PI3K pathway inhibition partially rescues the β1 ^KO MZ B cell phenotype. (A and B) Flow cytometry to identify the induction of CD1d and CD21 (A) and IgM and CD21 (B) expression on transitional B cells from β1^WT and β1^KO mice, cultured on OP9 or OP9-Dll1 cells for 3 d in the absence (DMSO) or presence of the PI3K inhibitor (PI3K-inh.). (C and D) Mean (±SD) frequencies (upper panel) and absolute numbers (lower panel) of the increase in CD1d^hi CD21^hi (C) and IgM⁺CD21^hi cells (D), as gated in A and B. (A–D)n = 3 mice. Each circle in the graphs represents data from one mouse. Data are representative of three independent experiments. Mean and SD are indicated by horizontal lines in the data points; significance is calculated by one-way ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001). (E) Scatter plots depict the gene-expression levels in DMSO-treated (x axis) and PI3K inhibitor–treated (y axis) β1^WT (left panel) and β1^KO (right panel) transitional B cells, cultured in OP9-Dll1 cells, as described in A and B. The unaltered (gray), up- (red), and downregulated (green) genes are highlighted. (F) The Venn diagram represents the overlap of the differentially expressed genes in the primary MZ B cells with the differentially expressed genes in OP9-Dll1 culture of WT cells treated with PI3K-inh. or DMSO. The up- and downregulated genes are grouped separately for each comparison. A 1.5-fold change was used as a cutoff for primary cells and a twofold change for in vitro data. (G) The overlap of differentially expressed genes in the comparison of β1^KO and β1^WT MZ B cells with the upregulated genes in the comparison of OP9-Dll1 culture of WT cells treated with PI3K inhibitor or DMSO. The differentially expressed genes in MZ B cells are ranked (y axis) according to the fold-change (x axis). The genes upregulated after treatment with PI3K inhibitor are indicated (right, red). The key genes of interest are highlighted in the figure. (H) Expression levels (FPKM) of differentially expressed key genes in DMSO or PI3K-inh.–treated transitional β1^WT and β1^KO B cells, cultured in the presence of OP9-Dll1 cells, as described in A and B. Error bars indicates SD; n = 2.