Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Basal pro− and anti−inflammatory gene expression in mixed−sex microglia is APOE genotype−dependent: ( A ) basal pro−inflammatory and ( B ) anti−inflammatory gene expression was evaluated in non−stimulated mixed−sex microglia from APOE3 and APOE4 genotypes. Gene expression between genotypes was analyzed using an unpaired Student’s t −test (N = 3 isolations, n = 6 plates). Data are represented as the mean ± standard deviation. *, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.001, and 0.0001, respectively. ns = not statistically significant ( p > 0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Gene Expression, Standard Deviation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Comparison of gene expression between APOE3 and APOE4 genotypes: ( A ) pro− and ( B ) anti−inflammatory gene expression was evaluated in mixed−sex microglia from APOE3 and APOE4 genotypes following treatment with LPS (10 ng/mL) and LPS (10 ng/mL) + IFNg (10 ng/mL) for 6 h. Gene expression between genotypes was assessed by two−way ANOVA followed by Tukey’s multiple comparisons test (N = 3 isolations, n = 6 plates). Each data point represents the average mRNA expression from one isolation. Data are represented as the mean ± standard deviation. # denotes a significant increase compared to the respective genotype control (data not included in the graphs, p < 0.05). *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ( C ) Heat map comparing the mean expression of pro− and anti−inflammatory genes in control, LPS, and LPS + IFNg treated APOE3 and APOE4 PMG. * and ^ denote a significant ( p < 0.05) increase and decrease, respectively, in APOE4 gene expression compared to APOE3.

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Comparison, Gene Expression, Expressing, Isolation, Standard Deviation, Control

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: APOE4 genotype exacerbates release of pro− and anti−inflammatory cytokines: cytokine levels were measured following treatment with LPS or LPS + IFNg for 24 h. ( A ) Average levels of nitrite (±standard deviation) accumulated in the media were measured by GRIESS assay. Using two−way ANOVA, significant treatment and genotype effects were observed. # denotes a significant increase compared to the respective genotype control ( p < 0.05). ( B ) Basal levels of secreted cytokines were measured in non−stimulated microglia after 24 h by an MSD multiplex assay. Comparisons between genotypes were made using an unpaired Student’s t −test. ( C ) Cytokine levels in the media were measured after treatment with LPS or a combination of LPS and IFNg for 24 h. Two−way ANOVA was used to assess cytokine release in mixed−sex microglia from APOE3 and APOE4 genotypes (N = 3−4 isolations, n = 6−8 plates). Data are represented as the mean ±standard deviation, and each data point denotes the average of each experimental plate. # denotes a significant increase compared to the respective genotype control (data not included in the graphs, p < 0.05). *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ns = not statistically significant ( p >0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Standard Deviation, Griess Assay, Control, Multiplex Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Basal pro− and anti−inflammatory gene expression in sex−specific microglia is dependent on both the APOE genotype and sex: ( A ) basal pro−inflammatory and ( B ) anti−inflammatory gene expression was evaluated in non−stimulated sex−specific microglia from APOE3 and APOE4 genotypes. Gene expression between genotypes and sexes was analyzed using two−way ANOVA (N = 3 isolations, n = 6 plates). Data are represented as the mean ± standard deviation. *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ns = not statistically significant ( p > 0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Gene Expression, Standard Deviation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Gene expression in sex-specific microglia is dependent on both the APOE genotype and sex: ( A ) pro− and ( B ) anti−inflammatory gene expression was evaluated in sex−specific microglia from APOE3 and APOE4 genotypes following LPS or LPS + IFNg treatment for 6 h. Gene expression between genotypes and sexes was analyzed using a three−way ANOVA (N = 3 isolations, n = 6 plates) followed by Tukey’s multiple comparisons test. Each data point represents the average mRNA expression from one isolation. Data are represented as the mean ± standard deviation. # denotes a significant increase compared to the respective genotype control (data not included in the graphs, p < 0.05). *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ns = not statistically significant ( p >0.05). ( C ) Heat map comparing the mean expression of pro− and anti−inflammatory genes in sex−specific APOE3 and APOE4 PMG treated with LPS and LPS + IFNg. * and ^ denote a significant ( p < 0.05) increase and decrease, respectively, in fold changes of gene expression compared to a genotype−matched male PMG.

