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. Author manuscript; available in PMC: 2020 Dec 15.
Published in final edited form as: Nat Cell Biol. 2020 May 18;22(6):701–715. doi: 10.1038/s41556-020-0514-z

Extended Data Fig. 5. GATA3 is required for maintenance of LOSS enhancers and expression of epithelial makers.

Extended Data Fig. 5

a, Our pyrosequencing analyses (bottom), and published DNA methylation data from three different endocrine-resistant MCF7-derived lines (TamR: tamoxifen-resistant, FASR: fulvestrant-resistant, MCF7X: estrogen deprivation-resistant) (top). DNA methylation level at GATA3 locus is significantly increased in endocrine-resistant lines. n=3 independent experiments, two-sided t-tests.

b, RT-qPCR showing transcript levels of GATA3 in MCF7P and TamR with or without 5-Aza treatment for 100 hours. n=2 independent experiments.

c, Heatmap generated by integrating TCGA data on GATA3 mRNA level, DNA methylation, and breast cancer subtype. High DNA methylation and low GATA3 expression are associated with invasive breast cancers (ER-/PR-/ HER2- and basal subtype).

d, Aggregate plots of normalized GRO-seq tag density in MCF7P with shCtrl or shGATA3.

e, Heatmap of ChIP-seq (bottom) showing that GATA3 OE in TamR can re-activate LOSS enhancers. Western blot confirms GATA3 overexpression (top).

f, Heatmap depiction of the downregulation of epithelial genes after KD GATA3 in MCF7P. n=2 independent experiments.

g, Western blot of the indicated epithelial markers in MCF7P cells upon GATA3 KD.

h, CCK8 assays with 4-OHT treatment for 5 days. Dox-induced GATA3 overexpression in TamR re-sensitizes them to 4-OHT. n=3 independent experiments, mean ± s.d., two-sided t-tests.

i, j, Tumor growth curves (i) and representative tumor images at end point (j) of orthotopic xenografts of manipulated TamR cells in nude mice (n=4/group). After tumors reached ~200mm3, tumor sizes were measured once a week upon starting doxycycline water diet and subcutaneous injections of tamoxifen (1 mg/mouse, three times/week). Mean ± s.d., two-sided t-tests.

k, Relapse free survival (RFS) curves generated from kmplot website according to BCL2 levels in patients receiving endocrine therapy. P values were determined by log-rank test.

Immunoblots are representative of two independent experiments. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 5. Statistical source data are available in Statistical Source Data Extended Data Fig. 5.