Detection of apoptosis through TUNEL assay

The HUVECs were divided into three groups: i) Control: Cells treated with neither LPS nor BAI; ii) LPS: Cells treated with LPS (10 µM); iii) LPS + BAI: Cells treated with LPS (10 µM) and BAI (6.25 µM). Apoptosis was identified through the DNA fragmentation in the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (14). The TUNEL assay works through the incorporation of modified dUTPs at the 3′-OH ends of fragmented DNA by the enzyme terminal deoxynucleotidyl transferase (TdT), which subsequently tags cells with damaged DNA (15). After the TUNEL assay treatment, damaged DNA is stained green, whereas intact DNA is not stained. HUVECs were detected using the Apo-Direct TUNEL Assay kit (EMD Millipore, Billerica, MA, USA). All steps were conducted in strict accordance with the specifications and photographed using a fluorescent microscope.