human endometrial stromal cells  (ATCC)

Journal: bioRxiv

Article Title: Deciphering ESR1-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape

doi: 10.1101/2025.04.23.650343

Figure Lengend Snippet: (A) Schematic for engineering cells with split plasmid CRISPR activation system using dCas9-VPR-Blast. (B) RT-qPCR analysis of dCas9-VPR expression in non-transduced cells, dCas9-VPR transduced cells cultured in blasticidin for 8 days, and dCas9-VPR transduced cells cultured in the absence of blasticidin for 8 days. Error bars are shown as mean with SEM. (C) UCSC genome browser view showing the location of the six gRNAs designed to target near alternate ESR1 transcription start sites annotated in the NCBI Refseq database. Tracks show H3K27ac ChIP-seq in myometrial tissue (orange) and H3K27ac Cut&Run in THESC (red) (unpublished data). (D) RT-qPCR analysis of ESR1 expression in THESC dCas9-VPR transduced with ESR1-3 gRNA (THESC ESR1 ) in comparison to control NT gRNA (THESC NT ). (E) Western blot analysis of ESR1 and GAPDH expression in THESC ESR1 and THESC NT . (G) RT-qPCR analysis of ESR1 target genes in THESC ESR1 and THESC NT treated with vehicle or 10 nM E2 for 24 hours. Statistical significance was determined by student’s t-test for two comparisons, and one-way ANOVA for more than two comparisons, with significance defined as p-value < 0.05. THESC = immortalized human endometrial stromal cells; SEM = standard error of the mean; TSS = transcription start site; THESC = immortalized human endometrial stromal cells; E2 = 10 nM estradiol.

Article Snippet: Telomerase-transformed human endometrial stromal cells, THESC ( ) (ATCC, CRL-4003), were maintained in DMEM/F-12 (Gibco Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco Thermo Fisher Scientific) and antibiotics (10 000 IU/mL penicillin, 10 000 IU/ mL streptomycin; Life Technologies, Grand Island, NY, USA), termed regular hESC media.

Techniques: Plasmid Preparation, CRISPR, Activation Assay, Quantitative RT-PCR, Expressing, Cell Culture, ChIP-sequencing, Transduction, Comparison, Control, Western Blot

Journal: bioRxiv

Article Title: Deciphering ESR1-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape

doi: 10.1101/2025.04.23.650343

Figure Lengend Snippet: (A) Schematic of comparisons to investigate E2-dependent and independent actions in THESC by bulk RNA-seq. (B) Volcano plot representation and (C) IPA pathway analysis of differentially expressed genes in response to unliganded ESR1 by comparing vehicle-treated THESC ESR1 and THESC NT . (D) Volcano plot representation and (E) select pathways from IPA pathway analysis of differentially expressed genes in response to liganded ESR1 by comparing THESC ESR1 treated with 10 nM E2 or vehicle. (F) Bar graph showing the number and percentage of E2-independent and dependent DEGs that are active (FPKM > 1) in human endometrial biopsies at the proliferative stage from published RNA-seq data (GSE132713). THESC = immortalized human endometrial stromal cells; THESC ESR1 = ESR1 activated cells; THESC NT = control non-targeting cells; E2 = 10 nM estradiol; vehicle = 0.1% ethanol; IPA = ingenuity pathway analysis

Article Snippet: Telomerase-transformed human endometrial stromal cells, THESC ( ) (ATCC, CRL-4003), were maintained in DMEM/F-12 (Gibco Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco Thermo Fisher Scientific) and antibiotics (10 000 IU/mL penicillin, 10 000 IU/ mL streptomycin; Life Technologies, Grand Island, NY, USA), termed regular hESC media.

Techniques: RNA Sequencing, Control

Journal: bioRxiv

Article Title: Deciphering ESR1-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape

doi: 10.1101/2025.04.23.650343

Figure Lengend Snippet: ESR1 E2-dependent and E2-independent cistrome in engineered THESC. (A) Table showing the number of ESR1 peaks in THESC ESR1 after treatment with E2 or vehicle for 1, 3, or 6 hours. Experiment was performed in biological duplicates and only peaks present in both duplicates were reported. (B) Venn diagram comparing number and location of ESR1 peaks after treatment with E2 for 1, 3, or 6 hours. (C) Venn diagram comparing number and location of ESR1 peaks in the presence or absence of E2 at the 3-hour timepoint. (D) Distance of ESR1 peaks (treated with E2 for 3 hours) from the nearest TSS, and location relative to annotated genes. (E) Venn diagram comparing number and location of ESR1 peaks in THESC ESR1 (treated with E2 for 3 hours) and ESR1 ChIP-seq peaks in proliferative endometrial biopsies from previously published data (GSE200807). (F) Summary of the top HOMER known motifs enriched in ESR1 peaks in THESC ESR1 treated with E2 for 3 hours. (G) Venn diagram comparing all genes whose TSS is within 100 kb of ESR1 peaks in THESC ESR1 (treated with E2 for 3 hours) and genes regulated by ESR1/E2 in THESC ESR1 (E2-dependent DEGs). THESC = immortalized human endometrial stromal cells; THESC ESR1 = ESR1 activated cells; E2 = 10 nM estradiol; vehicle = 0.1% ethanol; TSS = transcription start site.

