Microcon filter

All melanoma cells were grown in a DMEM except for IPC‐298 which was grown in a RPMI medium, complemented with essential amino acids Arg and Lys, containing naturally occurring atoms (Sigma) (the light medium) or two of their stable‐isotope counterparts (the medium and heavy media) (Cambridge Isotope Laboratories, Inc.; CIL). The medium culture contained arginine (l‐Arg ¹³C₆‐¹⁴N₄) and lysine (l‐Lys ¹³C₆‐¹⁵N₂), and the heavy culture contained arginine (l‐Arg ¹³C₆‐¹⁵N₄) and lysine (l‐Lys ¹³C₆‐¹⁵N₂) amino acids. After five cell divisions to obtain full incorporation of the labeled amino acids into the proteome, cells were counted and equal numbers of cells were split to 15‐cm dishes at roughly 50% confluence. Once cell lines reached ~70% confluence, one 15‐cm dish of each cell line was washed 3 × with PBS and 3 × with serum‐free media. Cells were starved in serum‐free media for 18 h, and the conditioned media (CM) were centrifuged (800 g, 3 min), filtered (0.22 μm) to remove detached cells, and concentrated via centrifugation at 4,500 g in 10 kDa molecular weight cutoff concentrating columns. Then, 500 μl of concentrated medium was filtered by microcon filters with 10 kDa cutoff (Millipore) and buffer was exchanged with 8 M Urea 100 mM Tris or PBS.

Nucleotide sequences of the resistance genes were determined using the DNA sequencing primers listed in Table 2 [26 (link),27 (link)]. PCR products were purified using Microcon filters (Millipore Corporation, Bedford, MA) and were sequenced using a BigDye Terminator Cycle Sequencing Reaction v. 3.1 kit (Applied Biosystems, Carlsbad, CA) on a 3500 Genetic Analyzer (Applied Biosystems). The complementary sense and antisense sequences were aligned using the SeqManII program within the Lasergene software package (DNASTAR, Madison, WI). The DNA sequences and deduced amino acid sequences were examined using the BLAST program at the DNA Data Bank of Japan (DDBJ) [28 ], and the “β-Lactamase Classification and Amino Acid Sequences for TEM, SHV, and OXA Extended-Spectrum and Inhibitor Resistant Enzymes” website [29 ].

Twenty milligrams of each mouse liver were washed with PBS, homogenized in 200 μL lysis buffer (4% SDS in 0.1 M Tris-HCl pH 8.5) and centrifuged at 3000× g at 4 °C for 5 min. Total protein content was measured according to the BCA method [25 ]. A total protein amount of 100 μg for each sample was used for tryptic digestion according to the common FASP protocol [26 (link)]. Briefly, detergents in the samples were exchanged with 100 mM Tris-HCl (pH 8.5) using Microcon filters (10 kDa cut off, Millipore, Bedford, MA, USA). Protein disulfide bridges were reduced with 100 mM 1,4-dithiothreitol in 100 mM Tris-HCl (pH 8.5), alkylation of thiols was performed with 55 mM iodacetamide in 8 M urea/100 mM Tris-HCl (pH 8.5). Tryptic digestion with a trypsin (Sequencing Grade Modified, Promega, Madison, WI, USA) to protein ratio of 1:100 was carried out overnight at 37 °C in a 50 mM tetraethylammonium bicarbonate (pH 8.5) followed by an additional digestion step under the same conditions with 2 h duration.

Ethanol quantification was carried with the aid of the EnzyChrom^® Ethanol Assay kit, according to the manufacturer’s specifications (BioAssay Systems, Hayward, CA, USA). Briefly, 1-mL aliquots of Z. mobilis cultures (grown in MM medium) were collected at different timepoints, centrifuged at 8000× g for 1 min and the resulting supernatant was filtered through 0.22 µm Microcon^® filters (Millipore, Burlington, VT, USA). Cells present in the centrifugation pellet were eluted in 1 × saline solution and used to quantify total protein present in the bacterial biomass with the aid of Bradford reagent, after treatment with 0.2 mM NaOH, for 30 min, according to the manufacturer’s specifications (Sigma-Aldrich/Merck). The filtered supernatant was submitted to serial dilution and 10 µL of each dilution was distributed into individual wells of a 96-well microplate, along with 90 µL of freshly prepared EnzyChrom^® reaction mixture (provided in the kit). The contents were mixed and incubated at room temperature for 30 min and reactions were interrupted by the addition of 100 µL of Stop Solution (provided in the kit). OD₅₉₅ readings were performed in a Multiskan Go microplate reader (Thermo Fisher Scientific) and ethanol content was determined by interpolating the results with a standard curve, made with samples containing pre-defined ethanol concentrations.