B-cell subsets and characteristics of the conditional mouse model harboring Notch1-activated mature B cells. (A) Schematic structure of the Tg and KI alleles of the Aicda-Cre^Tg/−/conditional NICD1^Tg/−/Rosa26-reporter RFP^KI/WT mice (NICD1 mice). The NICD1 transgene is preceded by a transcriptional stopper (CAT) flanked by a pair of loxP sites (white triangles). The reporter RFP locus harbors 2 pairs of loxP sites (white and pink triangles) that are pairwise compatible. Constitutive expression of NICD1 and RFP was achieved via Cre-dependent recombination steps involving deletion of the stoppers and inversion of the RFP gene. (B) The percentage of RFP-expressing cells within the splenic CD19⁺ B-cell, CD19⁺CD95⁺ B-cell, and CD3⁺ T-cell populations of NICD1 mice and controls was evaluated by flow cytometry. (C) NICD1 protein expression in splenic CD19⁺RFP⁺ B cells and CD19⁺RFP⁻ B cells from NICD1 mice and controls, as examined by intracellular flow cytometric analysis. (D) Expression of mRNA encoding NRARP and Notch3 in splenic CD19⁺RFP⁺ B cells isolated from NICD1 mice and controls, as evaluated by quantitative PCR. Expression levels are expressed relative to those detected in RFP⁺ B cells from control mice. (E) CD21 vs CD23 and IgM vs IgD profiles of splenic B220⁺CD19⁺ B cells from NICD1 mice and controls, as examined by flow cytometry. (F) RFP vs CD95 and CD80 profiles of splenic B220⁺CD19⁺ B cells, and RFP vs CD138 profiles of splenocytes from NICD1 mice and controls. (G) Flow cytometric analysis of apoptotic splenic CD19⁺CD95⁺ GC B cells from NICD1 mice and controls, as detected by staining for Annexin V and DAPI. In panels B and E-G, the mean percentage of cells from 3 individual mice per group is shown. In panels D and G, data in the bar graphs are expressed as the mean and standard error of the mean (SEM) from 3 individual mice per group. In panel D, **P < .01 and ***P < .001 (Tukey-Kramer test). In panels F-G, *P < .05 (Student t test). FO, follicular B cell; M, mature B cell; MZ, marginal zone B cell; T1, transitional 1 B cell; T2, transitional 2 B cell.