Effects of pterostilbene on endothelial-to-mesenchymal transition in human primary pulmonary arterial endothelial cells
Description
Pterostilbene is a stilbene compound originally isolated from the deciduous tree Pterocarpus marsupium native to India, which has a broad range of biological activities, including anti-oxidant, anti-inflammatory, hypoglycemic, hypolipidemic, and anti-tumor effects. Here we investigated the effects of pterostilbene on the endothelial-to-mesenchymal transition (EndMT) response induced in human primary pulmonary arterial endothelial cells in vitro.
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Human primary pulmonary arterial endothelial cells (hPAECs) (#HUM-iCell-a008) were purchased from Cellverse (Shanghai, China), and cultured in complete ICell Primary Endothelial Cell Culture Medium (#PriMed-iCell-002 from Cellverse). Cells between passages 2 to 5 were used for experimentation. Experimental design: 3 treatment groups with 3 independent replicates in each group 1. Vehicle control group (column title c1 to c3); 2. EndMT group (column title a1 to a3): treated with TGF-β1 (5 ng/ml) plus TNF-α (5 ng/ml) plus IL-1β (0.1 ng/ml) for 3 days; 3. EndMT + pterostilbene co-treatment group (column title pte1 to pte3): the concentration of pterostilbene was 20 µM. Total RNA was extracted using TRIZOL Reagent (Thermo Fisher, Waltham, MA, USA) following the manufacturer's instructions. RNA integrity was checked with an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Qualified total RNA was further purified by RNAClean XP Kit (Beckman Coulter, Brea, CA, USA) and RNase-Free DNase Set (QIAGEN, Germany). RNA concentration was determined using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher). Library construction was performed using VAHTS Stranded mRNA-seq Library Prep Kit for Illumina (from Vazyme, Nanjing, Jiangsu Province, China). RNA sequencing was performed using a NextSeq Illumina550 platform (Illumina, San Diego, CA, USA) in a paired-end manner. RNA processing, library construction and sequencing services were provided by LC-BIO Co., Ltd (Hangzhou, Zhejiang Province, China). High-quality clean reads were obtained by filtering with Cutadapt (version 1.9) (https://cutadapt.readthedocs.io/en/stable/) and verified with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Read alignment to GRCh38 human reference genome was performed using HISAT2 package (version 2.0.4) (https://daehwankimlab.github.io/hisat2). StringTie (version 1.3.4d) (http://ccb.jhu.edu/software/stringtie) was used for read assembly and estimation of the relative expression levels (FPKM values).