Journal:

Article Title: Sustained Phenotypic Correction in a Mouse Model of Hypoalphalipoproteinemia with a Helper-Dependent Adenovirus Vector

doi: 10.1038/sj.gt.3302819

Figure Lengend Snippet: Structures of adenoviral vectors used for co-injection

Article Snippet: For APOE3 immunoblot, goat anti-APOE antibody (1:4000, Calbiochem) or mouse monoclonal anti-human APOE3 (1:1000, SIGNET Lab) were used.

Techniques: Control

Journal:

Article Title: Sustained Phenotypic Correction in a Mouse Model of Hypoalphalipoproteinemia with a Helper-Dependent Adenovirus Vector

doi: 10.1038/sj.gt.3302819

Figure Lengend Snippet: Co-injection of FGAd vectors suppresses hAPOA1 expression mediated by HDAd-AI vector. (a) Plasma human APOA1 levels in APOA1−/− mice treated with Ad vectors. *p<0.05 vs. PBS, **p<0.01. (b) Plasma cholesterol levels in APOA1−/− mice after intravenous injection of Ad vectors. *p<0.05 vs. PBS, **p<0.01. (c) Immunoblot analysis of transgene expression. 0.1 μl of plasma collected from mice 8 weeks after treatment with various Ad vectors was separated by SDS-PAGE, and the presence of transgene products was detected by immunoblot. Upper panel: goat anti-human APOA1 antibody which does not cross react with mouse APOA1; middle panel: goat anti-apoE antibody; lower panel: mouse monoclonal antibody against human APOE3. Lane 1: Human plasma; lane 2: APOE−/− mouse; lane 3: APOE−/− mouse treated with HDAd-E3; lane 4: APOA1−/− mouse treated with FGAd-AI + HDAd-0; lane 5: APOE−/− mouse treated with HDAd-mouse APOE; lane 6: APOA1−/− mouse treated with HDAd-AI + HDAd-0; lane 7: APOA1−/− mouse treated with HDAd-AI and HDAd-E3; lane 8: APOA1−/− mouse treated with HDAd-AI + FGAd-E3. (d) Generation of anti-hAPOA1 antibodies after intravenous co-injection of various Ad vectors. *p<0.05 (vs. day 0).

Article Snippet: For APOE3 immunoblot, goat anti-APOE antibody (1:4000, Calbiochem) or mouse monoclonal anti-human APOE3 (1:1000, SIGNET Lab) were used.

Techniques: Injection, Expressing, Plasmid Preparation, Clinical Proteomics, Western Blot, SDS Page

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 binds recombinant and serum ApoE. ( A ) Recombinant CLCF1 (200 ng) and ApoE2, 3 or 4 (250 ng) were incubated alone or in combination for 16 h. The samples were subjected to immunoprecipitation (IP) with anti-CLCF1 and protein G agarose. Immunoprecipitated proteins were analyzed by WB using mAbs specific for ApoE (upper panel) or CLCF1 (lower panel). Lanes “CLC 2 ng” and “ApoE3 20 ng”, show results of recombinant proteins directly subjected to SDS-PAGE and WB blot analysis. ( B ) CLCF1 and ApoE form a complex when co-expressed in HEK-293 cells. The cell culture medium from stable transfectants expressing the indicated proteins in combination with CRLF1 or from cells transfected with empty expression vector (lane D5) was subjected to immunoaffinity chromatography with rat-anti-human ApoE and anti-rat Ig Agarose. The eluates were analysed by WB using anti-ApoE or anti-CLC mAbs (upper panels). Supernatants of the stable transfectants were analyzed for the presence of ApoE or CLCF1 by WB (lower panels). ( C and D ) CLCF1 binds mouse and human serum ApoE. ( C ) Mouse serum samples isolated from WT or ApoE −/− mice were diluted 1:20, mixed with biotinylated mouse CLCF1 and the complexes immunoprecipitated with rabbit anti-mouse ApoE and anti-rabbit Ig agarose. The eluates were analysed for ApoE or CLCF1 by WB using anti-mouse ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated mouse CLCF1 or ApoE included as control. ( D ) Human serum samples were diluted 1:20, mixed with biotinylated human CLCF1 and the complexes immunoprecipitated with rat anti-ApoE and anti-rat Ig agarose. The eluates were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated human CLCF1 or ApoE included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: ApoE2, 3 or 4 (0–100 nM) were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with rat IgG anti-ApoE (3 nM) (R&D systems, Cedarlane) and anti-rat IgG acceptor beads (10 μg/mL).

