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. 2018 Dec 3;215(12):3094–3114. doi: 10.1084/jem.20180570

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© 2018 Ariës et al.

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Figure 4. — Haploinsufficiency for the core PRC2 components Ezh2 or Eed is sufficient to induce mitochondrial apoptosis resistance in mouse thymocytes. (A) Schema of experimental design. (B) FACS gating and sorting strategy to isolate thymocytes at the indicated developmental stages. Negative sorting was also performed to exclude cells expressing B cell, NK cell, red blood cell, or granulocytic markers. (C and D) Mx1:Cre transgenic, Ezh2 fl/WT (n = 5) or WT/WT (n = 6) siblings were treated with pIpC, and thymocytes were harvested as indicated in A and stained with a panel of antibodies to determine T cell developmental stage (B). PCR was performed on genomic DNA to verify appropriate deletion of Ezh2 (C, top). Stained thymocytes were subjected to BH3 profiling analysis using 1 µM BIM peptide for 25 min, and cytochrome c release was assessed by stage of T cell development using FACS analysis (C, bottom). Results are shown for all stages of T cell development that consistently yielded >250 FACS events for analysis. Significance was assessed by two-way ANOVA with Sidak adjustment for multiple comparisons between WT/WT versus fl/WT only for each stage of T cell development. P = 0.0066 for DN3, 0.049 for DN4, 0.44 for immature single positive (ISP), 0.96 for double positive (DP), 1.0 for CD8 SP, and 0.94 for CD4 SP. (D) Mx1:Cre transgenic, Eed fl/WT (n = 12) or WT/WT (n = 9) siblings were treated with pIpC, and thymocytes were harvested as indicated in A. PCR was performed to verify Eed deletion (D, top), and staining for T cell developmental markers and BH3 profiling analysis were performed as in D (bottom). Results are shown for all stages of T cell development that consistently yielded >250 FACS events for analysis. Significance was assessed by two-way ANOVA with Sidak adjustment for multiple comparisons between WT/WT versus fl/WT only for each stage of T cell development. P = 0.37 for DN3, 0.0017 for DN4, 0.76 for ISP, 1.0 for DP, 0.96 for CD8 SP, and 1.0 for CD4 single positive (SP). *, P ≤ 0.05; **, P ≤ 0.01; n.s., P > 0.05.