobic atmosphere in MRS medium and TS medium (Thermo Fisher), respectively. Bacteria in stationary-growth phase were harvested by centrifugation, washed with PBS, and resuspended in cell culture medium and water for cellular and animal experiments, respectively. To identify specific effectors, bacteria were incubated in m-ICcl2 medium for 16 h, and bacterial pellets were collected, subjected to heat treatment (110°C, 30 min), and resuspended. Culture supernatants (CS) were filtered (pore size, 0.22 m), Bacteria (multiplicity of infection of 100) were added in the upper compartment of the cell chamber, and, following 16 h of incubation, culture supernatants from the upper and lower compartments were collected and cells washed twice in phosphate-buffered saline (PBS) before lysis unless otherwise specified. Cell viability was monitored using the CytoTox 96 cytotoxicity assay (Promega)
, , vol.000, p.5
, RNA extractions from organs and cultured cells were performed using a NucleoSpin RNA II kit (Macherey-Nagel) before cDNA synthesis and RT-qPCR were performed with the primers listed in Table S6 in the supplemental material. Western blotting. Cells were lysed in 300 l of radioimmunoprecipitation assay (RIPA) buffer, and the lysate was heated in Laemmli buffer for 5 min at 90°C before migration in acrylamide SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, incubated with the primary antibodies (see the supplemental material) overnight at 4°C in 5% bovine serum albumin (BSA)-PBS, washed in PBS-Tween 0.1%, incubated with a peroxidase-labeled secondary antibody (1:10,000) for 1 h, and revealed by the use of ECL chemiluminescence reagent (Thermo Fisher). Image acquisitions and quantifications were performed with an Amersham Imager 600 system (GE Healthcare), RNA isolation and quantitative real-time PCR (RT-qPCR). Distal jejunum and liver left lobe were homogenized in 2-ml and 7-ml tubes containing a mix of 1.4-diameter and 2.8-mm-diameter glass beads and 1 ml and 2 ml Trizol, respectively, using a Precellys system (Bertin Technologies)
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