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. 2017 Jul 12;8:16037. doi: 10.1038/ncomms16037

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Copyright © 2017, The Author(s)

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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(a) qRT-PCR analysis for levels of Pea3 group genes in splenic CD4⁺ T cells from Cic^f/f (WT) and Cic^f/fCd4-Cre (cKO) mice. n=3 per each genotype. (b) Chromatin immunoprecipitation (ChIP)-qPCR analysis showing CIC promoter occupancy of Etv4 and Etv5 in CD4⁺ T cells. CD4⁺ T cells purified from spleen of Cic^f/f and Cic^f/fCd4-Cre mice were used. n=3 per each genotype. (c) qRT-PCR analysis for levels of Etv4 and Etv5 in sorted CD4⁺PD-1⁻CXCR5⁻ non-T_FH and CD4⁺PD-1⁺CXCR5⁺ T_FH cells. Splenocytes from 5 to 6 Cic^f/f mice or 3 to 4 Cic^f/fCd4-Cre mice were pooled and subjected to cell sorting. Three independent experiments were conducted. (d) Western blot analysis for levels of ETV4 and ETV5 in sorted splenic CD4⁺PD-1⁺CXCR5⁺ T_FH cells. The dash lines on blot images indicate cropping without image manipulation to either side. Three independent experiments were conducted. The graphs in a–d show data as mean±s.e.m. *P<0.05 and **P<0.01 (two-tailed two-sample unequal variance Student t-test). (e) Western blot analysis for levels of CIC and ETV5 in sorted non-T_FH and T_FH cells from spleen of WT C57BL/6 (B6) mice. The images are representative of two independent experiments. (f) Thy1.1⁺ OT-II cells infected with control or ETV5-expressing retrovirus were adoptively transferred to Thy1.2⁺ B6 recipient mice. Eight days after immunization with NP-OVA in alum, donor cells were analysed for T_FH cell differentiation using flow cytometry. Control-RV, n=12; ETV5-RV, n=9. ***P<0.001 (two-tailed two-sample unequal variance Student t-test). (g) Thy1.1⁺ OT-II cells infected with control (shNC) or Etv5 shRNA (shETV5) expressing retrovirus were subjected to adoptive transfer experiment. Eight days after immunization, donor cells were analysed for T_FH cell differentiation. shNC, n=6; shETV5, n=8. **P<0.01 (two-tailed two-sample unequal variance Student t-test). (h) WT and Cic cKO OT-II cells were prepared from spleens of Thy1.1⁺ OT-II Cic^f/f and Thy1.1⁺ OT-II Cic^f/fCd4-Cre mice, respectively. WT OT-II cells infected with shNC retrovirus and Cic cKO OT-II cells infected with shNC or shETV5-expressing retrovirus were transferred into Thy1.2⁺ B6 recipient mice. Seven days after immunization with NP-OVA in alum, donor cells were analysed for T_FH cell differentiation. WT:shNC, n=9; cKO:shNC, n=6; cKO:shETV5, n=6. ***P<0.001 (two-tailed two-sample unequal variance Student t-test).