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. 2019 Aug 13;10:3644. doi: 10.1038/s41467-019-11570-6

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — B-cell development is negatively affected in the absence of TOP2B. a Absence of TOP2B in B cells results in reduced CD19+ B cells in peripheral blood compared to littermates (n = 6 WT and 7 Top2b^−/− mb1 cre mice per group, mean ± SEM, ****p < 0.0001). b Splenic white pulp subcompartments are reduced in size in the absence of Top2b. Area of three sub-compartments were evaluated in at least ten follicles across three sections (500 μM apart) with marker indicating average per mouse. (Mean SEM, *p < 0.05). Representative follicles from wildtype (c–f) and Top2b^−/− mb1-cre spleens (g–j) show altered splenic architecture. More B220-expressing (red) cells are seen in the wildtype (c, f) than Top2b^−/− mb1-cre mice (g, j). The metallophilic macrophages (blue) indicating the border between marginal zone and follicular region. The periarteriolar lymphoid sheath (PALS) subcompartment contains T cells (green). Scale bar indicates 200 µm. Bone marrow (k) and spleen (l) cells were isolated from wildtype and Top2b^−/− mb1-cre mice. Flow cytometry demonstrates that the bone marrow of Top2b^−/− mb1-cre mice have fewer PrePro B cells (Fr. A, B220+, CD43−, BP1−, CD24−), Pre B cells (Fr. D, B220+, CD43−, IgD−, IgM−), and immature B cells (Fr. E, CD19+, B220+, CD43−, IgM+, IgD−) compared to controls (k). In the spleen (l), there are overall fewer B cells with reductions in marginal zone precursors (CD19+, B220+, IgMhi, IgDhi, CD21/35+), follicular (CD19+, B220+, IgMlo, IgDhi, CD38+), and activated (CD19+, B220+, IgDhi, IgMhi, Flt3+) B cells compared to controls. (n = 4 mice per group, mean ± SEM, absolute numbers calculated from whole tissue numbers). *p < 0.05; **p < 0.01. m qPCR analysis of kappa-deleting recombination excision circles demonstrate that B cells isolated from Top2b^−/− mb1-cre mice have reduced replication in the spleen (n = 6 WT or 9 Top2b^−/− mb1 cre mice, shown as mean ± SEM). *p < 0.05. n Pax5 is significantly decreased in B cells isolated from Top2b^−/− mb1-cre mice (n = 9 WT or 6 Top2b^−/− mb1- cre mice), whereas Rag1 expression (o) is unchanged (n = 5 WT and 4 Top2b^−/− mb1- cre mice per group shown as mean ± SEM, with technical triplicates), **p < 0.005). All by two-tailed Student’s t test