Journal: Journal of Lipid Research

Article Title: Amino acid residues L261, W264, F265, L268, and V269 of apolipoprotein E4 critically regulate adipose tissue metabolism

doi: 10.1016/j.jlr.2025.100958

Figure Lengend Snippet: Effects of APOE4 and APOE4mut1 on 3T3-L1 adipocyte differentiation. A: Representative Western blot analysis for APOE4, APOE4mut1 in samples of culture medium collected on the 10th day of 3T3-L1 cell differentiation as well as β-tubulin in the respective cell lysates. Samples for APOE were analysed on two separate blots. B: Semiquantitative analysis of APOE4 and APOE4mut1 levels shown in the blots of panel A, normalized for β-tubulin levels (C–E) Representative images at 10× magnification of differentiated 3T3-L1 adipocytes after Oil Red O staining. The cells were induced to differentiate after infection with: (C) AdGFP, (D) AdAPOE4, (E) AdAPOE4mut1. F: Relative absorbance of Oil Red O dye, eluted from cultures infected with recombinant adenovirus. The values were derived from the average of at least three cultures for each adenovirus used. The data were analyzed using one-way ANOVA, and the values are expressed as Mean ± SEM. G: Effect of APOE4 and APOE4mut1 on triglyceride accumulation in mature 3T3-L1 adipocytes. The values for each culture were normalized to the total protein levels of the cells present in the culture. The analysis of the results was performed using one-way ANOVA, and the values are presented as Mean ± SEM. H: mRNA expression levels of the lipogenic genes ppar γ , dgat1 , and fasn as well as ppar γ effector genes adiponectin , fabp4 , fabp5 , and lpl in differentiated 3T3-L1 adipocytes expressing APOE4 and APOE4mut1. The results were normalized to the expression levels of the housekeeping rps18 gene. The analysis was performed using two-way ANOVA, and the values are presented as Mean ± SEM. ∗∗ = P < 0.005 and ∗∗∗ = P < 0.0005, n = 3 per group. Unless indicated by a horizontal line between two groups, statistical significance refers to differences among all test groups.

Article Snippet: For the semiquantitative measurement of murine and human protein levels in the plasma and the lipoprotein fractions of the mice, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, followed by Western blot, using the following antibodies: Rabbit anti-Human and anti-Mouse APOE (D7I9N, cat# 10197, Cell Signaling), Goat anti APOB-48/100 HRP (cat# K34005G, Meridian Life Science), Rabbit anti-Mouse β-tubulin(cat# # 2146, Cell Signaling), Rabbit anti-Mouse Cytc (cat# 4272, Cell Signaling), Rabbit anti-Mouse Ucp1 (E9Z2V, cat#72298, Cell Signaling), Rabbit anti-Mouse Cox4 (cat# 4844, Cell Signaling), and Goat anti-rabbit IgG-HRP (cat#7074, Cell Signaling).

Techniques: Western Blot, Cell Differentiation, Staining, Infection, Recombinant, Derivative Assay, Expressing

Journal: Journal of Lipid Research

Article Title: Amino acid residues L261, W264, F265, L268, and V269 of apolipoprotein E4 critically regulate adipose tissue metabolism

doi: 10.1016/j.jlr.2025.100958

Figure Lengend Snippet: Plasma lipid and glucose levels of C57BL/6 mice fed high-fat diet for 8 weeks and then infected with the adenoviruses AdGFP, AdAPOE4, and AdAPOE4mut1. A: Representative Western blot of human and murine APOE five days after infection of mice with the adenoviruses, (B) plasma total cholesterol, (C) plasma total triglyceride, and (D) plasma glucose levels five days after infection of mice with the adenoviruses. The results were analyzed using one-way ANOVA. Values are expressed as Mean ± SEM. ∗ = P < 0.05, ∗∗ = P < 0.005 and ∗∗∗ = P < 0.0005, n = 3 per group.

