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

Journal: PLoS Biology

Article Title: APOE Stabilization by Exercise Prevents Aging Neurovascular Dysfunction and Complement Induction

doi: 10.1371/journal.pbio.1002279

Figure Lengend Snippet: (A) Representative sections of the FPC from young and aged brains hybridized with an Apoe riboprobe. (B) Quantification of Apoe hybridized area in the cortex shows a significant decrease of Apoe expression in the aged mice. (C) Representative sections of the hippocampal CA1 region from young and aged brains hybridized with an Apoe riboprobe. (D) Apoe hybridized area in the CA1 shows a significant decrease of Apoe expression in the aged mice. (E) Representative sections of the CC from young and aged brains hybridized with Apoe riboprobe. (F) A significant increase of Apoe hybridized area is measured on the aged CC. (G) Representative images of Apoe in situ hybridization in the cortex and CC showing decreased expression of Apoe in the cortex and increased expression in the CC in aged mice compared with young mice. (H) No differences in APOE protein levels were found by western blotting of whole brains from young and aged mice. Values in (B, D, and F) are relative mean + SEM to the young values, n = 6 per group. In (D) ** p < 0.001 and in (F) * p = 0.0364 by unpaired t test. Scale Bars: 600 μm (A), 50 μm (C), 100 μm (E and G). The data used to make this figure can be found in .

Article Snippet: The primary antibodies used for immunoblotting are: goat anti-mouse APOE (1:1,000, Millipore), rabbit anti-pan laminin (LAM, 1:1,000, Sigma-Aldrich), mouse anti-PSD95 (1:1,000, Millipore), and mouse anti-βActin (1:2,000, Abcam).

Techniques: Expressing, In Situ Hybridization, Western Blot

Journal: PLoS Biology

Article Title: APOE Stabilization by Exercise Prevents Aging Neurovascular Dysfunction and Complement Induction

doi: 10.1371/journal.pbio.1002279

Figure Lengend Snippet: (A–B) Representative sections and quantitative analysis of Apoe -hybridized cortex on aging Apoe -/- , aging, aged sedentary (Sd), and aged runner (Rn) mice showing a significant preservation of Apoe expression on runner mice. No hybridization signal with Apoe riboprobe was observed in the Apoe -/- mouse brain. (C–D) Representative sections and quantitative analysis of Apoe expression on aging hippocampal CA1. Down-regulation of Apoe expression in the CA1 region in aging mice is prevented by wheel running. Values in (B and D) are relative mean + SEM to the aging values, n = 6 per group. In (B) * p = 0.0347 and 0.0244, and in (D) ** p = 0.0019 and 0.0016 by ANOVA followed by Tukey’s posthoc tests. Scale Bars: 50 μm. The data used to make this figure can be found in .

Article Snippet: The primary antibodies used for immunoblotting are: goat anti-mouse APOE (1:1,000, Millipore), rabbit anti-pan laminin (LAM, 1:1,000, Sigma-Aldrich), mouse anti-PSD95 (1:1,000, Millipore), and mouse anti-βActin (1:2,000, Abcam).

Techniques: Preserving, Expressing, Hybridization

Journal: PLoS Biology

Article Title: APOE Stabilization by Exercise Prevents Aging Neurovascular Dysfunction and Complement Induction

doi: 10.1371/journal.pbio.1002279

Figure Lengend Snippet: (A and B) Voluntary running does not change vascular and extravascular deposition of FIBRIN (red) in aged Apoe -/- mice. Endothelial cells (cyan) are immunolabeled with CD31. (C and D) PDGFRβ + pericyte number (magenta) but not coverage, is only partially increased in aged Rn Apoe -/- mice compared to controls. (E and F) The number of IBA1 + microglia/monocytes (green) is not reduced by running in aged Apoe -/- mice. Values in (B, D, and F) are mean + SEM, (B) n = 6 and (E–G) n = 3–4 per group. In (D) **p = 0.0024, *p = 0.0468, *p = 0.0166, and *p = 0.0430, and in (F) ** p = 0.0022 and 0.0084, and * p = 0.0105 by ANOVA followed by Tukey’s posthoc tests. Scale Bars: 50 μm. The data used to make this figure can be found in .

Article Snippet: The primary antibodies used for immunoblotting are: goat anti-mouse APOE (1:1,000, Millipore), rabbit anti-pan laminin (LAM, 1:1,000, Sigma-Aldrich), mouse anti-PSD95 (1:1,000, Millipore), and mouse anti-βActin (1:2,000, Abcam).

Techniques: Immunolabeling

Journal: PLoS Biology

Article Title: APOE Stabilization by Exercise Prevents Aging Neurovascular Dysfunction and Complement Induction

doi: 10.1371/journal.pbio.1002279

Figure Lengend Snippet: In the aged cortex of sedentary mice, neurovascular dysfunction is evident by decreased numbers of pericytes, decline in BM coverage, and increased transcytosis on endothelial cells. Expression of AQP4 in astrocyte endfeet and down-regulation of Apoe (purple) are also found as part of the age-related dysfunction of the neurovascular unit. In addition, decrease in synaptic proteins such as synaptophysin (SYN) is found in aged neurons. The number of proinflammatory IBA1 + microglia/monocytes expressing high levels of C1qa RNA is also increased in the aged cortex and HP indicating age-related neuroinflammation in aged mice. These age-related changes were successfully prevented by 6 months of voluntary running during aging, indicating the important contribution of physical activity on preservation of cerebrovascular function during aging.

Article Snippet: The primary antibodies used for immunoblotting are: goat anti-mouse APOE (1:1,000, Millipore), rabbit anti-pan laminin (LAM, 1:1,000, Sigma-Aldrich), mouse anti-PSD95 (1:1,000, Millipore), and mouse anti-βActin (1:2,000, Abcam).

Techniques: Expressing, Activity Assay, Preserving