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Gene Expression, Expressing, Isolation, Standard Deviation, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: APOE genotype and sex-dependent inflammation are mediated through p65 activation: ( A ) representative images for immunocytochemistry of p65 in control and LPS + IFNg treated APOE3 and APOE4, male and female PMG (scale bar = 100 μm). ( B ) Quantification of total p65 and ( C ) quantification of nuclear p65. N = 3 isolations. Each data point denotes the average p65 integrated density/cell density for an individual isolation. A denotes a significant increase compared to the E3 male control ( p < 0.05). # denotes a significant increase compared to the respective genotype and sex control ( p < 0.05). Data are represented as the mean ± standard deviation. * and **** indicate statistical significance for the comparison and denote p < 0.05 and 0.0001, respectively. ns = not statistically significant ( p > 0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Activation Assay, Immunocytochemistry, Control, Isolation, Standard Deviation, Comparison

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components

doi: 10.1161/atvbaha.119.313169

Figure Lengend Snippet: Figure 1. Primary pericytes from ApoE (apolipoprotein E)-targeted replacement mice abundantly secrete lipidated apoE. A, Representative images of pericytes stained for NG2 (neural/glial antigen 2; left) and PDGFRβ (platelet-derived growth factor receptor- β, right) are shown. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). B, The amount of apoE in the conditioned media from primary cultures of endothelial cells (EC), pericytes (PC), and astrocytes (AS) was measured by ELISA and normalized against the total protein concentrations in cell lysates. C, Conditioned media from primary cultures of PC and AS were concentrated and subjected to size- exclusion chromatography run by FPLC using a Superose-6 column. The amount of apoE in each fraction was determined by ELISA. Values of 3 independent experiments were averaged and plotted against fraction numbers. D, Conditioned media from primary cultures of PC and AS were concentrated and subjected to an immunoprecipitation using an apoE-specific antibody. The amount of apoE-associated cholesterol was determined by Amplex Red cholesterol assay after immunoprecipitation with anti-apoE antibody. Data in (B) and (D) are presented as mean±SEM (N=4). Each dot in (B) and (D) represents a measurement from one independent primary cell culture prepared from brains of 4 male and 4 female mice. *P<0.05, apoE3-EC vs apoE3-PC, Mann-Whitney U test (B, top). *P<0.05, apoE4-EC vs apoE4-PC, Mann-Whitney U test (B, bottom). The amount of apoE in the conditioned media from primary AS is not included in the statistical analysis (B). *P<0.05, apoE3- PC vs apoE3-AS, Mann-Whitney U test (D, top); *P<0.05, apoE4-PC vs apoE4-AS, Mann-Whitney U test (D, bottom).

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 or apoE4 protein purchased from Fitzgerald (Cat. #30R-AA016, 30R-2382).

Techniques: Staining, Derivative Assay, Enzyme-linked Immunosorbent Assay, Size-exclusion Chromatography, Immunoprecipitation, Amplex Red Cholesterol Assay, Cell Culture, MANN-WHITNEY

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components

doi: 10.1161/atvbaha.119.313169

Figure Lengend Snippet: Figure 2 Continued. (G) in EC were determined by qRT-PCR and compared between EC monoculture, EC cocultured with apoE3-PC or apoE4-PC. Data in (B–G) are presented as mean±SEM (N=4). Each dot in (B–G) represents a measurement from one independent primary cell culture prepared from brains of 4 male and 4 female mice. **P<0.01, EC monoculture vs EC cocultured with apoE3-PC; *P<0.05, EC cocultured with apoE3-PC vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (B, left). Kruskal- Wallis test followed by Dunn multiple comparison tests (B, right). ***P<0.001, EC monoculture vs EC cocultured with apoE3-PC; *P<0.05, EC cocultured with apoE3-PC vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (C, left). ***P<0.001, EC monoculture vs EC cocultured with apoE3-PC; EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (C, right). **P<0.01, EC monoculture vs EC cocultured with apoE3-PC; Kruskal-Wallis test followed by Dunn multiple comparison tests (D, left). Kruskal-Wallis test followed by Dunn multiple comparison tests (D, right). **P<0.01, EC monoculture vs EC cocultured with apoE3-PC; Kruskal-Wallis test followed by Dunn multiple comparison tests (E, left). ****P<0.0001, EC monoculture vs EC cocultured with apoE3-PC; ***P<0.001, EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (E, right). **P<0.01, EC monoculture vs EC cocultured with apoE3-PC; Kruskal-Wallis test followed by Dunn multiple comparison tests (F, left). ***P<0.001, EC monoculture vs EC cocultured with apoE3-PC; *P<0.05, EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (F, right). ****P<0.0001, EC monoculture vs EC cocultured with apoE3-PC; **P<0.05, EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (G, left). **P<0.01, EC monoculture vs EC cocultured with apoE3-PC, EC monoculture vs EC cocultured with apoE4-PC; 1-way ANOVA followed by Tukey multiple comparison tests (G, right). N.S. indicates not significant.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 or apoE4 protein purchased from Fitzgerald (Cat. #30R-AA016, 30R-2382).

