| PMC full text: | Nat Immunol. Author manuscript; available in PMC 2016 May 18. Published in final edited form as:
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Fig. 2
(a) Effects of DZNep on EZH2 and H3K27me3 expression in T cells. T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days in the absence or presence of 5 μM DZNep. The expression of EZH2, H3K27me3, H3, and β-actin was measured with immunoblotting. One representative experiment of 4 is shown.
(b,c) Effects of DZNep on polyfunctionality of CD8+ T cells. CD8+ T cells were activated with anti-CD3 and anti-CD28 antibodies for 3 days in the absence or presence of 5 μM DZNep. Expression of IFN-γ, TNF, and granzyme B was analyzed by flow cytometry. The percentage of double (b) and triple-positive (c) cells is shown. Data presented as mean ± SEM, N=5, Wilcoxon rank-sum test, *P < 0.05.
(d–f) Effects of shEZH2 on EZH2 and H3K27me3 expression and polyfunctionality in CD8+ T cells. T cells transfected with shEZH2 or scramble vectors were activated for 3 days. The expression of EZH2 and H3K27me3 was measured with immunoblotting (d). Expression of IFN-γ, TNF, and granzyme B was analyzed by flow cytometry. The percentage of double (e) and triple-positive (f) cells is shown. 3 donors with duplicates. Student's t-test, *P < 0.05.
(g–i) Effects of GSK126 on H3K27me3 expression and polyfunctionality in CD8+ T cells. T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 4 days. 10 μM GSK126 was added on Day 0 and 2. The expression of EZH2, H3K27me3, H3, and β-actin was detected with immunoblotting in 3 donors with duplicates (g). Expression of IFN-γ, TNF, and granzyme B was analyzed by flow cytometry. The percentage of double (h) and triple-positive (i) cells is shown (mean ± SEM, N = 5, Wilcoxon rank-sum, *P < 0.05).
(j) Effects of EZH2 on T cell apoptosis. T cells were treated with 5 μM DZNep, 10 μM GSK126 or shEZH2 transfection and activated with anti-CD3 and anti-CD28 antibodies for 2 days. T cell apoptosis was evaluated with Annexin V and 7-AAD staining. Representative dotplots for one of 5 donors tested.
(k,l) Effects of EZH2 blockade on Bcl-2 expression. T cells were treated with 5 μM DZNep or shEZH2 transfection and activated for 3 days. Expression of Bcl-2, EZH2, and β-actin was detected with immunoblotting. One of 5 experiments is shown.
(m) Effect of EZH2 blockade on Bcl-2 promoter activity. 293T cells were co-transfected with vectors encoding shEZH2 or scramble and hBCL-2-EGFP vector for 2 days. The promoter activity was analyzed by flow cytometry and expressed as the percentage of GFP expression (mean ± SD). N = 4, *P < 0.05.
