RelB and cRel coordinate together to provide proper BAFF-mediated survival signals in vitro. (A) FACS plots of in vitro survival assay of whole splenic wild-type, Relb^−/−, cRel^−/−, and Relb^−/−cRel^−/− B cells stimulated with BAFF ligand for 70 hours. Numbers represent the percentage of live cells (7AAD⁻) found in culture. Graphical representation of the FACS plots (right), n = 3. (B) FACS plots of in vitro survival assay of FO (CD23⁺) wild-type and Relb^−/−cRel^−/− B cells stimulated with BAFF for 40 hours. (C) RNA-seq analysis from BAFF-stimulated CD23⁺wild-type, cRel^−/−, and Relb^−/−cRel^−/− B cells at the indicated time points. Genes (517) were upregulated in BAFF-stimulated follicular B cells; 289 of these showed substantial expression defect in B cells lacking both cRel and RelB (middle and bottom panels). Of these, 127 showed expression defects even in the single cRel knockout (middle panel); 162 showed expression defects only in the Relb^−/−cRel^−/− double knockout (bottom panel). (D) cRel-dependent genes protect cells against cell death. Gene ontology analysis identifies distinct process terms for cRel-dependent vs RelB/cRel-dependent gene clusters. Whereas RelB/cRel-dependent clusters are significantly associated with terms describing metabolic processes, the cRel-dependent cluster shows overrepresentation of negative regulation of cell death/apoptosis. *P < .05; **P < .005; ***P < .001. Also see supplemental Figure 1. (E) List of representative cRel-dependent and RelB/cRel-dependent genes identified as “negative regulators of cell death” and “metabolic process,” respectively, by gene ontology analysis.