FIGURE 1.

Overexpression of KLF6 potentiates TNFα- and IL-1β-induced NF-κB activation. A, identification of KLF6 (61A3) as a positive regulator of TNFα- and IL-1β-induced NF-κB activation. Human and mouse expression cDNA clones (Origene Inc.) (0.05 μg) were individually transfected into HEK293 cells (5 × 10⁴) together with an NF-κB luciferase reporter (0.02 μg). Twenty hours after transfection, cells were treated with TNFα (20 ng/ml) or IL-1β (10 ng/ml) or left untreated for 10 h before reporter assays were performed. B, KLF6 but not other members of the KLF family potentiated TNFα- and IL-1β-induced NF-κB activation. Experiments were performed as in A except that indicated plasmids were used. C, KLF6 but not its splicing variants potentiated TNFα- and IL-1β- induced NF-κB activation. HEK293 cells are transfected with KLF6-WT and KLF6 SV1/2/3 expression plasmid (0.05 μg) together with an NF-κB luciferase reporter (0.02 μg). Twenty hours after transfection, cells were treated with TNFα (20 ng/ml) or IL-1β (10 ng/ml) or left untreated for 5 or 10 h before reporter assays were performed. (WT: wild-type; SV1: splicing variant 1; SV2: splicing variant 2; SV3: splicing variant 3). D, overexpression of KLF6 potentiates TNFα- and IL-1β induced NF-κB activation in a dose-dependent manner. HEK293 cells (1 × 10⁵) were transfected with Flag-KLF6 expression plasmid (0, 0.01, 0.02, or 0.05 μg) together with an NF-κB luciferase reporter (0.02 μg). Twenty hours after transfection, cells were treated with TNFα (20 ng/ml) or IL-1β (10 ng/ml) or left untreated for 10 h before reporter assays were performed. E, overexpression of KLF6 potentiates TNFα- and IL-1β-induced activation of NF-κB in HeLa and HCT116 cells. HeLa or HCT116 cells (5 × 10⁵) were transfected with NF-κB luciferase reporter (0.04 μg) and Flag-KLF6 plasmid (0.05 μg) by lipofatamine 2000. Twenty hours after transfection, cells were treated with TNFα (20 ng/ml) or IL-1β (10 ng/ml) or left untreated for 10 h before reporter assays were performed. F, overexpression of KLF6 potentiates poly(I:C)- or LPS-induced activation of NF-κB in TLR3–293 or TLR4–293 cells. TLR3/4–293 cells were treated as in E except cells are stimulated with poly(I:C) (10 μg/ml) or LPS (1 μg/ml) for 6 h. G and H, overexpression of KLF6 potentiates TNFα- and IL-1β-induced expression of MCP1, CXCL2, and IL8. HEK293 cells (4 × 10⁵) were transfected with an empty control vector or Flag-KLF6 plasmid (0.5 μg). Twenty hours after transfection, cells were treated with TNFα (20 ng/ml) (G) or IL-1β (10 ng/ml) (H) or left untreated for the indicated time points before qPCR was performed.