Techniques: Quantitative RT-PCR, Cell Culture, Comparison

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components

doi: 10.1161/atvbaha.119.313169

Figure Lengend Snippet: Figure 5. Reduced collagen-IV deposition along cortical capillaries in ApoE4-targeted replacement (apoE4-TR) mice. A, Collagen IV, CD31, claudin-5, occludin, CD13, and AQP4 were stained in frozen cortical sections from apoE3-TR (male; N=4, female; N=4) or apoE4-TR mice (male; N=4, female; N=4) at the age of 22 mo. B, Total fluorescence intensity of collagen IV in cortical sections from those apoE-TR mice were quantified by ImageJ software (apoE, P=0.0034; sex, P=0.0011, apoE×sex, P=0.7080). C–F, The % of coverage against CD31-positive endothelial by claudin-5 (C, apoE, P=0.9304; sex, P=0.6093, apoE×sex, P=0.1227), occludin (D, apoE, P=0.2478; sex, P=0.1107, apoE×sex, P=0.3657), CD13 (E, apoE, P=0.1683; sex, P=0.0897, apoE×sex, P=0.3923) or AQP4 (F, apoE, P=0.1698; sex, P=0.9376, apoE×sex, P=0.3703) was quantified in cortical sections from the mice. Data in (B–F) are presented as mean±SEM. Each dot in (B–F) represents a measurement from one mouse. *P<0.05, ***P<0.001 by Tukey-Kramer post hoc analysis of 2-way ANOVA. N.S. indicates not significant among groups.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 or apoE4 protein purchased from Fitzgerald (Cat. #30R-AA016, 30R-2382).

Techniques: Staining, Fluorescence, Software

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components

doi: 10.1161/atvbaha.119.313169

Figure Lengend Snippet: Figure 6. Increased plasma protein leakage in the cortex of ApoE4-targeted replacement (apoE4-TR) mice. A–E, The levels of collagen IV (A; apoE [apolipoprotein E], P<0.0001; sex, P=0.4401, apoE×sex, P=0.7307), claudin-5 (B; apoE, P=0.4365; sex, P=0.5057, apoE×sex, P=0.4332), occludin (C, apoE, P=0.7529; sex, P=0.8528, apoE×sex, P=0.9210), fibrinogen (D, apoE, P=0.0002; sex, P=0.9759, apoE×sex, P=0.5057), and IgG (E, apoE, P=0.0428; sex, P=0.5767, apoE×sex, P=0.6582) were determined by ELISA in apoE3-TR (male; N=8, female; N=9) and apoE4-TR mice (male; N=8, female; N=8) at 22 mo of age. The measurement was normalized by protein concentration in each of the samples. Data in (A–E) are presented as mean±SEM. Each dot represents a measurement from one mouse. *P<0.05, **P<0.01 by Tukey-Kramer post hoc analysis of 2-way ANOVA. F and G, The correlations between the levels of leaked plasma protein (fibrinogen and IgG) and that of collagen IV calculated across the male or female apoE3-TR (red circle) and apoE4-TR (blue circle) mice were assessed through nonparametric Spearman correlation analysis. The correlation coefficient (R2) and P value are shown in each panel. N.S. indicates not significant among groups.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 or apoE4 protein purchased from Fitzgerald (Cat. #30R-AA016, 30R-2382).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Protein Concentration

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Ficolin-2 inhibits hepatitis C virus infection, whereas apolipoprotein E3 mediates viral immune escape.

doi: 10.4049/jimmunol.1302563

Figure Lengend Snippet: FIGURE 5. ApoE3 binds to both E1 and E2 glycoproteins. (A) ApoE3 did not bind to ficolin-2 by ELISA analysis. The Kd value of ApoE3–ficolin-2 was deter- mined by ELISA as described in Mate- rials and Methods. (B) ApoE3 bound to E2-HeLa and E1-HeLa cells but not to HeLa cells. The Kd values of ApoE3-E2/ E1 were determined by flow cytometry as described in Materials and Methods. (C) Ficolin-2 binds to E2-HeLa cells and E1-HeLa cells. The Kd values of ficolin- 2-E2/E1 were determined by flow cyto- metry. (D) GST protein did not bind to E1-HeLa cells, E2-HeLa cells, and un- transfected HeLa cells by flow cytometry analysis. (E) Analysis of different con- centrations of ApoE3 binding to E1- and E2-glycoprotein–expressing HeLa cells using flow cytometry and a PE–anti- ApoE3 mAb. (F) Analysis of the block- ing effects of mannan or GlcNAc on ApoE3 binding to E1- and E2-glycopro- tein–expressing HeLa cells using flow cytometry.

