Journal: Archives of biochemistry and biophysics

Article Title: Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

doi: 10.1016/j.abb.2019.05.025

Figure Lengend Snippet: Fig. 1. (A) Schematics of the 2D and 3D culture formats used throughout this work. Both formats contained 40,000 T47D cells that were either plated directly in a commercial 96 well plate or suspended in collagen I and seeded into a paper-based scaffold with a 1 × 10−3 cm3 culture zone. Once seeded, the paper-based scaffolds were also placed in a com- mercial 96 well plate. All experiments were in- cubated under normoxic (21% O2) or hypoxic (1% O2) conditions in E2-deprived (Veh) or -supple- mented (E2) medium for 24 h. (B, C) Average ± SEM of ERα protein levels for each ex- perimental condition were determined by ELISA. Protein levels were normalized to β-actin. Each bar represents n ≥12 replicate cultures from three dif- ferent cell passages. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001. (D) A representative Western blot of ERα protein levels with a β-actin loading control.

Article Snippet: The resulting lysates were clarified by centrifugation at 10,000×g at 4 °C for 10 min. For ELISA, ERα and HIF-1α concentrations were determined with commercial kits (R&D Systems, DYC-5715 and DYC1935); β-actin concentration was determined with a sandwich assay consisting of a βactin capture antibody (R&D Systems, AF4000) and an HRP-linked detection antibody (SantaCruz Biotechnology, sc-47778).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Archives of biochemistry and biophysics

Article Title: Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

doi: 10.1016/j.abb.2019.05.025

Figure Lengend Snippet: Fig. 3. 2D and 3D T47D cultures under normoxic (21% O2) or hypoxic (1% O2) conditions in E2-de- prived (Veh) or -supplemented (E2) medium for 24 h, and then probed with ELISA to quantify HIF-1α protein levels (A, B). Protein levels were normalized to β-actin. Data represent the average ± SEM, from n ≥12 replicate cultures from three different cell passages. Total RNA was extracted, and the relative expression of HIF1A (C, D) and VEGFA (E, F) for each experimental condition determined using the ΔΔCt method; β-actin served as the reference gene. Data represent the average ± SEM, from n = 9 replicate cultures from a total of three passages of cells. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤0.0001.

Article Snippet: The resulting lysates were clarified by centrifugation at 10,000×g at 4 °C for 10 min. For ELISA, ERα and HIF-1α concentrations were determined with commercial kits (R&D Systems, DYC-5715 and DYC1935); β-actin concentration was determined with a sandwich assay consisting of a βactin capture antibody (R&D Systems, AF4000) and an HRP-linked detection antibody (SantaCruz Biotechnology, sc-47778).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Archives of biochemistry and biophysics

Article Title: Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

doi: 10.1016/j.abb.2019.05.025

Figure Lengend Snippet: Fig. 4. 2D and 3D T47D cultures exposed to nor- moxic or hypoxic conditions in E2-deprived (Veh) or -supplemented (E2) medium for 24 h. Total RNA was extracted and relative expression of ESR1 (A, B) was determined using the ΔΔCt method; β-actin served as the reference gene. Data represent the average ± SEM, from n = 9 replicate cultures from three different cell passages. 2D and 3D T47D cul- tures were exposed to normoxic or hypoxic condi- tions in estrogen-deprived medium in the presence or absence of MG-132 (10 μM) for 8 h. HIF-1α (C, D) and ERα (E, F) protein levels were quantified with ELISA. Data represent the average ± SEM, from n ≥6 replicate cultures from two different cell passages. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001.

Article Snippet: The resulting lysates were clarified by centrifugation at 10,000×g at 4 °C for 10 min. For ELISA, ERα and HIF-1α concentrations were determined with commercial kits (R&D Systems, DYC-5715 and DYC1935); β-actin concentration was determined with a sandwich assay consisting of a βactin capture antibody (R&D Systems, AF4000) and an HRP-linked detection antibody (SantaCruz Biotechnology, sc-47778).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Archives of biochemistry and biophysics

Article Title: Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

doi: 10.1016/j.abb.2019.05.025

Figure Lengend Snippet: Fig. 5. ERα transcriptional activation is reduced under hypoxic conditions in both 2D and 3D culture formats. Both culture formats were incubated in E2- deprived (Veh) or -supplemented (E2) medium under normoxic or hypoxic conditions for 24 h. Luciferase activity was quantified using the ONE-Glo luciferase assay; the fold-change in luminescence relative to E2-deprived cultures under normoxia was plotted for 2D (A) and 3D (B) culture formats. Figures represent the average ± SEM, from n ≥6 replicates from three different cell passages. Total RNA was ex- tracted and the relative expression of PGR mRNA (C, D) was determined using the ΔΔCt method; β-actin served as the reference gene. Data represent the average ± SEM, from n = 9 replicate cultures from three different cell passages *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001.

Article Snippet: The resulting lysates were clarified by centrifugation at 10,000×g at 4 °C for 10 min. For ELISA, ERα and HIF-1α concentrations were determined with commercial kits (R&D Systems, DYC-5715 and DYC1935); β-actin concentration was determined with a sandwich assay consisting of a βactin capture antibody (R&D Systems, AF4000) and an HRP-linked detection antibody (SantaCruz Biotechnology, sc-47778).

Techniques: Activation Assay, Incubation, Luciferase, Activity Assay, Expressing

Journal: Archives of biochemistry and biophysics

Article Title: Hypoxia differentially regulates estrogen receptor alpha in 2D and 3D culture formats.

doi: 10.1016/j.abb.2019.05.025

Figure Lengend Snippet: Fig. 6. T47D cells in 2D and 3D culture formats were incubated in the absence or presence of 1 mM DMOG for 1 h before culture in E2-deprived (Veh) or -sup- plemented (E2) medium ( ± DMOG) for 24 h. ELISA was used to quantify HIF-1α (A, B) and ERα (C, D) protein levels which were normalized to β-actin. ERα transcriptional activity was measured using the ONE-Glo luciferase activity assay (E, F). Data represent the average ± SEM from n ≥6 replicate cultures from at least two different cell passages.

Article Snippet: The resulting lysates were clarified by centrifugation at 10,000×g at 4 °C for 10 min. For ELISA, ERα and HIF-1α concentrations were determined with commercial kits (R&D Systems, DYC-5715 and DYC1935); β-actin concentration was determined with a sandwich assay consisting of a βactin capture antibody (R&D Systems, AF4000) and an HRP-linked detection antibody (SantaCruz Biotechnology, sc-47778).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay, Luciferase