Quality one software

Pulsed field gel electrophoresis (PFGE) was carried out on Enterococcus isolates according to the method described previously with minor modifications (Liu et al., 2013 (link)). Enterococcal cells were treated with lysozyme at a final concentration of 10 mg/ml for 1 h at 37°C and then incubated with the restriction enzyme SmaI (Takara, Japan) for 4 h with shaking. Briefly, genomic DNA fragments were resolved on a 1% agarose (SeaKem Gold Agarose, Lonza) gel at 14°C, and electrophoresis was conducted using a CHEF-Mapper XA PFGE system (Bio-Rad, United States) at a pulse time gradient of 3.5–23.5 s and a total running time of 18 h within a molecular weight of 10–300 kb. The DNA band patterns were visualized by a Gel Doc XR gel imaging system (Bio-Rad, United States) and further analyzed by QualityOne software (Bio-Rad Laboratories, United States). Phylogenetic trees then allow the comparison of genetic relatedness and clonal assignment (Neoh et al., 2019 (link)). A similarity coefficient of 80% was selected to define the pulsed-field type (PFT) clusters (McDougal et al., 2003 (link)).

Western blot analyses were performed according to previous protocols [29 (link)]. Briefly, proteins were extracted from the cells or liver tissues by lysis buffer. Protein concentrations were determined, and the equal amounts of lysates were loaded, separated by SDS-PAGE gels, and transferred to PVDF membranes. The membranes were blocked and incubated with primary antibodies overnight at 4 °C followed by an incubation with HRP-conjugated secondary antibody. Signals were detected by an ECL system (GE Healthcare, Buckinghamshire, UK) and band intensities were quantified by densitometry using the Quality One software (Bio-Rad, Hercules, CA, USA). The primary antibodies against α-SMA (Cat#19245), CD36 (Cat#14347), P-JNK1/2 (Cat#4668), JNK1/2 (Cat#9252), P-NF-κB (Cat#3033), NF-κB (Cat#8242), and GAPDH (Cat#5174) were purchased from Cell Signaling Technology (Danvers, MA, USA), and the primary antibody against Col-1a1 (Cat#A1352) was purchased from ABclonal Technology (Wuhan, China).

Cells were lysed in RIPA lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) supplemented with 10 mM NEM (N-ethylmaleimide; Sigma, E3876) to obtain total protein lysates. Nuclei were lysed in RIPA lysis buffer supplemented with 10 mM NEM to obtain nuclear lysates. Protein concentrations were measured using an enhanced BCA protein assay kit (P0009, Beyotime Biotechnology, Shanghai, China). The protein lysates were separated by SDS-PAGE, and proteins transferred to PVDF membranes. Membranes were blocked with 5% fat-free dry milk in Tris-buffered saline (10 mM Tris-base, 150 mM NaCl, pH 7.6) supplemented with 0.1% Tween 20 and incubated with primary antibodies at 4 °C overnight. Protein bands were detected by incubation with horseradish peroxidase-conjugated antibodies (Kirkegaard and Perry Laboratories) and visualized with Clarity Western ECL Substrate (Bio-Rad). For immunoprecipitation, total or nuclear protein lysates were obtained as described and equal quantities of proteins were incubated with primary antibodies at 4 °C on a rocking platform overnight. Immune complexes were collected with protein A/G agarose beads (Beyotime Technology), washed five times with phosphate-buffered saline (PBS), and subjected to SDS-PAGE and western blot. Quantification relative to.
GAPDH/Histone3 by densitometric analysis using the Quality One software (Bio-Rad).

Cells were seeded at 1 × 10⁵/ml onto 60 mm dishes, treated, collected, lysed, and immunoblotted with indicated antibody. The detailed procedure was described in our previous publications [52 (link), 56 (link)]. Briefly, cell lysis was completed by sonication and the total protein was separated on an SDS-PAGE, transferred onto a PVDF membrane (Millipore), and immunoblotted with indicated primary antibody. Antibodies to PD-L1 (cat no. A19135) and to NKG2D (cat no. A6123) were purchased from Abclonal. Antibodies to IDO1 (cat no. 51851) and (cat no. 86630) were purchased from Cell Signaling Technology. Antibody to TDO2 (cat no. NBP2-45,995) was purchased from Novus Biologicals. Antibody to IDO2 (cat no.703150) was purchased from ThermoFisher Scientific. All antibody dilutions were at 1:1000, except for β-Actin (Sigma, cat no. A1978) which was diluted at 1:10,000. Bands were measured using a molecular imager Chemidoc system with Quality One software (Bio-Rad Laboratories).