Article Snippet: Levels of ApoE3 were normalized to a standard curve generated using ApoE3 recombinant protein obtained from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Cytometry, Transfection, Binding Assay, Expressing, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Ficolin-2 inhibits hepatitis C virus infection, whereas apolipoprotein E3 mediates viral immune escape.

doi: 10.4049/jimmunol.1302563

Figure Lengend Snippet: FIGURE 6. ApoE3 competitively blocks the interaction between ficolin-2 and HCV-E2. (A) ApoE3 significantly blocked ficolin-2 binding to purified extra-HCV (HCVcc) in a dose-dependent manner measured by ELISA. (B) Analysis of the ApoE3 blocking effect on the binding of ficolin-2 to E2- glycoproteins using flow cytometry. (C) ApoE3 blocks ficolin-2 binding to E2, even when ficolin-2 is preincubated with E2-HeLa cells. All data are presented as means 6 SEM from three independent experiments. (D) Representative data showing ApoE3 blocking the binding of ficolin-2 to E1- and E2- glycoproteins expressing HeLa cells using flow cytometry.

Article Snippet: Levels of ApoE3 were normalized to a standard curve generated using ApoE3 recombinant protein obtained from R&D Systems.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cytometry, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Ficolin-2 inhibits hepatitis C virus infection, whereas apolipoprotein E3 mediates viral immune escape.

doi: 10.4049/jimmunol.1302563

Figure Lengend Snippet: FIGURE 7. ApoE3 promotes HCV infection and blocks the anti-HCV effect of ficolin-2. (A) Western blot analysis of the effects of ApoE3 and ficolin-2 on HCV-NS3 expression in HCVcc-infected Huh7.5.1 cells using an anti-NS3 mAb. (B and C) Real-time RT-PCR analysis of HCV-NS3 expression in the presence of GST–ficolin-2 or ApoE3 or GST–ficolin-2 plus ApoE3 in HCVcc-infected Huh7.5.1 cells. (D) Plasmids pcDNA 3.1- ApoE3, -ApoE2, and -ApoE4 were used to transfect Huh7.5.1 cells. After 72 h of transfection, cells were harvested and the expressions of ApoE3, ApoE2, and ApoE4 in the cell lysates and supernatants of Huh7.5.1 cells were determined using a polyclonal anti-ApoE by Western blotting analysis. (E) Densities of ApoE3, ApoE2, and ApoE4 proteins expressed in the supernatants shown in (D) were normalized based on the density of the housekeeping protein b-actin. (F) The cell culture supernatants were collected at 72 h and the secreted ApoE3, ApoE2, and ApoE4 proteins were determined by specific ELISA. The effects of ApoE3, ApoE2, and ApoE4 on ficolin-2 anti-HCV infection activity were analyzed by real-time qRT-PCR (G) and Western blotting using anti–HCV-NS3 mAb (H). (I) Densities of NS3 protein shown in (H) were normalized based on the density of the housekeeping protein b-actin. All data shown are the means 6 SEM of three independent experiments.

Article Snippet: Levels of ApoE3 were normalized to a standard curve generated using ApoE3 recombinant protein obtained from R&D Systems.

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Ficolin-2 inhibits hepatitis C virus infection, whereas apolipoprotein E3 mediates viral immune escape.

doi: 10.4049/jimmunol.1302563

Figure Lengend Snippet: FIGURE 8. ApoE3 blocks the effects of ficolin-2 and restores HCVcc binding to LDLR and SR-B1. (A and B) Immunoflu- orescent detection of E2 and SR-B1 or LDLR receptor expression in SR-B1 or LDLR plasmid-transfected HeLa cells in the presence of ficolin-2 or ficolin-2 plus ApoE3 using confocal fluorescence mi- croscopy. Original magnification 3600. (C and D) Exogenous ApoE3 restored HCV binding to its receptor and reversed the blocking effects of ficolin-2, as shown by ELISA. **p , 0.01.

