Hsp70

Human umbilical vein endothelial cells (HUVECs) (ATCC-CRL-1730) were cultured in F-12K medium (ATCC-100-010) with 10% FBS (ATCC-30-2020) according to the instructions provided by ATCC.
HUVECs were transfected with 1 mg of luciferase reporter constructs, and either 20 nmoles of either 328a-3p microRNA mimic (Qiagen, Hilden, Germany, cat # 219600-MSY0000752-5 ‘CUGGCCCUCUCUGCCCUUCCGU), control mimic (Qiagen cat # 339173 -YM00479902), inhibitor (Qiagen, cat# 339121 (YI04101608), 5 ‘CUGGCCCUCUCUGCCCUUCCGU) or control inhibitor (Qiagen cat# 339125-YI00199006) using FuGENE 6 reagent according to the manufacturer’s instructions (Roche, Indianapolis, IN, USA). The media was changed after 1–2 h to serum-free media and the cells were harvested 24 h post-transfection.
Fetal bovine serum (FBS)–starved HUVECs (for 24 h) in 1 mL of medium were treated for 24 h with either 25, 50, or 100 μL of pooled human plasma from normal controls, patients with either CAD or ectasia. In separate experiments, FBS-starved HUVECs were also treated with endotoxin-free (according to the manufacturer) human recombinant HSP70 (0.5 ng/mL, StressMarq Biosciences, Victoria, BC, Australia), S100B (0.1 ng/mL) or HMGB1 (10 ng/mL, R&D Systems, Minneapolis, MN, USA).

Proteins were extracted by washing cells with phosphate-buffered saline and lysed with sodium dodecyl sulphate buffer. Equal amounts of each total protein (20 μg) lysate sample were dissolved in 12.5% polyacrylamide gels (ATTO corporation, Tokyo, Japan) and transferred onto Immobilon-P membranes (Merck, Darmstadt, Germany). The membranes were subsequently probed with horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence kit (Global life science technologies, Tokyo, Japan). Antibodies against HSP70 (Stress Marq Biosciences Inc., Victoria, BC, Canada) and HIKESHI (Proteintech, Rosemont, IL, USA) were used to assess protein expression levels. GAPDH (Cell Signaling Technology, Massachusetts, USA) and anti-Histone H3 (Abcam, Cambridge, UK) were used as a loading control.