FIGURE 2.

Knockdown of KLF6 impairs TNFα- and IL-1β-triggered NF-κB activation. A, efficiencies of KLF6-RNAi plasmids on KLF6 expression. In the upper panels, HEK293 cells (4 × 10⁵) were transfected with expression plasmids for Flag-KLF6 (0.2 μg), Flag-MMP5 (0.05 μg) and the indicated RNAi plasmids (2 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblots with anti-Flag. In the lower panels, HCT116 cells (1 × 10⁶) were transfected with a control or the indicated KLF6-RNAi plasmids (10 μg each). Thirty-six hours later, cells were lysed, and the cell lysates were analyzed by immunoblots with anti-KLF6 or anti-β-actin. B, knockdown of KLF6 inhibits TNFα- and IL-1β-induced NF-κB activation. HEK293 cells (1 × 10⁵) were transfected with the indicated KLF6-RNAi (0.5 μg each) and NF-κB luciferase reporter (0.02 μg) plasmids. Thirty-six hours after transfection, cells were treated with TNFα (20 ng/ml) or IL-1β (10 ng/ml) or left untreated for 10 h before luciferase assays were performed. C, knockdown of KLF6 potentiates poly(I:C)- or LPS-induced activation of NF-κB in TLR3–293 or TLR4–293 cells. TLR3/4–293 cells (5 × 10⁵) were transfected with the indicated KLF6-RNAi (1 μg each) and NF-κB luciferase reporter (0.02 μg) plasmids by lipofectamine 2000. Thirty-six hours after transfection, cells were treated with poly(I:C) (10 μg/ml) or LPS (1 μg/ml) for 6 h before luciferase assays were performed. D, knockdown of KLF6 inhibits TNFα- and IL-1β-induced activation of NF-κB in HeLa and HCT116 cells. HeLa and HCT116 cells were treated as in C except cells are stimulated for TNFα (20 ng/ml) or IL-1β (10 ng/ml) for 10 h. E and F, knockdown of KLF6 inhibits TNFα- and IL-1β-induced expression of MCP1, CXCL2, and IL8. HEK293 cells (4 × 10⁵) were transfected with a control or KLF6-RNAi plasmid (3 μg). Thirty-six hours after transfection, cells were left untreated or treated with TNFα (20 ng/ml) (E) or IL-1β (10 ng/ml) (F) for the indicated time points before qPCR was performed.