Figure 3.

TRIB1 mediates expression of CCND1 via regulation of NFκB and AP1. A. Schematic representation of -1080-Cyclin D1 (D1) pGL3 basic luciferase reporter constructs and deletion mutants. AP1, EtsA/B (both Ets A and B were deleted), and both NFκB sites (κB1 and κB2) were eliminated as shown. B. Cells were treated with NC or TRIB1 siRNA pools for 24h followed by transfection with 1μg of CCND1-WT, AP1-mutant, EtsA/B-mutant, or κB1/2-mutant promoter-reporter constructs. Luciferase activity was measured and standardized by co-transfection with SEAP expression vectors. Results represent the means and standard deviations from three independent experiments. (RLU, relative light units). C. TRIB1 regulates NFκB-responsive promoter activity. Cells were treated as in (B) and transfected with NFκB-responsive reporter vector and SEAP expression control vector, and treated with TNFα for 4 h. Cells were co-transfected with 1μg vector-EGFP or TRIB1-EGFP and with NFκB -promoter vector/SEAP and stimulated with TNFα for 4 h. D. TRIB1 knockdown leads to downregulation of NFκB target genes. Levels of CXCL1, CSF2, and IL8 proteins were measured using Luminex assay for secreted cytokines. E. TRIB1 knockdown inhibits NFκB signaling via activation of IκBa and inhibition of p50 and pIKKα. IB analysis of cells treated with NC or TRIB1 siRNA pools with indicated antibodies.