Restoration of KLF2 or KLF4 protects endothelial cells from APLA-mediated activation. (A) Endothelial cells were transfected with expression vectors containing scrambled DNA (Scr DNA, control), or KLF2 or KLF4 cDNA. Transfected cells were then incubated with medium alone, β₂GPI (100nM) and anti-β₂GPI antibodies (600nM), or TNF-α (10 ng/mL) for 5 hours, after which endothelial cell activation was measured using an E-selectin enzyme-linked immunosorbent assay. Activation was blocked in cells transfected with KLF2 or KLF4, but not scrambled DNA (**P < .01 and *P < 0.05 for KLF2 or KLF4, respectively, vs control). KLF2 and KLF 4 also blocked cellular activation in response to TNF-α. Error bars represent the mean ± SEM of quadruplicate points, and data are representative of 4 experiments. (B) Cells were treated as in panel A but cotransfected with an E-selectin-luciferase reporter and Renilla luciferase (to control for transfection efficiency). Endothelial cell activation was measured as a function of E-selectin luciferase activity normalized to Renilla. KLF2 and KLF4 inhibited E-selectin transcription compared with cells transfected with the control vector (*P < .02 for each) Error bars represent the mean ± SEM of triplicate points, and data are representative of 3 experiments. (C) Cells were transfected with KLF expression plasmids as described in panel A and incubated with medium alone (control) or β₂GPI and anti–human β₂GPI antibodies for 5 hours. Total RNA was then isolated, and E-selectin mRNA expression was analyzed by quantitative real-time PCR. Expression of KLF2 or KLF4 reduced the induction of E-selectin mRNA expression in response to β₂GPI and anti-β₂GPI antibodies by 6.7- and 2.6-fold, respectively (**P < .001 and *P < 0.01, respectively). Error bars represent the mean ± SEM of triplicate points.