Figure 5.

TAARD activates the TLR-1-NF-κB and TLR3-STAT3 signaling pathways in natural killer (NK) cells. NKL cells were infected with concentrated lentivirus of pSIH1-puro-STAT3 shRNA or pSIH1-puro-control shRNA and selected with puromycin. The knockdown of NKL cells were treated with or without TAARD for 18 h. (A) STAT3 knockdown was confirmed by immunoblotting. (B) Cell-free supernatants were collected for analysis by enzyme-linked immunosorbent assay (ELISA). (C) Cell pellets were harvested for real-time RT-PCR. (D–F) Primary human NK cells and NKL cells were pretreated with the NF-κB inhibitor tosyl-l-phenylalaninechloromethyl ketone (TPCK) (10 µM) and then exposed to TAARD for 18 h. NF-κB phosphorylation inhibition was confirmed by immunoblotting (D) and the supernatants from primary NK (E) and NKL (F) cells were assayed for interferon (IFN)-γ secretion. (G) STAT3-knockdown NKL cells were pretreated with TPCK and then exposed to TAARD for 18 h. The supernatants were collected and analyzed by ELISA. (H–J) Human NK cells were pretreated with 10 µg/mL of a non-specific rat or mouse IgG or neutralizing antibodies against TLR1, TLR3, TLR6, or their different combinations for 1 h. Cells were then treated with 0.1 µM of TAARD for an additional 18 h. Supernatants were assayed for IFN-γ secretion and cells were harvested and lysed for immunoblotting of phosphorylated NF-κB and STAT3. The data represent plots of three donors with similar results. Data from one of three independent experiments with similar results are shown. *p < 0.05, **p < 0.01, and ***p < 0.001 denote statistical comparison between the two marked treatment groups.