Techniques: Recombinant, Incubation, Immunoprecipitation, SDS Page, Cell Culture, Expressing, Transfection, Plasmid Preparation, Chromatography, Isolation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: AlphaLISA proximity assay shows CLCF1-ApoE complex formation that is increased by the lipid DMPC. ( A ) ApoE2, 3 or 4 were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with IgG rat anti-ApoE (3 nM) and anti-IgG rat acceptor beads (10 μg/mL). Streptavidin donor beads (40 μg/mL) were further added for 1 h and 615 nm fluorescence signal assessed. ( B ) The indicated ApoE isotypes (30 nM) were mixed at 37 °C with increasing amount of DMPC for 1 h. ApoE-DMPC mix were further incubated with CLCF1 and subjected to AlpahLISA proximity assay. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: ApoE2, 3 or 4 (0–100 nM) were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with rat IgG anti-ApoE (3 nM) (R&D systems, Cedarlane) and anti-rat IgG acceptor beads (10 μg/mL).

Techniques: Proximity Assay, Fluorescence, Incubation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 co-purifies or co-elutes with serum lipoprotein complexes. ( A ) WT mouse, ( B ) human or ( C ) ApoE −/− mouse sera were mixed with corresponding biotinylated CLCF1. The mixtures were fractionated by FPLC using a Superose 6 HR 10/ column in order to obtain 40 fractions. The fractions were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The right lanes show the signals obtained with 40 ng of ApoE or biotinylated CLCF1 included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: ApoE2, 3 or 4 (0–100 nM) were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with rat IgG anti-ApoE (3 nM) (R&D systems, Cedarlane) and anti-rat IgG acceptor beads (10 μg/mL).

Techniques:

Journal: Journal of Molecular Endocrinology

Article Title: Upregulation of hepatic LRP1 by rosiglitazone: a possible novel mechanism of the beneficial effect of thiazolidinediones on atherogenic dyslipidemia

doi: 10.1530/jme-12-0119

Figure Lengend Snippet: Figure 3 The effect of rosiglitazone on ApoE uptake in HepG2 cells. HepG2 cells were treated with indicated concentrations of rosiglitazone for 48 h. Human recombinant ApoE3 was added to culture media and cells were incubated for 1 h. ApoE3 was reconstituted with lipid using DMPC before the treatment on HepG2 cells. Three independent experiments were performed for the representative figures. (A) Western blot analysis of ApoE3 in HepG2 cells incubated with or without added ApoE3. (B) Western blot analysis of ApoE3 in HepG2 cells incubated with added ApoE3. HepG2 cells were transfected with siRNA targeting human LRP1 (siLRP1) and non-targeting negative siRNA (siCTRL) before the rosiglitazone treatment and adding ApoE3.

Article Snippet: The lysates were subjected to SDS–PAGE using anti-human ApoE3 antibody at a 1:150 000 dilution (R&D Systems).

Techniques: Recombinant, Incubation, Western Blot, Transfection

Journal: eLife

Article Title: APOE2 is associated with longevity independent of Alzheimer’s disease

doi: 10.7554/elife.62199

Figure Lengend Snippet: Figure 1. APOE associate with lifespan irrespective of AD in clinical cohorts. (A) Kaplan-Meier survival curve according to APOE genotype. (B) Effects of APOE4 (e3/e4 or e4/e4) or APOE2 (e2/e3 or e2/e2) on survival compared to APOE3 (e3/e3) as a reference in all subjects (‘All subjects’), when stratifying by AD diagnosis at last visit (‘No AD’ or ‘AD’), in subjects with neuropathologically-assessment (‘Patho-assessed’), and subjects with minimal amyloid pathology (‘Minimal amyloid’). HR = hazard ratio. CI = confidence interval. HRs and 95% CIs result from Cox proportional hazards regression models. Models for all subjects were adjusted for sex, race, cognitive status at last visit, presence of AD at last visit, and cardiovascular factors. The models for subjects with neuropathological assessment were adjusted for sex, race, CERAD diffuse plaque score, CERAD neuritic plaque score, Braak NFT stage, presence of vascular pathology, and cardiovascular factors. All other models were adjusted for sex, race, and cardiovascular factors.

