ESRRB cooperates with GR to potentiate dexamethasone-induced gene expression. RNA was isolated from mouse T-ALL cells infected with NS, Nr3c1, or Esrrb shRNAs treated with vehicle (DMSO) or dexamethasone (100 nM) for 6 hours and sequenced using BGI-seq 500. RSEM was used to quantify RNA-sequencing results. (A) The heat map represents differentially expressed genes between vehicle- and dexamethasone-treated NS cells using log 1.5-fold cutoff (n = 3 replicates). (B) Gene set enrichment analysis (top) on dexamethasone-induced gene set shows significant negative correlation in Esrrb-knockdown leukemic cells treated with dexamethasone (P < .001). Distribution of log fold change across detected genes (bottom). The NS cells exhibit the highest average log fold change when treated with dexamethasone, whereas Nr3c1 or Esrrb knockdown interferes with optimal changes in gene expression. Nr3c1, Bcl2l11, and Tsc22d3 are GR-regulated genes dependent on ESRRB for their expression. (C) Control or Esrrb-knockdown cells (2 independent shRNAs) were treated with dexamethasone for 6 hours, and Nr3c1, Bcl2l11, and Tsc22d3 mRNA expression was analyzed by qRT-PCR. The copy number was normalized to β-actin using the ΔΔCT method. (D) BIM protein expression was analyzed by immunoblot after 12 hours of dexamethasone (100 nM) treatment in NS or Nr3c1- or Esrrb-knockdown cells. Protein was quantified using densitometry and normalized to actin. The results are averages of 3 independent experiments; error bars represent SEM. **P < .01; ***P < .001.