Gel electrophoresis

Three‐month‐old and 12‐month‐old mice tissues were flash frozen in liquid nitrogen and stored at −80°C before use. Tissues were briefly homogenized in T‐PER (Thermo Fisher) and centrifuged at 10,000 × g for 5 min to remove insoluble material. Protein concentration was determined by Bradford assay. Protein extracts were denatured at 95°C for 5 min and separated by gel electrophoresis (Thermo Fisher) and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk in 1X PBST for 1 hour at room temperature. Membranes were incubated overnight at 4°C with primary antibodies specific to PRODH (Abcam ab203875), ALDH4A1 (Abcam ab181256), Histone H3 (Sigma H0164), Actin (Sigma A3853), washed and incubated with goat anti‐rabbit secondary antibody (Abcam) at room temperature for 1 h, and protein visualized with SuperSignal (Thermo Fisher) chemiluminescent substrate.

The HBV S gene was detected by nested PCR as described previously [25 (link)]. HBV DNA was extracted from about 30 mg (obtained from approximately 20 sections, 5 μm thick, not attached to glass slides) of paraffin-embedded synovium with the RecoverAll™ total nucleic acid isolation kit (Life Technologies). Liver tissue from patients with HBV-related hepatocellular carcinoma was included as a positive control. Amplification was carried out in a 50-μl reaction volume containing 3 μl of forward and reverse primers (10 μM), 40 ng of DNA template, and 25 μl of 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, MA, USA). The following thermocycles were used: 95 °C for 3 minutes, followed by 35 cycles of 98 °C for 20 seconds, 65 °C for 15 seconds, and 72 °C for 1 minute, with a final extension at 72 °C for 1 minute. The PCR products were then resolved by gel electrophoresis (Life Technologies). DNA bands were visualized by ultraviolet fluorescence. PCR products were sequenced in both directions on an ABI 3730 XL Automated DNA Sequencer with the ABI BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). The sequences were aligned using the Basic Local Alignment Search Tool (National Center for Biotechnology Information website https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm the identity of the HBV S gene.

Fecal DNA was isolated from the pellets obtained by centrifuging 500 µL of fecal slurry using the Zymo Research kit (Quick-DNA^TM Fecal/Soil Microbe MiniPrep Kit) following the manufacturer’s protocol (Zymo Research, Irvine, CA). Enzymatic lysis of DNA was performed using lysozyme (20 mg) and mutanolysine (10 KU) (Sigma-Aldrich, Oakville, Canada). The resulting DNA extracts were eluted in 1X TE buffer (Tris and EDTA) and stored at −20 °C until sequencing. DNA quality was assessed by gel electrophoresis (1.2% w/v agarose) (Life Technologies, Madrid, Spain). DNA concentrations were determined using a Qubit (Thermo Fisher Scientific, Waltham, US). DNA samples were stored at −20 °C until preparation of the 16 S rRNA library.

Western blot was carried out as previously described.^{44 (link)} Briefly, fresh tissue or cells were lysed in 1 × lysis buffer (Cell Signaling Technology). Fifty micrograms of total protein were separated via gel electrophoresis (Life Technologies, Carlsbad, CA, USA), and transferred onto a nitrocellulose membrane, immunoblotted with primary antibodies. β-Actin served as a loading control. All western blots were quantified by gray densitometry assay using Multi Gauge V3.0 software (Fuji Film, Tokyo, Japan). Values under each blot are shown as the ratios of target protein/Actin, and present the mean of three independent assays.

SHIME® suspensions from ileum and colon vessels (500 μL) were stained with 1.25 μL of propidium monoazide (PMA, 50 μM) (Biotium, Fremont, Canada) to inactivate dead bacterial DNA, as previously described.^{53 (link)} DNA was pelleted (8 minutes, 18 000 × g, 4°C), and extracted according to Geirnaert et al.^{54 (link)} In the mucus microcosm samples, an additional step was performed to increase DNA yield and break the disulfide bonds from the mucins. Sputolysin 0.1 M (250 μL, Sigma-Aldrich, St. Louis, US) was added to 125 mg mucus sample from ileum or 250 mg mucus from colons. Samples were incubated in a water bath for 30 minutes at 37°C prior to the DNA extraction procedure. DNA extracts were eluted in 1X TE buffer (Tris and EDTA) and stored at −20°C until sequencing. The quality of DNA was analyzed by gel electrophoresis (1.2% w/v agarose) (Life technologies, Madrid, Spain). Concentrations were measured by the Qubit (Thermo Fisher Scientific, Waltham, US) and the DNA were stored at −20°C, until 16S rDNA library preparation.