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. 2018 Apr 9;128(5):1937–1955. doi: 10.1172/JCI95089

Figure 12. Acetylation of ZMYND8 by p300 is necessary for HIF activation and breast tumor progression.

Figure 12

(A and B) Acetylation of ZMYND8-V5 (A) and WT or mutant FLAG-ZMYND8 (B) in HEK293T cells treated with TSA or DMSO (–) for 6 hours (n = 3). (C) Co-IP assays of BRD4 and WT or mutant FLAG-ZMYND8 in transfected HEK293T cells (n = 3). (D and E) In vitro acetylation assays of WT or K1007/1034R FLAG-ZMYND8 by purified FLAG-p300, FLAG-PCAF, or FLAG-GCN5 (n = 3). (F) Co-IP assays of endogenous ZMYND8 and p300 in MCF-7 cells (n = 3). (G) Acetylation of endogenous ZMYND8 in SC and p300-KD MCF-7 cells (n = 3). (H) Co-IP assays of endogenous ZMYND8 and BRD4 in SC and p300-KD MCF-7 cells (n = 3). (I) RT-qPCR analysis of indicated mRNAs in ZMYND8-rescued MDA-MB-231 cells exposed to 20% or 1% O2 for 24 hours (mean ± SEM, n = 3). *P < 0.05, **P < 0.01, by 2-way ANOVA with Tukey’s t test. (J–L) Clonogenic assays (J), migration assays (K), and invasion assays (L) in ZMYND8-rescued MDA-MB-231 cells exposed to 20% or 1% O2 for 12 days (J), 16 hours (K), or 24 hours (L) (mean ± SEM, n = 3). *P < 0.05, ***P < 0.001, ****P < 0.0001, by 2-way ANOVA with Tukey’s t test. (M–P) Growth of ZMYND8-rescued MDA-MB-231 tumors in mice (M, mean ± SEM, n = 5). Endomucin-positive areas (N) and CC3-positive cell numbers (O) in tumors and lung metastasis (P) were quantified (mean ± SEM, n = 5). **P < 0.01, ***P < 0.001, ****P < 0.0001, by 2-way ANOVA with Tukey’s t test.