Trizol reagent

RAW 264.7 cells were seeded in a 12-well plate (3 × 10⁵ cells/each well) and incubated at 37 °C with 5% CO₂ for 24 h. and stimulated with LPS (1 μg/mL) for 18 h after a 6-h pre-treatment with 5-HMF at different concentrations (0, 31.5, 63.0 and 126.0 μg/mL). Total cellular RNA was isolated from cultured RAW 264.7 cells using Trizol reagent (Sangon Biotech, Shanghai, China). The RNA was reverse-transcribed into cDNA using ReverTra Ace^® qPCR RT Master Mix with gDNA Remover (TOYOBO LIFE SCIENCE, Shanghai, China) in T100™ Thermal Cycler (BIO RAD, Hercules, CA, USA). The sample mixture was prepared with 1.5 μL of cDNA in 10 μL of ChamQ SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China), primers and ddH₂O for a final volume of 20 μL. The primers were supplied from Sangon Biotech (Shanghai, China) and the sequences are listed in Table 1. The qPCR was carried out using the LightCycler^® 96 Real-Time PCR System (Roche, Switzerland) under the standard thermal cycle conditions: 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by 95 °C for 10 s, 65 °C for 60 s and 97 °C for 1 s. The threshold cycle (CT) was calculated as the fractional cycle number at which the amount of the amplified target gene reached a fixed threshold. β-actin was used as the housekeeping gene. Each reaction in at least three independent experiments was performed in triplicate.

Total RNA was extracted from PA2 and PA4 leaves using the TRIzol reagent (Sangon, Shanghai, China). Primers were designed using Beacon Designer (version 7.7) (Premier Biosoft International, Palo Alto, CA, USA) (S1 Table). PCR mixtures with a final volume of 20 μL contained 10 μL SYBR Green PCR mix, 1 μL cDNA, 0.4 μM forward primer, 0.4 μM reverse primer, and 7.0 μL sterile ddH2O. The qRT-PCR was completed using a CFX96 Real-Time System (Bio-Rad) and SYBR Premix Ex Taq II (Takara, Dalian, China). The PCR program was as follows: 95°C for 1 min; 40 cycles of 95°C for 10 s and 55°C for 15 s. Three replicates were analyzed for each gene. Relative gene expression levels were calculated using the 2^−ΔΔCt method, and the 18S rRNA of Paulownia was chosen as a reference gene for normalization. Student’s t test was used to detect differences between PA4 and PA2 at a significance level of p = 0.05.

Total RNA was extracted using TRIzol reagent (Sangon, Shanghai, China) from homogenized tissues or cells. ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) was used to generate cDNA from mRNA and SYBR Premix Ex Taq (Takara, Kyoto, Japan) was used for real‐time qPCR with the ABI 7500 Real‐time PCR system following the manufacturer's instructions. Primers for mouse VCAM‐1, ICAM‐1, E‐selectin, IL‐1, IL‐6, TNF‐α and human VCAM‐1, ICAM‐1, E‐selectin are listed in Table S1.

RNA was isolated using Trizol reagent (Sangon Inc.). Reverse transcription was applied using a PrimeScript™ RT reagent kit (Takara). qRT-PCR was conducted with SYBR green PCR Master Mix (TOYOBO) with the ABI 7500 system. Primers are listed in Table S1. Relative levels of genes were determined using the 2^-ΔΔCt method. All of these assays were conducted based on the supplier’s instructions.

Total RNA was extracted from Aortic vessel using TRIzol reagent (Sangon Biotech, B511311). The initial cDNA was synthesized using reverse transcriptase (Yeasen, 11141ES60) according to the manufacturer’s instructions. The cDNA was used for PCR amplification and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. The Sequences was shown in Table 1.

Total RNA was extracted from root tissues using TRIzol reagent (Sangon, China) at 1 dpi with FO according to the manufacturer’s instructions. Genomic DNA was removed by DNase treatment (BBI, Canada). One milligram of total RNA was reverse-transcribed using ReverTra Ace qPCR RT Kit (Toyobo, Japan) according to the manufacturer’s instruction. The iCycler iQ™ real-time PCR detection system (Bio-Rad, Hercules, CA, USA) was used for quantitative real-time PCR. Twenty-five-microliter reaction mixtures consisted of 12.5 µl SYBR Green PCR Master Mix (Takara, Japan), 1 µl of diluted cDNA, and 0.2 µM of forward and reverse primers. The conditions for PCR cycling were as follows: 95°C for 3 min and 40 cycles of 95°C for 10 s 58°C for 45 s. The gene-specific primers used for the amplification were determined on the basis of gene or EST sequences and are listed in Supplementary Table S1. mRNA levels were quantified according to the method of Livak and Schmittgen (2001) (link). To obtain a ΔCt value, the threshold cycle (Ct) value of actin was subtracted from that value of the gene of interest.