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. 2017 Nov 21;7:15928. doi: 10.1038/s41598-017-15918-0

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© The Author(s) 2017

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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Mature NCOR1-null SP cells display elevated CD127 and BCL2 levels. (a) Immunoblot showing the three BIM isoforms (BIM_EL, BIM_L, BIM_S) and BCL-xL expression in 4 independent NCOR1 cKO^Cd4 (lane 1–4) and 3 independent WT (lane 5–7) thymocyte samples. Whole cell lysates from ~5 × 10⁶ cells were loaded and ERK1/2 was used as loading control. The pictures were cropped and uncropped pictures are shown in Supplementary Fig. 3. (b) Histograms depict pan BIM expression in WT and NCOR1 cKO^Cd4 DP, CD4SP and CD8SP thymocytes. (c) Flow cytometry analysis of CD69 and TCRβ expression on DP thymocytes isolated from WT and NCOR1 cKO^Cd4 mice. Histograms on the right show surface expression of CD127 and intracellular expression of BCL2 expression in the indicated subsets. (d) Flow cytometry analysis of BCL2 and CD127 expression on TCRβ^loCD69⁺ DP thymocytes isolated from WT and NCOR1 cKO^Cd4 mice. (e) The diagram indicates the percentage of BCL2⁺CD127⁺ TCRβ^loCD69⁺ DP thymocytes. (f) Histograms depict CD127 (upper panel) and BCL2 (lower panel) expression in TCRβ^hiCD69⁻ thymocyte subsets (total, CD4SP and CD8SP) in WT and NCOR1 cKO^Cd4 mice. (g) The diagrams indicate geometric mean fluorescence intensity (gMFI) of CD127 and BCL2 expression on the particular subset, and show the summary of 3 independent experiments (2–5 mice were analyzed per group in an experiment, average gMFI for each experiment is shown). The lines connect samples analyzed in the same experiment. (e) *P < 0.01, unpaired two-tailed Student’s t-test. (g) *P < 0.01, n.s., not significant, one sample t-test (columns statistics; WT was set as 1 and relative NCOR1 cKO^Cd4 values were calculated, diagrams show absolute gMFI levels for each experiment). Data are representative (a,b,c,d,f) or show the summary (e,g) of 7 (a), 7–8 (b), 9–10 (d,e) mice that were analyzed in 2 (a,b) and 3 (c,d,e) independent experiments. Histograms (c,f) are representative of 9–10 mice analyzed in 3 independent experiments.