Fig. 2: CAR expression in endothelial cells is regulated by the flow-responsive KLF2-AP-1 axis.
HUVECs were transfected with siRNA against Krüppel-like factor 2 (KLF2; 50 nM) (a, f) or plasmid-encoding KLF2 (5 μg) (b, e) and then exposed to FSS for 4 h or 24 h. KLF2 and CAR protein levels were measured by western blotting. a n = 5; *P < 0.05, static vs. LSS; #P < 0.05, LSS vs. siKLF2 + LSS. b n = 5; *P < 0.05, static vs. LSS or OSS; &P < 0.05, LSS vs. OSS; #P < 0.05, OSS vs. pKLF2 + OSS. c HUVECs were exposed to FSS for 24 h after pretreatment with the AP-1 inhibitor SR11302 for 1 h. CAR protein levels were detected by western blotting (n = 7; *P < 0.05, static vs. LSS or OSS; &P < 0.05, LSS vs. OSS; #P < 0.05, OSS vs. SR11302 + OSS). d HUVECs transfected with siRNAs against c-Fos (100 nM) and c-Jun (100 nM) were exposed to FSS for 24 h. The protein levels of CAR, c-Fos, and c-Jun were measured by western blotting (n = 5; *P < 0.05, static vs. LSS or OSS; &P < 0.05, LSS vs. OSS; #P < 0.05, OSS vs. siAP-1 + OSS). e, f Transfected HUVECs were exposed to FSS for 4 h. Levels of mRNAs encoding AP-1 components (c-Jun and c-Fos) and GAPDH were measured by RT-PCR. e n = 4; *P < 0.05, static vs. LSS or OSS; &P < 0.05, LSS vs. OSS; #P < 0.05, OSS vs. pKLF2 + OSS. f n = 4; *P < 0.05, static vs. LSS or OSS; &P < 0.05, LSS vs. OSS; #P < 0.05, LSS vs. siKLF2 + LSS. g AP-1 promoter activity was measured by luciferase reporter assay (n = 5; *P < 0.05, static vs. LSS or OSS; &P < 0.05, LSS vs. OSS; #P < 0.05, OSS vs. pKLF2 + OSS).
