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. 2018 Mar 8;9:1006. doi: 10.1038/s41467-018-03455-x

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© The Author(s) 2018

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 — Impaired γH2AX levels in spleen B cells from CD19-cre-Usp22 KO mice. a AID mRNA measured by qPCR of d3-LPS stimulated spleen B cells from CD19-cre-Usp22 KO or WT littermates (left panel; n = 5 mice per group). AID blot analysis of d3-LPS stimulated spleen B cells from Mb1-cre or CD19-cre Usp22 KO or their WT littermates (right panel; n = 2–3 mice per group); AID-KO cells were used as control and data represent two or three independent experiments. b Spleen B cells from CD19-cre-Usp22 KO and WT littermates were stimulated for IgG3 CSR (by LPS) or for IgA CSR (by LPS+ IL-4+ TGF-β+ IL-5+ anti-IgD dextran) for 2.5 days, exposed to γ-radiation, and then collected for γH2AX immunofluorescent staining. Line within micrograph represents 10 μm. The number of γH2AX foci per cell was analyzed 1 or 4 h post irradiation for Usp22-WT (black circles) and CD19-cre-Usp22-KO (white circles) (bottom panel), and representative images for IgG3-CSR group (γH2AX in green and DAPI in blue) are shown in the top panel. Approximately 30–50 cells were analyzed per sample and data represent two or three independent experiments. c Representative flow cytometry plots (left panel) and compiled cell cycle analysis (right panel) of Usp22-WT and CD19-cre-Usp22-KO spleen B cells. Cells were stimulated with LPS for 2.5 days and then incubated with EdU for 4 h, before FACS staining (n = 3 samples per group). d Purified splenic B cells from CD19-cre Usp22-KO or WT littermates was stimulated with LPS and growth of these cells was monitored over time (n = 3 samples per group). Data were tested by two-way ANOVA. e CFSE-pulsed spleen B cells from CD19-cre-Usp22 KO or WT littermates were stimulated by LPS plus IL-4 for CSR to IgG1 for 4 days. The gating (G2 to G11) is based on the intensity of CFSE and the percentage of IgG1 switch per gate is summarized (n = 3 samples per group). Data in c–e represent two independent experiments; data in a–c and e were analyzed using two-tailed unpaired Student’s t-test. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001