Fig. 2. Characterizations of CU-T12-9 as a TLR1/2 agonist, not TLR2/6.

(A) HEK-Blue hTLR2 cells were treated with CU-T12-9 and anti-hTLR1, anti-hTLR2, or anti-hTLR6 antibodies for 24 hours. CU-T12-9 strongly activates QUANTI-Blue SEAP signaling at 60 nM, and anti-hTLR1–IgG (immunoglobulin G) and anti-hTLR2–IgA antibodies can dose-dependently inhibit CU-T12-9–triggered SEAP signaling, whereas anti-hTLR6–IgG has no influence. This demonstrates that CU-T12-9 can activate TLR1/2 signaling, with no activation of TLR2/6. (B) The positive control of Pam₃CSK₄, a TLR1/2 agonist, showed similar activation to CU-T12-9. (C) The positive control of Pam₂CSK₄, a TLR2/6 agonist, showed that anti-hTLR2–IgA and anti-hTLR6–IgG dose-dependently inhibited the Pam₂CSK₄-induced TLR2 and TLR6 QUANTI-Blue SEAP signaling, whereas anti-hTLR1–IgG had no influence. (D) The human monocyte cell line U937 was stably transfected with a GFP-labeled NF-κB reporter gene. The cells sensitive to TLR1/2 activation were sorted using a MoFlo Cytomation fluorescence-activated cell sorter, and 10% of activated cells were collected. The flow cytometric analysis of 0, 1, and 5 μM CU-T12-9 and 66 nM (100 ng/ml) Pam₃CSK₄ triggered NF-κB expression in U937 cells. (E) Flow cytometric analysis for 20 μM CU-T12-9 and GA, along with the blank and positive control in U937 cells. The result demonstrated that CU-T12-9 had excellent NF-κB activation compared with GA. (F) The NF-κB activation of CU-T12-9 can be inhibited by an NF-κB inhibitor, triptolide, in HEK-Blue hTLR2 cells.