Pires2 egfp vector

Total RNA from Caco2 was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Total RNA samples (with A₂₆₀/A₂₈₀ ratios between 1.8 and 2.0) were quantified spectrophotometrically by absorbance at 260 nm. Less than 2 μg total RNA was used to generate cDNA using a reverse transcription kit (TaKaRa, Dalian, China).
The complete CDS of human STC1 mRNA (NM_003155.2) was amplified using the following primers: sense, 5′-ATC AAG CTT ATG CTC CAA AAC TCA G-3′; antisense, 5′-ATG GAT CCT TAT GCA CTC TCA TGG-3′ (HindIII and BamH I cleavage sites are underlined). Amplicons were subjected to 1% agarose gel electrophoresis, purified using a DNA gel extraction kit (Axygen, Union City, CA, USA), and cloned into a pMD-18T TA-clone vector (TaKaRa) for sequencing and subsequent digestion by restriction endonucleases. The gene fragments of interest were then subcloned into the pIRES2-EGFP vector (Invitrogen) using T4 DNA ligase (TaKaRa). DNA sequencing was performed with an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster, CA, USA). The recombinant plasmid was named pIRES-STC1. Endotoxin-free plasmid DNA was prepared from overnight cultures of E. coli DH5α (Invitrogen) containing pIRES-STC1 using the EZNA plasmid max kit (Omega, Doraville, GA, USA).

PC12 cells were purchased from the Wuhan cell collection center and cultured in RPMI-1640 medium (Gibco) with 10% fetal bovine serum (Gibco) in a 5% CO₂ incubator at 37°C. The ALDH2 gene was amplified by PCR and subcloned into the pIRES2-EGFP vector (Invitrogen). The control vectors or vectors carrying the ALDH2 gene were transfected into PC12 cell using Lipofectamine LTX with Plus (Invitrogen) according to the manufacturer's instructions. The cells were divided into 6 groups (n = 8 per group): (1) control group; (2) 4-HNE group, PC12 cells treated with 0.1 mM 4-HNE; (3) +ALDH2(O) group, PC12 cells overexpressing ALDH2 +0.1 mM 4-HNE; (4) +εV1-2 group, PC12 cell overexpressing ALDH2 +0.01 mM 4-HNE +10⁻⁶ M εV1–2; (5) +Vector group, PC12 transfected with vector +0.1 mM 4-HNE; and (6) +Vehicle, PC12 cells treated with 0.1 mM 4-HNE +0.1% DMSO.