Article Snippet: Telomerase-transformed human endometrial stromal cells, THESC ( ) (ATCC, CRL-4003), were maintained in DMEM/F-12 (Gibco Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco Thermo Fisher Scientific) and antibiotics (10 000 IU/mL penicillin, 10 000 IU/ mL streptomycin; Life Technologies, Grand Island, NY, USA), termed regular hESC media.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: Deciphering ESR1-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape

doi: 10.1101/2025.04.23.650343

Figure Lengend Snippet: (A) Table showing the number of genes with proximal or anchored ESR1 binding (identified through integration of H3K27ac HiChIP and ESR1 Cut&Run), as well as overlap with 369 genes regulated by ESR1/E2 in THESC ESR1 (E2-dependent DEGs). (B) Heatmap generated via unsupervised hierarchical clustering of 80 genes regulated by ESR1/E2 with proximal or anchored ESR1 binding at the promoter. (C) Comparison between genes regulated by ESR1/E2 with proximal or anchored ESR1 binding at the promoter, and genes differentially expressed in response to EPC from previously published RNA-seq (GSE205481). (D) Bar chart comparing RNA-seq log2CPM normalized counts for FOXO1 and IL6R in THESC ESR1 treated with E2 or vehicle for 24 hours. Error bars are shown as mean with SEM. (E) UCSC genome browser views around the FOXO1 and IL6R loci. Tracks show ESR1 Cut&Run in THESC ESR1 treated with E2 for 3 hours, with IgG background subtracted (black), H3K27ac Cut&Run in THESC (red), and H3K27ac HiChIP loops in EPC treated hESC (blue). The promoter of the gene of interest is highlighted in pink, ESR1 binding peaks are highlighted in blue, and the HiChIP loop that connects the gene promoter with the ESR1 binding peak is highlighted with a blue loop. THESC = immortalized human endometrial stromal cells; EPC = 10 nM E2, 1 uM medroxyprogesterone acetate, and 100 uM cAMP; THESC ESR1 = ESR1 activated cells; E2 = 10 nM estradiol; CPM = counts per million; SEM = standard error of the mean.

Article Snippet: Telomerase-transformed human endometrial stromal cells, THESC ( ) (ATCC, CRL-4003), were maintained in DMEM/F-12 (Gibco Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco Thermo Fisher Scientific) and antibiotics (10 000 IU/mL penicillin, 10 000 IU/ mL streptomycin; Life Technologies, Grand Island, NY, USA), termed regular hESC media.

Techniques: Binding Assay, HiChIP, Generated, Comparison, RNA Sequencing

Journal: bioRxiv

Article Title: Deciphering ESR1-driven transcription in human endometrial stromal cells via transcriptome, cistrome, and integration with chromatin landscape

doi: 10.1101/2025.04.23.650343

Figure Lengend Snippet: (A) Table showing the number of genes with proximal or anchored ESR1 binding (identified through integration of H3K27ac vehicle HiChIP and ESR1 Cut&Run), as well as overlap with 369 genes regulated by ESR1/E2 in engineered THESC (E2-dependent DEGs). (B) Select pathways from metascape pathway analysis of 75 genes regulated by ESR1/E2 with proximal or anchored ESR1 binding at the promoter. (C) Bar chart comparing RNA-seq log2CPM normalized counts for ERRFI1, NRIP1, and EPAS1 in THESC ESR1 treated with E2 or vehicle for 24 hours. Error bars are shown as mean with SEM. (D) UCSC genome browser views around the ERRFI1, NRIP1, and EPAS1 loci. Tracks show ESR1 Cut&Run in THESC ESR1 treated with E2 for 3 hours, with IgG background subtracted (black), H3K27ac Cut&Run in THESC (red), and H3K27ac HiChIP loops in vehicle treated hESC (blue). The promoter of the gene of interest is highlighted in pink, ESR1 binding peaks are highlighted in blue, and the HiChIP loop that connects the gene promoter with the ESR1 binding peak is highlighted with a blue loop. (E) Cell viability assessed by MTT assay in THESC ESR1 and THESC NT treated with vehicle or E2 for 4 days. Statistical significance was determined by one-way ANOVA with significance defined as p-value < 0.05. Error bars are shown as mean with SEM. (F) Microscopic images and quantification of density of migrated THESC ESR1 and THESC NT treated with vehicle or E2 for 72 hours. Cells were stained with 1% crystal violet solution, and cell density was quantified using ImageJ. Statistical significance was determined by one-way ANOVA with significance defined as p-value < 0.05. Error bars are shown as mean with SEM. THESC = immortalized human endometrial stromal cells; THESC ESR1 = ESR1 activated cells; SEM = standard error of the mean; THESC NT = control non-targeting cells; E2 = 10 nM estradiol; vehicle = 0.1% ethanol; EPC = 10 nM E2, 1 uM medroxyprogesterone acetate, and 100 uM cAMP; CPM = counts per million; SEM = standard error of the mean.

Article Snippet: Telomerase-transformed human endometrial stromal cells, THESC ( ) (ATCC, CRL-4003), were maintained in DMEM/F-12 (Gibco Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco Thermo Fisher Scientific) and antibiotics (10 000 IU/mL penicillin, 10 000 IU/ mL streptomycin; Life Technologies, Grand Island, NY, USA), termed regular hESC media.

Techniques: Binding Assay, HiChIP, RNA Sequencing, MTT Assay, Staining, Control