Amicon ultra 0.5 filter

Cecal digesta was weighted to ≈150 mg. Metabolomics buffer (0.125 M KH₂PO₄, 0.00375 M NaN₃, and 0.375 M KF; pH 7.4) was added to the cecal digesta samples at a ratio of 2:1 (v/w) and homogenized using a Bullet Blender tissue homogenizer (Next Advance; Troy, NY, USA) with 150 mg of 2-mm-diameter zirconium oxide beads (Next Advance) at setting eight for 5 min. Samples were centrifuged for 5 min at 14,000× g, and supernatants were filtered through a 3000 MWCO Amicon Ultra-0.5 filter (Millipore Sigma, Oakville, ON, Canada) by centrifugation at 14,000× g for 30 min at 4 °C. An aliquot of 360 µL was mixed with 200 µL of metabolomics buffer and 140 µL of deuterium oxide containing 0.05% (v/v) trimethylsilylpropanoic acid (TSP) for a final volume of 700 µL; TSP was used as a chemical shift reference for ¹H-NMR spectroscopy. The solutions were vortexed and centrifuged at 12,000× g for 5 min at 4 °C, and an aliquot of 550 µL of the supernatant of each sample was loaded in a 5 mm NMR tube. Spectra were obtained as previously described [26 (link)].

The N-terminal half of POLE that encodes the DNA polymerase and exonuclease domains of human Polϵ (residues 1–1189, 140kDa) was cloned into a pHB-CMV-MCS-EF1-Puro vector as previously described (29 (link)). The plasmids encoding Polϵ-P286R, R375Q, V411L, and P452L were constructed using the primers summarized in Supplementary Table 2. A FLAG tag was added to the N-terminus via PCR. The recombinant FLAG-protein was expressed in 293T cells. Co-immunoprecipitation (Co-IP) was performed to isolate the FLAG-conjugated catalytic subunit using the FLAG-beads (Sigma-Aldrich, St. Louise, MO, USA) following the manufacturer’s instruction. The FLAG-tagged protein band was visualized using Coomassie blue staining and Western blot analysis with an anti-FLAG antibody (#2368T; 1:1000; Cell Signaling Technology, Danvers, MA, USA). The FLAG-tagged wildtype and mutant catalytic fragments were purified from the immunoprecipitated complexes using the 100 kDa Amicon^®Ultra-0.5 filter (MilliporeSigma, Burlington, MA, USA).

Livers of the control rats and DBDCT-treated rats were suspended in pyrolysis liquid (7 M urea, 2 M thiourea, 65 mM dithioerythritol, and 0.1% protease inhibitor) for 0.5 h on ice. The mixture was centrifuged at 12,000 rpm for 15 min at 4 °C to remove solid impurities (TD5A-WS, Changsha Xiangyi Centrifugal Machine Company, Changsha, China). Then the sample was redissolved in 0.1% formic acid. For the quantitative proteomics analysis, samples were subjected to trypsin digestion. The tryptic digest extracts were analyzed by a LC-MS/MS system according to a previous study [25 (link)]. The samples were reduced with 20 mM dithiothreitol (DTT; 56 °C, 30 min), alkylated with 50 mM iodoacetamide (26 °C, 20 min in the dark) and digested with sequencing-grade modified trypsin (1:50 w/w; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 24 h. The solution was filtered by centrifugation at 11,000× g for 90 min at 4 °C in a 10 k Amicon Ultra-0.5 filter (Merck Millipore, Bedford, MA, USA).

Casein zymography (β-casein, Sigma-Aldrich, St. Louis, MO, USA) was performed as described elsewhere [23 (link)]. Mark12™ unstained protein standard (LC5677, Thermo Fisher Scientific, Schwerte, Germany) was run in a parallel lane. After electrophoresis, the gels were incubated in 50 mM Tris-HCl (pH 7.5) containing 10 mM CaCl₂, 1 µM ZnCl₂ and 0.1% Triton X-100 with or without the selective MMP-3 inhibitor UK370106 (R&D Systems, Minneapolis, MN, USA) for 72 h at 37 °C.
Gelatin zymography was used to analyze the MMP-2 content in 4× concentrated (Amicon Ultra-0.5 filter, Merck-Millipore, Darmstadt, Germany) conditioned media [23 (link)]. rmMMP-2 (924-MP, R&D Systems, Minneapolis, MN, USA) was run at 2 ng in parallel to estimate the MMP-2 content of the samples by the image analysis of digitized gels [47 (link)].