Article Snippet: Levels of ApoE3 were normalized to a standard curve generated using ApoE3 recombinant protein obtained from R&D Systems.

Techniques: Binding Assay, Expressing, Plasmid Preparation, Transfection, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Ficolin-2 inhibits hepatitis C virus infection, whereas apolipoprotein E3 mediates viral immune escape.

doi: 10.4049/jimmunol.1302563

Figure Lengend Snippet: FIGURE 9. A potential model of the ficolin-2 antiviral effects and ApoE3-mediated immune escape.

Article Snippet: Levels of ApoE3 were normalized to a standard curve generated using ApoE3 recombinant protein obtained from R&D Systems.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

doi: 10.1074/jbc.m114.625251

Figure Lengend Snippet: FIGURE 1. Human brain vascular pericytes produce lipidated apoE. A, conditioned media and cell lysates were collected from human brain vascular pericytes with the APOE 3/3 genotype or immortalized astrocytes from apoE3-TR mice. ApoE concentrations in the conditioned media were normalized againstthetotalproteinconcentrationsincelllysatesandcomparedbetweenpericytesandastrocytes.B,conditionedmediafrompericytesorastrocyteswere concentrated and subjected to fractionation by a Superose-6 size exclusion column run on FPLC. Fractions were subjected to analysis for apoE concentration by ELISA. The amount of apoE in each fraction was plotted as percent of total apoE, calculated by combining those in all fractions. C, the cholesterol levels associating with apoE produced by pericytes or astrocytes were analyzed by Amplex Red cholesterol assay after immunoprecipitation. The amount of cholesterol in each fraction was plotted as percent of total cholesterol, calculated by combining those in all fractions. D, the fractions used in B were further subjected to analysis for cholesterol level by Amplex Red cholesterol assay.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 protein (Fitzgerald).

Techniques: Fractionation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Produced, Amplex Red Cholesterol Assay, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

doi: 10.1074/jbc.m114.625251

Figure Lengend Snippet: FIGURE 4. ApoE suppresses two-dimensional pericyte migration in an isoform-dependent manner. A, a wound healing assay was performed in control and apoE knockdown pericytes in the presence or absence of purified apoE3 or apoE4 particles (5 ng/ml). Cells from half of each well from the mid- line (dashed line) were removed and left to heal for 24 h before reimaging. B, migrated cell numbers were calculated as the number of cells beyond the scratch line. Data are plotted as mean S.D. (n 7). *, p 0.05; ***, p 0.001; n.s., not significant.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 protein (Fitzgerald).

Techniques: Migration, Wound Healing Assay, Control, Knockdown, Purification

Journal: The FASEB Journal

Article Title: APOE4 genotype exacerbates the impact of menopause on cognition and synaptic plasticity in APOE‐TR mice

doi: 10.1096/fj.202002621rr

Figure Lengend Snippet: FIGURE 1 Experimental overview. At 4 months of age APOE4 and APOE3 TR mice were provided with a high-fat diet to model a western style diet. At 8 months of age mice received i.p. injections with 160 mg/kg VCD for 14 days in order to deplete ovarian follicles and mimic menopause by 12 months of age. At 12 months of age, mice were assessed cognitively through a battery of behavioral tests including Barnes maze, Y-maze, and object recognition. Upon completing the behavioral tests animals were immediately sacrificed. Created with BioRender.com

Article Snippet: Forty eight C57BL/6 background female humanized APOE3 (B6.129P2- Apoetm2(APOE*3)Mae N8) and APOE4 (B6.129P2- Apoetm2(APOE*4)Mae N8)- targeted replacement mice homozygous for the human APOE3 or APOE4 gene (Taconic, Germantown, NY, USA) were used in these experiments.12,20,21 Mice were maintained in a controlled environment (21 ± 2°C; 12- h light- dark cycle; light from 07:00 hours) and fed ad libitum on a standard chow diet (RM3- P, Special Diet Services, Essex, UK) until the age of 4 months, ensuring normal development.

