Figure 7. Tet1/2 are required for resistance to BET inhibitors.

(a) Population doublings of JQ1-treated ESCs relative to DMSO controls during day 4-6 of treatment with 500 nM JQ1. All cells express Nanog and are cultured in S/L. (b) Quantification of number of alkaline phosphatase (AP)-positive colonies with mixed or undifferentiated morphology formed in the presence of continuous JQ1 treatment in control or Tet1/2 double mutant (DKO) ESCs expressing Nanog. (c and d) Population doublings of JQ1-treated ESCs relative to DMSO controls during day 4-6 of treatment with 500 nM JQ1. Cells are cultured in S/L+2i (c) or 2i/L (d). (e and f) Images (e) and quantification (f) of alkaline phosphatase (AP)-positive colonies formed in the presence of continuous JQ1 treatment in control or Tet1/2 DKO ESCs cultured in S/L+2i. (g) Relative chromatin accessibility in JQ1-treated ESCs assessed by ATAC-seq. All peaks associated with each gene were summed and plotted as the log₂ fold change of accessibility with each treatment relative to control (Nanog overexpression or 2i culture). (h and i) Expression of pluripotency genes in Nanog expressing (h) or S/L+2i cultured (i) ESCs treated with 500 nM JQ1 for 72 h relative to DMSO controls. (j) Nanog-GFP expression in JQ1-treated ESCs cultured in S/L+2i in full medium (20 mM glucose, 2 mM glutamine) or medium with reduced glucose (included as a control; Low Glu, 2 mM) or reduced glutamine (Low Gln, 200 μM). Data are presented as mean ± SEM (b, f, h, i) or ± SD (a, c, d, j) of n=3 independent samples. *, p < 0.05; **, p < 0.005; ***, p < 0.0005; ****, p < 0.0001 by 1-way ANOVA (a, c, d, f, j) or 2-way ANOVA (h, i) with Tukey’s multiple comparison post-test.