hbec cultures  (Lonza)

Journal: bioRxiv

Article Title: Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

doi: 10.1101/2025.03.13.643100

Figure Lengend Snippet: A ) Vector map of pIND- ESR1 . rtTA and GFP are constitutively expressed by EF1α promoter and ESR1 is expressed by TRE2 promoter. B) ERα protein levels in 76N TERT- ESR1 , MCF10A- ESR1 , HMECC- ESR1 and ME16C2- ESR1 . GFP + cells pooled and treated with 100ng/ml doxycycline show similar levels of ERα for each HBEC- ESR1 as MCF7.

Article Snippet: HBEC- ESR1 cells were plated in 8-chambered slides (CELLTREAT #229168) at 30,000 cells per chamber in F media.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

doi: 10.1101/2025.03.13.643100

Figure Lengend Snippet: ERα transactivation with 3X ERE-TATA-Luc luciferase reporter assays were performed in A) 76N TERT- ESR1 , B) MCF10A- ESR1 , C ) HME-CC- ESR1 , and D) ME16C2- ESR1 cell lines. Control response (grey bar) was compared with E2 response (blue bar) and ICI response (black bars). Without any ERα induction (No Dox) E2 responses are not significantly different from Control in 76N TERT- ESR1 (A) and HME-CC- ESR1 (C) but significantly increased in MCF10A- ESR1 ( B ) and ME16C2- ESR1 (D) . Increase in doxycycline levels led to higher transactivation in all cells. ICI response was similar to Control in all levels of doxycycline. (* p < 0.05). E) Expression of TFF1 and GREB1 are ERα inducible in HBEC- ESR1 lines. In No Dox condition, no expression of TFF1 and GREB1 was detected in qRT-PCR. 76N TERT- ESR1 and HME-CC- ESR1 expressed TFF1 and GREB1 with Dox+Control. All 4 cell lines showed strong induction of TFF1 and GREB1 following with Dox+E2 treatment. Conversely, Dox+ICI treatment repressed both gene expression.

Article Snippet: HBEC- ESR1 cells were plated in 8-chambered slides (CELLTREAT #229168) at 30,000 cells per chamber in F media.

Techniques: Luciferase, Control, Expressing, Quantitative RT-PCR, Gene Expression

Journal: bioRxiv

Article Title: Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

doi: 10.1101/2025.03.13.643100

Figure Lengend Snippet: Cell proliferation of HBEC- ESR1 after treatment with Dox+Control, Dox+E2 or Dox+ICI. A ) 76N TERT- ESR1 , B ) MCF10A- ESR1 , C ) HME-CC- ESR1 show increase in proliferation with Dox+E2. Dox+ICI shows same level of proliferation as Dox+Control. D ) ME16C2- ESR1 showed no difference in cell proliferation between treatments. (* p < 0.05). Error bars indicate SEM.

Article Snippet: HBEC- ESR1 cells were plated in 8-chambered slides (CELLTREAT #229168) at 30,000 cells per chamber in F media.

Techniques: Control

Journal: bioRxiv

Article Title: Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

doi: 10.1101/2025.03.13.643100

Figure Lengend Snippet: A ) Experimental design for conditioned growth experiment. Donor HBEC- ESR1 (i.e. either 76N TERT- ESR1 or MCF10A- ESR1 ) were treated with Dox+Control, Dox+E2 or Dox +ICI. Conditioned media (CM) from these cells were collected at 48h and 72h, filtered, diluted 1:1 with minimal growth media and added to the receiver cell as shown in B-D . Proliferation of the receiver cells were monitored for 5 days with Alamar blue reduction. B ) Proliferation of parental 76N TERT cell line with conditioned media from 76N TERT- ESR1 cells. C ) Proliferation of parental MCF10A cell line with conditioned media from MCF10A- ESR1 cells. D ) Proliferation of T47D cells treated with conditioned media from MCF10A- ESR1 cells.

