Figure 3. CLIP170 attenuates TLR4- but not TLR9-induced NF-κB activation.

(a) HEK293 cells expressing human TLR4/CD14/MD2 were transfected with increasing concentrations of HA-CLIP170 (50, 100, and 250 ng), pNF-κB-Luc reporter plasmid (100 ng), and pRL-TK (50 ng). (b) For TLR9 assay, HEK293T cells were co-transfected with mTLR9 (200 ng) plasmid along with other plasmids mentioned above. The total amount of DNA was made constant by adding empty vector. Twenty four hours post-transfection, cells were challenged overnight with LPS (300 ng/ml) and ODN (1 μg/ml), respectively. Luciferase activity was measured using the Dual-Luciferase Reporter assay system; the lower panels are immunoblots demonstrating the overexpression of HA-CLIP170. Actin was used as the loading control; (c) Overexpression of CLIP170 in mouse macrophages. J774 cells were transduced with lentiviral particles harboring CLIP170 or empty vector followed by immunoblotting and qPCR. The blot was probed with anti-CLIP170 antibody to examine the overexpression of CLIP170. Actin served as the loading control; (d) CLIP170 suppresses LPS-induced activation of IL-6 and TNF-α in mouse macrophages. J774 cells transduced with CLIP170 expression plasmid or empty vector were challenged with LPS (100 ng/ml) for indicated time points, followed by ELISA and qPCR to quantify the levels of IL-6 and TNF-α. (e - h) Effect of CLIP170 on Pam3CSK4-, ODN-, TNF-α-induced pro-inflammatory cytokines and LPS-induced IFN-β secretion. CLIP170 weakly suppressed Pam3CSK4-induced IL-6 and TNF-α levels (e) whereas it did not affect ODN- (f), TNF-α (g)-induced pro-inflammatory cytokines or LPS-induced IFN-β (h) levels. Data are presented as mean ± SD from at least three independent experiments (*P < 0.05, **P < 0.01 and ***P < 0.001); ns: not significant.