Fig 2: Genome-wide identification of ESR2 binding regions in the Granta-519-ESR2 cells. (A) The genome-wide heatmap shows ESR2 peaks’ coverage ±3 kb from the peak center. Each row represents a peak, and the blue color intensity indicates the depth of the signal. (B) Genomic distribution of the ESR2 binding regions. (C) Top enriched DNA motifs of the ESR2-bound sequences identified using HOMER de novo motif analysis and sorted by p-value. (D) Venn diagram comparing distribution and co-occurrence of ERE motif, ERRγ motif, and NFκB-p65 motif. (E) GO BP enrichment analysis of genes located nearest to ESR2 binding regions with ERE motif, ERRγ motif, and NFκB-p65 motif, respectively. The number in a circle represents the number of genes that belong to this BP.

Fig 3: Female reproductive tissues of Esr2-depleted mice are negative for estrogen receptor beta (ERβ) expression. Ovaries (A) and uterine tissue (B) of Esr2 knockout (Esr2−/−) and wild-type (Esr2+/+) animals from the same mouse line were stained with PPZ0506 antibody or with isotype-specific IgG2b negative control. Only granulosa cells (Gr) in Esr2+/+ ovaries strongly express ERβ, whereas in Esr2−/− ovary and uterine tissue no specific nuclear staining was observed. Specific staining is shown by arrowheads in Esr2+/+ and # indicates diffuse non-specific background staining. Scale bar 250 µm (100×) or 50 µm (400×).

Fig 4: ESR2 binding occurs to the DPN-regulated genes in the Granta-519 tumor cells. (A) Venn diagram of ESR2 cistrome and transcriptome data identified 210 differentially regulated genes that showed direct ESR2 binding. (B) Bar graph showing the log2-fold change of genes from tumor cells after DPN treatment with peak scores >30 in the ChIP-seq analysis. Upregulated genes are marked in red, and downregulated genes are marked in blue. (C) The top enriched DNA motifs in the 411 ESR2-bound sequences present in the 210 genes were identified using HOMER de novo motif analysis and sorted by p-value. (D) ESR2 ChIP-seq enrichment signals and localization for FOS, FOSB, NEAT1, and VEGFA binding sites visualized by UCSC genome browser. (E) Venn diagram of ESR2 cistrome with ERE motif and NFκB-p65 motif. (F) GO BP enrichment analysis based on the 210 genes that are differentially regulated and have ESR2 binding regions with ERE motif and/or p65 motif, respectively. The number in a circle represents the number of genes that belong to this BP. (G) Gene concept network depicting the linkages of significant genes and GO terms. Upregulated genes are marked in orange, and downregulated genes are marked in blue. The size of a GO BP circle represents the number of genes attributed to it.

Fig 5: The ESR2 selective-agonist DPN inhibits growth in vivo of both ibrutinib-sensitive and -resistant MCL grafted to mice. (A) Male NSG mice were injected subcutaneously with Granta-519 cells and treated subcutaneously daily with vehicle or the ESR2-selective agonist DPN (12.5 mmol/kg body weight). Both groups consisted of 7 mice. Tumor size was measured daily. (B) Mino, Maver-1, Z-138, or JeKo-1 cells were engrafted into male NSG mice and treated subcutaneously daily with vehicle, ibrutinib (5 mg/kg body weight), DPN (12.5 mmol/kg body weight), or with both drugs. Tumor size was measured daily. Each group consisted of 6–9 mice, depending on the grafted tumor cell. (C) Ki67 immunostaining in sectioned Granta-519 tumors at the size of 1.3 cm3. (D) TUNEL staining in sectioned Granta-519 tumors at the size of 1.3 cm3. The percent of stained cells from 10 randomly chosen fields (at 200× magnification) were counted for each sample. Data are shown as means ± SD. Unpaired two-tailed t-test was used for statistical analysis between the two groups, * p < 0.05, ** p < 0.01, *** p < 0.001.