Article Snippet: HBEC- ESR1 cells were plated in 8-chambered slides (CELLTREAT #229168) at 30,000 cells per chamber in F media.

Techniques: Control

Journal: bioRxiv

Article Title: Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

doi: 10.1101/2025.03.13.643100

Figure Lengend Snippet: A) PCA of top 1000 variant genes of estrogen treatment (Dox+E2) vs vehicle control (Dox+Control) with all HBEC- ESR1 show 682 genes which are differentially expressed (red circle) (FRD < 0.05). Top 10 differentially expressed genes (DGE) are labelled with Gene symbols. D) Overlap of HBEC- ESR1 estrogen response DGEs with MCF7 and T47D. E) Heatmap of 682 HBEC- ESR1 DEGs showing inverse trend in E2 and ICI treatments, and similar trend in BC cell lines (MCF7 and T47D). F) GSEA analysis of HBEC- ESR1 Dox+E2 vs Dox+Control expression data identified 8 pathways positively enriched and 1 negatively enriched in Dox+E2 treated cells (FDR 0.25). G) Enrichment plot of DNA repair pathway comparing Dox+E2 vs Dox vs Dox+Control.

Article Snippet: HBEC- ESR1 cells were plated in 8-chambered slides (CELLTREAT #229168) at 30,000 cells per chamber in F media.

Techniques: Variant Assay, Control, Expressing

Journal: bioRxiv

Article Title: Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

doi: 10.1101/2025.03.13.643100

Figure Lengend Snippet: A) Immunofluorescence staining of γH2AX (red) and nucleus (blue) in HBEC- ESR1 cells treated with Dox+Control or Dox+E2. B) Quantification of nuclear γH2AX in A . 76N TERT- ESR1 , MCF10A- ESR1 and ME16C2- ESR1 showed increased nuclear γH2AX foci Dox+E2 over Dox+Control. No increase in γH2AX was observed in HME-CC- ESR1 . C) Immunofluorescence staining of nucleus(blue), γH2AX(red) and CyclinA2 (white) in MCF10A- ESR1 showing DNA damage in CyclinA2 high (S/G2) and CyclinA2 low (G1)cells. D ) Quantification of C showing increase in nuclear γH2AX in Dox+E2 over Dox+Control in both S/G2 and G1 cells. S/G2 cells show 10-fold higher DNA damage over G1 Dox+E2 treated cells.

Article Snippet: HBEC- ESR1 cells were plated in 8-chambered slides (CELLTREAT #229168) at 30,000 cells per chamber in F media.

Techniques: Immunofluorescence, Staining, Control

Journal: bioRxiv

Article Title: Inducible estrogen receptor alpha in normal breast epithelial cells demonstrate estrogen receptor-dependent DNA damage

doi: 10.1101/2025.03.13.643100

Figure Lengend Snippet: A ) Immunofluorescence staining of γH2AX(red) and nucleus(blue) MCF10A- ESR1 cells showing DNA damage following treatments with 24h of E2 and 4h of DNA repair pathway inhibitors. B ) Quantification of nuclear γH2AX in MCF10A- ESR1 shows increase in DNA damage following MRE11 inhibitor Mirin and DNA-PKC inhibitor NU7441 treatment. C ) Quantification of nuclear γH2AX in ME16C2- ESR1 showing the similar levels of increase in DSB following treatment with Mirin or NU7441. D ) Immunofluorescence staining of γH2AX(red) and nucleus(blue) of MCF10A- ESR1 and MCF10A- BRCA1 +/185delAG - ESR1 . E ) Quantification of A showing comparable levels of nuclear γH2AX foci in Dox+Control treated cells, but higher in Dox+E2 treated MCF10A- BRCA1 +/185delAG - ESR1 compared to Dox+E2 treated MCF10A- ESR1 .

Article Snippet: HBEC- ESR1 cells were plated in 8-chambered slides (CELLTREAT #229168) at 30,000 cells per chamber in F media.

Techniques: Immunofluorescence, Staining, Control