EpH4-Ev Cell Culture and Treatment

EpH4-Ev cells (mouse mammary epithelial cell line) were grown at 37°C in DMEM supplemented with 10% FBS in 6-well plates until they reached 70–80% confluency. The resulting monolayers were cultured in FBS-free DMEM for 4 h and then further cultured in FBS-free DMEM for 24 h, with or without 45 mmol/L taurine. Next, both groups of cells were infected with S. uberis at an MOI of 10 as before(16) for 1, 2, or 3 h for metabolomics analysis. In other experiments, EpH4-Ev cells were exposed to S. uberis for 3 h.

Cell samples prepared for GC–TOF-MS analysis were collected as described previously (32). Briefly, cells (plated at approximately 5 × 10⁶ cells/well) were infected with S. uberis for 1, 2, or 3 h. The cell supernatants were discarded, and the cells were washed three times with cold phosphate-buffered saline (PBS), quenched with 400 µL cold methanol (precooled at -80°C), and held at -80°C for 30 min. An additional 400 µL double distilled water was added to the plates, and the cells were scraped off separate plates. The cell suspensions were stored at -80°C and then used to prepare samples for metabolomics analysis.

For extracellular flux analysis, EpH4-Ev cells were challenged with 10 μg/mL LPS for 12 h, or with inactivated S. uberis or E. coli NJ-17 at a MOI of 100 for 3 h, at 37°C.

For the inhibition and activation experiments, cells were pretreated as described below before S. uberis infection. Specifically, EpH4-Ev cells were grown at 37°C in DMEM with 10% FBS in 6-well plates and grown to 70–80% confluency. After culturing the cells in FBS-free DMEM for 4 h, the monolayers were treated with 5 mM 2-DG (Selleckchem, USA) for 1 h, 25 μM CPI-613 (Selleckchem, USA) for 12 h, 100 nM MHY1485 (Selleckchem, USA) for 24 h, or 10 µM Compound C (Selleckchem, USA) for 1 h. All the inhibitors or activators used in this study had been determined by cell viability and they were nonpoisonous to cells.