Figure 1.
Figure 1. 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-CreTg/−/conditional NICD1Tg/−/Rosa26-reporter RFPKI/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.

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-CreTg/−/conditional NICD1Tg/−/Rosa26-reporter RFPKI/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.

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