Chemicdoctm mp imaging system

A human Th1/Th2/Th17 cytokines antibody array kit (AB169809; Abcam, Cambridge, UK) was used to compare and analyze the secretion profile of Th1/Th2/Th17-related cytokines by OMC and MSC. To this end, membranes were either incubated into fresh media, or OMC-S and MSC-S (3 independent batches) and processed following the instructions of the manufacturer (see Protocol Booklet for Array Map). Chemiluminescence signal was captured with a ChemicDoc^TM MP Imaging System (Bio-Rad, Hercules, CA, USA) and quantified with the Image Lab 6.0.1 software (Bio-Rad). Exposure time was set using positive control spots in fresh media-incubated membrane (see Array Map). Cytokine signals were measured into a fixed circle area drawn around the spots. Basal signals for cytokines produced into a membrane incubated in fresh media was also calculated. The mean background signal for each PVDF membrane was also calculated into 5 areas lacking spot antibodies. Specific cytokine signal produced by OMC-S (n = 3) and MSC-S (n = 3) were finally calculated after subtracting non-specific basal and background signals. A grid (Figure 2) was drawn and added to the membrane’s pictures to delimitate in a single rectangle each cytokine (detected in duplicated dots).

Western blot was performed following the method described previously [29 (link),30 (link),31 (link)]. An equal amount of denatured proteins was separated by SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk powder in a PBST solution for 1 h, and were incubated with peroxisome proliferator-activated receptor-γ (PPAR-γ) (Santa Cruz Biotechnology, CA, USA), sterol regulatory element-binding protein-1 (SREBP-1) (Santa Cruz Biotechnology, CA, USA), stearoyl-CoA desaturase -1 (SCD-1) (Cell Signaling Technology, MA, USA) and β-Actin (Proteintech^TM, Wuhan, Hubei, China) at 4 °C overnight, then incubated with appropriate secondary antibodies (Proteintech^TM, Wuhan, Hubei, China) for 1 h at room temperature. Protein bands were detected by an enhanced-chemiluminescent (ECL) reagent (Vazyme Biotech Co., Ltd., Nanjing, China) and analyzed using a ChemicDoc^TM MP Imaging System (Bio-Rad, Hercules, CA, USA) with a supporting system (ImageLab., Bio-Rad, Hercules, CA, USA).

Western blot was performed following the method described previously in [52 (link),53 (link),54 (link)]. In brief, the frozen liver tissues were homogenized using a 2 × SDS buffer. Equal amounts of denatured proteins were separated by SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk powder in PBS-T solution for 1 h and were incubated with fatty acid synthase (FAS) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), sterol regulatory element-binding protein-1 (SREBP-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-Actin (Proteintech^TM, Wuhan, Hubei, China) at 4 °C overnight, then incubated with appropriate secondary antibodies (Proteintech^TM, Wuhan, Hubei, China) for 1 h at room temperature. Protein bands were detected by enhanced chemiluminescent (ECL) reagent (Vazyme Biotech Co., Ltd., Nanjing, China) and analyzed using the ChemicDoc^TM MP Imaging System (Bio-Rad, Hercules, CA, USA) with supporting system (ImageLab., Bio-Rad, Hercules, CA, USA).