Inhibition of AP-1 complexes impairs the viability of cell lines derived from ABC DLBCL. (A) Schematic representation of the structure of transcriptionally active Jun/ATF and inactive Jun/A-Fos complexes illustrating the dominant-negative function of A-Fos. (B) Jurkat T cells were lentivirally transduced with a FLAG-tagged expression construct for A-Fos or an empty vector (mock) as control. Cells were treated with PMA and ionomycin (PI) for the indicated times. Lysates were immunoprecipitated using anti-FLAG sepharose beads and analyzed by immunoblot with the indicated antibodies. (C) Jurkat T cells were electroporated with an IL-2 firefly luciferase reporter and a renilla luciferase reporter, stimulated with PMA and ionomycin for 14 hours and the relative luciferase activity of the cell lysates was determined. (D) Viability of ABC DLBCL and GCB DLBCL cell lines transduced with constructs coexpressing GFP with FLAG-tagged A-Fos (top panel) or DN-IκBα (bottom panel), assessed by flow cytometry. (E-F) HBL-1 cells were transduced with indicated silencing constructs and cell viability was assessed using a PMS/MTS (phenazine methosulfate/3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Efficiency of protein silencing and equal loading (tubulin) was verified by western blot. *A nonspecific band recognized by anti-ATF7. Three independent shRNAs were used for ATF3. (C,E,F) Bars represent means ± standard deviation (SD); differences were statistically significant with **P < .01, ***P < .001 (unpaired Student t test). Data in panels B-E are representative of at least 2 independent experiments.