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus. musculus) Apoetm1(APOE*2)Mae or apoE2-TR Sullivan et al., 1998 RRID:MGI:3695702 Genetic reagent (Mus. musculus) Apoetm2(APOE*3)Mae or apoE3-TR Sullivan et al., 1997 RRID:MGI:3695698 Genetic reagent (Mus. musculus) Apoetm3(APOE*4)Mae or apoE4-TR Knouff et al., 1999 RRID:MGI:4355228 Genetic reagent (Mus. musculus) Apoetm1Unc or Apoe-KO Piedrahita et al., 1992 RRID:MGI:4358709 Antibody Goat polyclonal anti-ApoE-biotin Meridian Life Science Cat#: K74180B ELISA detection (1:4000) Antibody Goat polyclonal antiApolipoprotein E Antibody Millipore Cat#: AB947 ELISA capture (1:4000) Peptide, recombinant protein ApoE3 Fitzgerald Cat#: 30 R-2381 ELISA standard Commercial assay or kit Cholesterol E Wako Cat. #: 999–02601 Commercial assay or kit L-Type Triglyceride M Wako Cat. #: 994–02891 Commercial assay or kit HDL-Cholesterol E Wako Cat. #: 997–01301 Software, algorithm SAS SAS Institute, Inc version 9.4 Software, algorithm R Statistical Software R Foundation for Statistical Computing version 3.2.3 Software, algorithm JMP Pro SAS Institute, Inc version 12 Software, algorithm AnyMaze software Stoelting Co Animal behavior test Human clinical and neuropathological data The clinical data from NACC, which were collected by the 34 past and present Alzheimer’s Disease Centers (ADCs) from September 2005 to November 2016 as the longitudinal Uniform Data Set (Weintraub, 2009), were assessed in this study.

Techniques: Biomarker Discovery

Journal: eLife

Article Title: APOE2 is associated with longevity independent of Alzheimer’s disease

doi: 10.7554/elife.62199

Figure Lengend Snippet: Figure 3. APOE2 is associated with preserved activity in clinical cohorts. (A) Odds ratio with 95% CIs of APOE2 or APOE4 on ‘dropped activities and interests’, ‘total GDS score’, and ‘memory problem’ compared to APOE3, as calculated by logistic regression models that were adjusted for sex, race, and age at the time of the GDS questionnaire. (B) Kaplan–Meier survival curve of subjects with/without ‘dropped activities and interests’; dropped activities and interests were associated with poorer survival (Hazard ratio = 1.20, p=0.010). *p<0.05; calculated by Cox proportional hazards regression analysis adjusting for sex, race, and APOE genotype group.

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Mus. musculus) Apoetm1(APOE*2)Mae or apoE2-TR Sullivan et al., 1998 RRID:MGI:3695702 Genetic reagent (Mus. musculus) Apoetm2(APOE*3)Mae or apoE3-TR Sullivan et al., 1997 RRID:MGI:3695698 Genetic reagent (Mus. musculus) Apoetm3(APOE*4)Mae or apoE4-TR Knouff et al., 1999 RRID:MGI:4355228 Genetic reagent (Mus. musculus) Apoetm1Unc or Apoe-KO Piedrahita et al., 1992 RRID:MGI:4358709 Antibody Goat polyclonal anti-ApoE-biotin Meridian Life Science Cat#: K74180B ELISA detection (1:4000) Antibody Goat polyclonal antiApolipoprotein E Antibody Millipore Cat#: AB947 ELISA capture (1:4000) Peptide, recombinant protein ApoE3 Fitzgerald Cat#: 30 R-2381 ELISA standard Commercial assay or kit Cholesterol E Wako Cat. #: 999–02601 Commercial assay or kit L-Type Triglyceride M Wako Cat. #: 994–02891 Commercial assay or kit HDL-Cholesterol E Wako Cat. #: 997–01301 Software, algorithm SAS SAS Institute, Inc version 9.4 Software, algorithm R Statistical Software R Foundation for Statistical Computing version 3.2.3 Software, algorithm JMP Pro SAS Institute, Inc version 12 Software, algorithm AnyMaze software Stoelting Co Animal behavior test Human clinical and neuropathological data The clinical data from NACC, which were collected by the 34 past and present Alzheimer’s Disease Centers (ADCs) from September 2005 to November 2016 as the longitudinal Uniform Data Set (Weintraub, 2009), were assessed in this study.

