Genechip expression assay

Liver DNAs were extracted using DNeasy Blood & Tissue Kit (n = 3–4) from Pemt−/− and Pemt+/+ mice fed with HFHS or STD chow for 25 weeks. One hundred ng of DNA was subjected to Imprint Methylation DNA Quantification Kit (SIGMA) to measure the methylation of DNA. Pemt−/− (n = 4) and Pemt+/+ (n = 4) mice were fed with HFHS chow and liver samples were obtained at 1, 2, 3 and 4 weeks. Methylated DNA was enriched with EpiXplore Methylated DNA Enrichment Kit (Clontech) according to manufacturer’s protocol. Genomic DNA libraries was prepared by TruSeq ChIP Sample Preparation Kit (Illumina) and sequenced by Genome Analyzer IIx. For DNA microarray, total RNAs were extracted by using RNeasy Midi Kit (QIAGEN) and subjected to GeneChip Expression Assay (Affymetrix). The whole raw and processed data are freely available in the Gene Expression Omnibus (GEO) under the accession number GSE67791, GSE67792 and GSE67793.

To detect the microRNA expression in primary human umbilical vein endothelial cells (HUVECs), the HUVECs were treated with NaHS (30 μmol/L) or the vehicle for 12 h in the first instance. Then, the RNA was isolated from the HUVECs using Trizol (Thermo, Waltham, MA, USA), in accordance with the protocol of the manufacturer. The microRNA expression was tested by an Affymetrix Gene Chip Expression Assay (Affymetrix, Santa Clara, CA, USA).