TLR4 mediates LPS-induced chemokine up-regulation and monocytes chemotaxis in LECs. LECs were stimulated with LPS (500 ng/mL) for indicated times (A), 4 hours (C), or 48 hours (E,H), or stimulated with indicated amounts of LPS for 4 hours (B) or 48 hours (D,G). Prior to LPS stimulation, LECs were transfected with control (C), scTLR4 (scT4), or siTLR4 (siT4) (C,E,H). (A-C) The CCL2, CCL5, and CX3CL1 mRNA levels were analyzed by quantitative RT-PCR. The mRNA level of each gene were normalized to GAPDH and presented as a fold increase compared with the control. (D,E) CCL2 and CCL5 protein levels in the LEC culture supernatant were measured by ELISA. (F) Schematic diagram of the monocyte chemotactic migration assay. (G,H) The number of THP-1 monocytes that migrated from the hanging transwell to the culture supernatants of the LPS-stimulated LECs in the bottom were counted and presented as a percentage of the number of initially seeded cells. Bars represent mean plus or minus SD (n = 4). *P < .05 versus LPS−; #P < .05 versus scT4+ LPS+.