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. 2018 Jul 11;24(2):391–405. doi: 10.1016/j.celrep.2018.06.035

Figure 2.

Figure 2

B Cells from Pten-Deficient Mice Are Committed to Terminal Differentiation

(A) Splenocytes from mice of the indicated genotypes were left untreated (n. t. = non-treated) or stimulated with 2.5 μg/mL LPS and analyzed after 3 and 5 days for B220 and CD138 expression by flow cytometry (pre-gated on CD19+ cells). Bar diagrams besides the representative FACS plots show quantification of CD138+ B cells in LPS- and non-treated splenocytes at days 3 (top) and 5 (bottom) post stimulation. Ptenf/f (n = 7), Ptenf/f × mb1-cre (n = 6) and Ptenf/f × CD19-cre mice (n = 5), mean ± SD. Statistical significance was calculated by using the two-tailed paired (n. t. versus LPS) and otherwise the two-tailed unpaired t test.

(B) Intracellular flow cytometric analysis of pS6, Irf4, and Blimp-1 expression in splenocytes from Figure 2A at day 3. Numbers in the histogram plots indicate the percentages of positive cells. Data are representative of at least 2 mice per genotype.

(C) Immunoblot analysis of Blimp-1, Irf4, pS6, Irf8, and Bach2 expression in splenocytes from Figure 2A at day 3. n. t. = non-treated; Gapdh served as loading control. Data are representative of at least 2 mice per genotype.

(D) Fo.B (CD21+/CD23+, blue), MZ.B (CD21hi/CD23lo/−, red), and CD21lo/CD23− B cells (green) from control (n = 11), Ptenf/f × mb1-cre (n = 5) and Ptenf/f × CD19-cre mice (n = 4) were FACS-purified (for the gating strategy and re-analysis, see Figures S3A, S3B, and S3F) and expression levels of Blimp-1 in the respective B cell populations were determined by quantitative reverse transcriptase (RT)-PCR. Results are shown as relative units (RU) normalized to levels measured in bone-marrow (BM)-derived pro-B cells (CD19+/B220+/IgM−/IgD−/CD25−), mean + SD.

(E) Concentrations of IgM in the sera of Ptenf/f and Ptenf/f × mb1-cre mice were measured by α-IgM ELISA, (n = 29; mean ± SD) (left). Levels of autoreactive antibodies in the respective sera were determined by α-dsDNA-IgM ELISA (Ptenf/f, n = 21; Ptenf/f × mb1-cre, n = 19; mean ± SD) (right).

See also Figures S1–S3.