Techniques: Activity Assay

Journal: Cell reports

Article Title: PICALM Rescues Endocytic Defects Caused by the Alzheimer's Disease Risk Factor APOE4.

doi: 10.1016/j.celrep.2020.108224

Figure Lengend Snippet: Figure 1. APOE4-Expressing iPSC-Derived Astrocytes Exhibit a Defect in Early Endocytosis (A) Differentiation of iPSCs to astrocytes. (B) Astrocytes stained for S100b and GFAP. Scale bar, 100 mm. (C) Schematic of endocytosis steps probed in human APOE astrocytes. (D) Levels of Rab5 (left) and EEA1 (right) staining an isogenic pair of APOE astrocytes. Mean ± SD shown of 80–100 cells per sample for n = 3 samples. (E and F) Representative images of (E) Rab5 staining and (F) EEA1 staining in APOE astrocytes. Scale bar, 50 mm. (G) Quantification of microscopy images of the number of EGF puncta per cell (left) or the internalization of transferrin per cell (right) in one isogenic pair of APOE astrocytes (APOE3 parental line). Mean ± SD shown of 30–50 cells per sample for n = 12 samples. (H and I) Representative images displaying (H) EGF endocytosis and (I) transferrin endocytosis in human APOE astrocytes. Scale bar, 50 mm. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-S100B Atlas Antibodies AMAB91038 Cat#: AMAb91038, RRID:AB_2665776 Goat polyclonal anti-GFAP Abcam ab53554 Cat# ab53554, RRID:AB_880202 Mouse monoclonal anti-Rab5 CST 46449 Cat# 46449, RRID:AB_2799303 Mouse monoclonal anti-EEA1 BD Transduction Laboratories 610457 Cat# 610457, RRID:AB_397830 Rabbit monoclonal anti-Rab7 CST 9367 Cat# 9367, RRID:AB_1904103 Rabbit monoclonal anti-Rab11 CST 5589 Cat# 5589, RRID:AB_10693925 Goat polyclonal anti-APOE Millipore Sigma AB947 Cat# AB947, RRID:AB_2258475 Rabbit polyclonal anti-PGK1 Antibodies-online.com ABIN568371 Rat monoclonal anti-HA (conjugated to HRP) Roche 12 013 819 001 Cat# 12013819001, RRID:AB_390917 Bacterial and Virus Strains pLVX-GFAP-PICALM-HA, PICALM overexpression lentivirus This study N/A Chemicals, Peptides, and Recombinant Proteins Transferrin-647 Invitrogen T23366 EGF-555 Invitrogen E35350 Lysotracker Red Invitrogen L7528 B-Estradiol Sigma Aldrich E8875-5G Propidium iodide Sigma Aldrich P4864 CSM lacking uracil Sunrise Science Products 1004-100 CSM lacking uracil and histidine Sunrise Science Products 1009-100 Experimental Models: Cell Lines APOE3/APOE3 parental human iPSC line Coriell AG09173 APOE4/APOE4 isogenic human iPSC line Lin et al., 2018 Lin et al., 2018 APOE4/APOE4 parental human iPSC line Coriell AG10788 APOE3/APOE3 isogenic human iPSC line Lin et al., 2018 Lin et al., 2018 Experimental Models: Organisms/Strains S. cerevisiae: Strain background: W303 ATCC 201238 Recombinant DNA Invertase GSS reporter construct Conibear lab Burston et al., 2009 pNH603-ApoE3 This study N/A pNH603-ApoE4 This study N/A pNH604-ZEM (pJMB102) Kayatekin et al., 2018 Kayatekin et al., 2018 pBY011-YAP1802 (FLEXGene library clone) DNASU.org ScCD00094178 Software and Algorithms GraphPad Prism GraphPad Software https://www.graphpad.com/scientific-software/prism/ Imaris Oxford Instruments https://imaris.oxinst.com/ ImageJ Schneider et al., 2012 https://imagej.net/Welcome FlowJo v8 FlowJo https://www.flowjo.com/solutions/ flowjo/downloads

Techniques: Expressing, Derivative Assay, Staining, Microscopy