Nucleic acid protein detector

Total RNA from BEND cells was extracted using a Total RNA Extraction Kit (TaKaRa Biotechnology Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. The RNA concentration and purity were determined using a nucleic acid protein detector (Bio-Rad Instruments). Reverse transcription of the extracted RNA into single-stranded cDNA was carried out using a reverse transcription kit (TaKaRa Biotechnology Co., Ltd.) according to the manufacturer’s instructions. Quantitative real-time PCR (RT-PCR) was performed using SYBR Premix Ex Taq II (TaKaRa Biotechnology Co., Ltd.) to assess mRNA expression levels of TNF-α, IL-6, IL-8, and NF-κB p65. All amplifications were repeated three times. RT-PCR data were calculated using the gene expression formula 2^−ΔΔCt. The relative expression of each gene was normalized to β-actin levels. The primers used for RT-PCR are listed in Table 1. Primers for IL-8 and TNF-α were designed using Primer Express Version 5.0 software (New York, NY, USA), and primers for NF-κB p65, IL-6, and β-actin were identical to those used by Shi et al. [40 (link)]. All primers were commercially synthesized by Sangon Biotech Institute Co., Ltd. (Beijing, China).

Cells in six-well plate were harvested from each group on day 8 of differentiation (n = 6) for qPCR analysis. The total RNA was extracted by RNAiso plus (Takara, Tokyo, Japan). RNA concentration was quantified at 260 nm by a nucleic acid protein detector (Bio-Rad, Hercules, CA, USA). An amount of 1 μg total RNA was used to synthesize cDNA using a PrimeScript RT reagent kit. qPCR was conducted by a SYBR GreenⅡqPCR kit (Takara, Tokyo, Japan). The 2^−ΔΔCt method was adopted to calculate the relative gene expression level normalized to GAPDH. Primers are shown in Table S1.