TLR4 in LECs is a functional receptor mediating LPS-induced NF-κB signaling. (A) p65 immunostaining (a subunit of NF-κB; green) in LECs in the absence (LPS−) and presence (LPS+) of LPS stimulation (50 ng/mL for 30 minutes). Scale bars indicate 10 μm. (B) Immunoblotting analysis for phosphorylated IκB-α, IκB-α, and β-actin proteins in LECs after LPS (50 ng/mL) stimulation for indicated times. (C,D) p65 immunostaining in BECs and LECs after LPS stimulation (500 ng/mL for 60 minutes). Scale bars indicate 100 μm. (D) Cells positive for p65 intranuclear staining were counted; the values are presented as a percentage of the total cell number. Bars represent mean plus or minus SD (n = 4). *P < .05 versus BECs. (E) TLR4 knock-down by siTLR4 (siT4) but not scTLR4 (scT4) was confirmed by semiquantitative RT-PCR in LECs. (F,G) LECs were transfected with C (control transfection), scT4, and siT4, stimulated with LPS (500 ng/mL for 60 minutes), and immunostained for p65. Scale bars indicate 100 μm. Cells positive for p65 intranuclear staining were counted and presented as a percentage of total cell number. Bars represent means plus or minus SD (n = 4). *P < .05 versus LPS−; #P < .05 versus scT4 plus LPS+.