Oligonucleotide primers

DNA extraction kits, DNA polymerase for PCR, and T4 DNA ligase were purchased from Beyotime Biotech Co. Ltd. (Haimen, China). The PET-28(a) expression plasmid, restriction endonucleases, oligonucleotide primers, and competent cells, including E. coli DH5α and BL21 (DE3), were provided from Sangon Bioengineering Co. Ltd. (Shanghai, China). A nickel-nitrilotriacetic acid (Ni-NTA) superflow column was supplied by TransGen Biotech Co. Ltd. (Beijing, China). The DNA ladder and protein molecular weight marker were obtained from Detai Biologics Co. Ltd. (Nanjing, China). CS-A from the bovine trachea, CS-C from shark cartilage, and hyaluronic acid (HA) sodium salt from Streptococcus equi were purchased from Sigma Co. Ltd. (St. Louis, MO, USA). Kanamycin sulfate, IPTG, and all other reagents used in the experiments were of analytical grade and ordered from Aladdin Co. Ltd. (Shanghai, China).

Genomic DNA collected from tumor samples were examined for six polymorphic microsatellite markers including: D10S1239, D7S1824, D2S1363, D6S1056, D15S822, and D22S689. The oligonucleotide primers corresponding to each microsatellite marker were purchased from Sangon Biotech (Shanghai, China) Co., Ltd. For PCR, the reaction samples were each prepared to a total volume of 25 μL as follows: 1 μL each primer, 1 μL template DNA, 9.5 μL nuclease-free water, and 12.5 μL PCR Mix (2X). Amplifications were carried out in a Perkin Elmer Thermocycler (Perkin Elmer, Waltham, USA) using a step-cycle program consisting of 39 cycles of 95°C for denaturing (30 s), 55°C for annealing (30 s), and 72°C for extension (1 min). PCR products were fractionated on 6% polyacrylamide gels containing Tris-borate/EDTA (pH 8.0) and visualized by ethidium bromide staining.

Total RNA from the adipocytes treated with LQG and/or RAP was isolated utilizing a Trizol reagent and 1 μg RNA was converted to cDNA using HiScriptIIQ RT SuperMix (Vazyme, Nanjing, China). Gene expression was quantified using Hieff UNICON^® qPCR SYBR Green Master Mix (Yeasen Biotech, Shanghai, China) and a LightCycler 480 II (Roche, Basel, Switzerland). RT-PCR reactions were run in triplicate for each sample, and relative gene expressions were calculated using the 2^−ΔΔCT method after values were normalized to β-actin. The oligonucleotide primers (Sangon Biotech, Shanghai, China) used for amplification are shown in Table 1.

High-fidelity KOD DNA polymerase (TOYOBO) was used for PCR amplification using the cDNA of D. salina as a template. The amplified gene was cloned into the target vector using Gibson assembly (44 (link)). Restriction endonucleases were purchased from Thermo Fisher Scientific, and Gibson assembly reagents were purchased from New England BioLabs (NEB). Oligonucleotide primers were purchased from Sangon Biotech. Table S3 (available at https://www.biosynnatlab.com/wp-content/uploads/2022/12/Supplemental-Material.pdf) lists the primers used for cloning.

Total RNA was isolated from ileum tissue using TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA). cDNA was synthesized from 1 μg of total RNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time quantitative polymerase chain reaction (qPCR) was performed using the ABI 7500 Real-Time PCR system and SYBR Premix Ex Taq™ II Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. Oligonucleotide primers for mouse zonula occludens- (ZO-) 1 (forward 5′-ACTCCCACTTCCCCAAAAAC-3′ and reverse 5′-CCACAGCTGAAGGACTCACA-3′) and β-actin (forward 5′-ACAGGCATTGTGATGGACTC-3′ and reverse 5′-ATTTCCCTCTCAGCTGTGGT-3′) were synthesized by Sangon Biotech.