GroEL1, from Chlamydia pneumoniae, Induces Vascular Adhesion Molecule 1 Expression by p37AUF1 in Endothelial Cells and Hypercholesterolemic Rabbit
Figure 4
The 5′ UTR flanking sequence of VCAM-1 mRNA conferred P37AUF1-responsiveness in the BAECs.
(A) The BAECs were transfected with the 4His-A-AUF1-p37 plasmid (p37AUF1), 4His-A-AUF1-p40 plasmid (p40AUF1), 4His-A-AUF1-p42 plasmid (p42AUF1), or 4His-A-AUF1-p45 plasmid (p45AUF1). The level of VCAM-1 mRNA were analyzed using real-time PCR after transfectiuon for 24 hours. (B) The VCAM-1 mRNA stability was analyzed using an actinomycin D chase experiment in the AUF1-transfected BAECs. (C) Schematic representation of the various plasmids containing the luciferase and UTR of the VCAM-1 mRNA. Control plasmid: pcDNA™ 3.1 plasmid; construct A, CMV-Luciferase plasmid; construct B, CMV-Luciferase-VCAM1 5′UTR (sense) plasmid; construct C, CMV-Luciferase-VCAM1 5′UTR (antisense) plasmid; construct D, CMV-Luciferase-VCAM1 3′UTR (sense) plasmid; construct E, CMV-Luciferase-VCAM1 3′UTR (antisense) plasmid. (D), BAECs were co-transfected with the CMV-Luciferase-VCAM1 UTR plasmid, the β-galactosidase reporter plasmid, and the 4His-A-AUF1 plasmid. Uniform transfection efficiencies were confirmed using a β-galactosidase reporter plasmid. The luciferase activity was quantified by luminometry. Data are expressed as relative luciferase units, presented as the mean ± SEM and represent the results of three independent experiments (*P<0.05 was considered significant and n = 3).