Techniques: Western Blot, Battery

Journal: Journal of Biological Chemistry

Article Title: Apolipoprotein E Inhibits Cerebrovascular Pericyte Mobility through a RhoA Protein-mediated Pathway

doi: 10.1074/jbc.m114.625251

Figure Lengend Snippet: FIGURE 1. Human brain vascular pericytes produce lipidated apoE. A, conditioned media and cell lysates were collected from human brain vascular pericytes with the APOE 3/3 genotype or immortalized astrocytes from apoE3-TR mice. ApoE concentrations in the conditioned media were normalized againstthetotalproteinconcentrationsincelllysatesandcomparedbetweenpericytesandastrocytes.B,conditionedmediafrompericytesorastrocyteswere concentrated and subjected to fractionation by a Superose-6 size exclusion column run on FPLC. Fractions were subjected to analysis for apoE concentration by ELISA. The amount of apoE in each fraction was plotted as percent of total apoE, calculated by combining those in all fractions. C, the cholesterol levels associating with apoE produced by pericytes or astrocytes were analyzed by Amplex Red cholesterol assay after immunoprecipitation. The amount of cholesterol in each fraction was plotted as percent of total cholesterol, calculated by combining those in all fractions. D, the fractions used in B were further subjected to analysis for cholesterol level by Amplex Red cholesterol assay.

Article Snippet: ApoE concentration of each sample was calculated against a standard curve derived from serial dilutions of recombinant human apoE3 protein (Fitzgerald).

Techniques: Fractionation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Produced, Amplex Red Cholesterol Assay, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Basal pro− and anti−inflammatory gene expression in mixed−sex microglia is APOE genotype−dependent: ( A ) basal pro−inflammatory and ( B ) anti−inflammatory gene expression was evaluated in non−stimulated mixed−sex microglia from APOE3 and APOE4 genotypes. Gene expression between genotypes was analyzed using an unpaired Student’s t −test (N = 3 isolations, n = 6 plates). Data are represented as the mean ± standard deviation. *, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.001, and 0.0001, respectively. ns = not statistically significant ( p > 0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Gene Expression, Standard Deviation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Basal pro− and anti−inflammatory gene expression in sex−specific microglia is dependent on both the APOE genotype and sex: ( A ) basal pro−inflammatory and ( B ) anti−inflammatory gene expression was evaluated in non−stimulated sex−specific microglia from APOE3 and APOE4 genotypes. Gene expression between genotypes and sexes was analyzed using two−way ANOVA (N = 3 isolations, n = 6 plates). Data are represented as the mean ± standard deviation. *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ns = not statistically significant ( p > 0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Gene Expression, Standard Deviation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: Gene expression in sex-specific microglia is dependent on both the APOE genotype and sex: ( A ) pro− and ( B ) anti−inflammatory gene expression was evaluated in sex−specific microglia from APOE3 and APOE4 genotypes following LPS or LPS + IFNg treatment for 6 h. Gene expression between genotypes and sexes was analyzed using a three−way ANOVA (N = 3 isolations, n = 6 plates) followed by Tukey’s multiple comparisons test. Each data point represents the average mRNA expression from one isolation. Data are represented as the mean ± standard deviation. # denotes a significant increase compared to the respective genotype control (data not included in the graphs, p < 0.05). *, **, ***, and **** indicate statistical significance for the comparison and denote p < 0.05, 0.01, 0.001, and 0.0001, respectively. ns = not statistically significant ( p >0.05). ( C ) Heat map comparing the mean expression of pro− and anti−inflammatory genes in sex−specific APOE3 and APOE4 PMG treated with LPS and LPS + IFNg. * and ^ denote a significant ( p < 0.05) increase and decrease, respectively, in fold changes of gene expression compared to a genotype−matched male PMG.

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Gene Expression, Expressing, Isolation, Standard Deviation, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice

doi: 10.3390/ijms23179829

Figure Lengend Snippet: APOE genotype and sex-dependent inflammation are mediated through p65 activation: ( A ) representative images for immunocytochemistry of p65 in control and LPS + IFNg treated APOE3 and APOE4, male and female PMG (scale bar = 100 μm). ( B ) Quantification of total p65 and ( C ) quantification of nuclear p65. N = 3 isolations. Each data point denotes the average p65 integrated density/cell density for an individual isolation. A denotes a significant increase compared to the E3 male control ( p < 0.05). # denotes a significant increase compared to the respective genotype and sex control ( p < 0.05). Data are represented as the mean ± standard deviation. * and **** indicate statistical significance for the comparison and denote p < 0.05 and 0.0001, respectively. ns = not statistically significant ( p > 0.05).

Article Snippet: TR-APOE breeder mice with a C57BL/6 background, with the mouse APOE gene replaced by a homozygous human APOE3 or APOE4 gene [ ], were purchased from Taconic and bred in-house.

Techniques: Activation Assay, Immunocytochemistry, Control, Isolation, Standard Deviation, Comparison