Accumulation of T1 B cells in TSC1KO mice is cell intrinsic.

<p>Sublethally irradiated <i>Rag-1</i>-deficient mice were reconstituted with mixed CD45.1⁺ WT and CD45.2⁺ TSC1KO BM cells at 1:1 ratio. Recipient mice were analyzed similar to the method shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127527#pone.0127527.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127527#pone.0127527.g002" target="_blank">2</a>, two months after reconstitution. (A) Assessment of B cell development in the BM. Developing B cells (R1) and mature B cells (R2) in the BM were identified by B220 and CD93 expression. IgM^-IgD^- cells (R3) from R1-gated cells were further analyzed for CD45.1 and CD45.2 expression. Bar graph shows mean ± SEM of percentages of CD45.1 and CD45.2 positive cells in the indicated populations. (B) Assessment of B cell development in the spleen. Splenic developing (R1) and mature (R2) B cell compartments were identified by B220 and CD93 expression. T1/T2 B cells identified by R1 and R3 gating were further divided into CD45.1⁺ and CD45.2⁺ populations (top panels). Mature B cell compartment (R2) was further divided by IgM/IgD expression into mature follicular (R6) and marginal zone (R7) B cell compartments. CD45.1/CD45.2 expressions in each compartment are shown. Bar graphs are mean ± SEM of CD45.1 and CD45.2 percentages in the indicated populations (n = 4). (C) Assessment of <i>TSC1</i> deletion in B cell subsets. Genomic DNA from sorted B cell subsets was used to quantify <i>TSC1</i> gene in WT configuration by real-time qPCR. Bar graphs represent relative genomic DNA levels of undeleted <i>TSC1</i> gene. Data shown represent or are calculated from two experiments. *p<0.01; **p<0.01; ***p<0.001.