TLR4 is necessary, but not sufficient to confer A2t responsiveness. (A-B) Intracellular TNF-α staining of 3-day-old human monocytes (A) or THP-1 cells differentiated with 15nM PMA for 3 days (B) after 6-hour treatment with indicated stimuli. (C) HEK293 cells stably transfected with a NF-κB–driven GFP reporter plasmid were transiently transfected with plasmids containing no insert (mock), or encoding TLR4 or TLR4 along with MD-2, then treated with the indicated stimuli (A2t = 100nM, LPS = 100 ng/mL, PMA = 5mM), and GFP fluorescence was measured by FACS 24 hours after treatment. Data are expressed as percentage of GFP induction compared with the PMA-induced fluorescence for the corresponding transfection and experiment. Results are the mean ± SEM of 3 experiments.