Article Snippet: For the semiquantitative measurement of murine and human protein levels in the plasma and the lipoprotein fractions of the mice, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, followed by Western blot, using the following antibodies: Rabbit anti-Human and anti-Mouse APOE (D7I9N, cat# 10197, Cell Signaling), Goat anti APOB-48/100 HRP (cat# K34005G, Meridian Life Science), Rabbit anti-Mouse β-tubulin(cat# # 2146, Cell Signaling), Rabbit anti-Mouse Cytc (cat# 4272, Cell Signaling), Rabbit anti-Mouse Ucp1 (E9Z2V, cat#72298, Cell Signaling), Rabbit anti-Mouse Cox4 (cat# 4844, Cell Signaling), and Goat anti-rabbit IgG-HRP (cat#7074, Cell Signaling).

Techniques: Clinical Proteomics, Infection, Western Blot

Journal: Journal of Lipid Research

Article Title: Amino acid residues L261, W264, F265, L268, and V269 of apolipoprotein E4 critically regulate adipose tissue metabolism

doi: 10.1016/j.jlr.2025.100958

Figure Lengend Snippet: Plasma lipoprotein lipid levels of C57BL/6 mice fed high-fat diet for 8 weeks and then infected with the adenoviruses AdGFP, AdAPOE4, and AdAPOE4mut1. Lipoproteins were fractionated by KBr density gradient ultracentrifugation. A, C–E: Total cholesterol and (F–I) triglyceride levels of lipoprotein fractions 5 days after infection of mice with the adenoviruses. Panel B shows the murine APOE, human APOE4 and APOE4mut1 distribution among lipoprotein fractions. The results were analyzed using one-way ANOVA, and values are expressed as Mean ± SEM. ∗∗∗ = P < 0.0005, n = 3 per group. Statistical significance refers to differences among all test groups.

Article Snippet: For the semiquantitative measurement of murine and human protein levels in the plasma and the lipoprotein fractions of the mice, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, followed by Western blot, using the following antibodies: Rabbit anti-Human and anti-Mouse APOE (D7I9N, cat# 10197, Cell Signaling), Goat anti APOB-48/100 HRP (cat# K34005G, Meridian Life Science), Rabbit anti-Mouse β-tubulin(cat# # 2146, Cell Signaling), Rabbit anti-Mouse Cytc (cat# 4272, Cell Signaling), Rabbit anti-Mouse Ucp1 (E9Z2V, cat#72298, Cell Signaling), Rabbit anti-Mouse Cox4 (cat# 4844, Cell Signaling), and Goat anti-rabbit IgG-HRP (cat#7074, Cell Signaling).

Techniques: Clinical Proteomics, Infection

Journal: The American journal of pathology

Article Title: The bradykinin B1 receptor regulates Aβ deposition and neuroinflammation in Tg-SwDI mice.

doi: 10.1016/j.ajpath.2013.01.021

Figure Lengend Snippet: Figure 3 B1R blockade is not associated with changes in APP processing or in levels of LRP1, APOE, and neprilysin in Tg-SwDI mice. Represen- tative blots (A) and quantitative results (B) of Western blot analysis showing that R715 admin- istration (1 mg/kg, s.c., 8 weeks) causes no changes in the levels of APP, APP C-terminal fragments C99 and C83, or APP-cleaving enzymes BACE1, ADAM10, and ADAM17, compared with vehicle-treated animals. C and D: Effect of R715 treatment on the expression of LRP1, APOE, and neprilysin. GAPDH levels were used as loading controls. The values represent means SEM (N Z 6 to 8).

Article Snippet: Western Blot Analysis Equal protein amounts were separated on a 4% to 12% SDSPAGE gradient, transferred to a nitrocellulose membrane, and incubated overnight with primary antibodies at 4 C. The following primary antibodies were used: human APP-CT20, 1741 Passos et al disintegrin and metalloproteinase domainecontaining protein (ADAM)10, ADAM17, b-site APP-cleaving enzyme 1 (BACE1; Calbiochem, San Diego, CA), glyceraldehyde-3phosphate dehydrogenase (GAPDH), B1R (Santa Cruz Biotechnology, Santa Cruz, CA), low-density lipoprotein receptorerelated protein 1 (LRP1), apolipoprotein E (APOE), neprilysin (Abcam, Cambridge, MA), phosphorylated-p65 NF-kB, liver X receptor, peroxisome proliferator-activated receptor g, and matrix metalloproteinase-9 (Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: ATP-binding Cassette Transporter A1 Mediates the Beneficial Effects of the Liver X Receptor Agonist GW3965 on Object Recognition Memory and Amyloid Burden in Amyloid Precursor Protein/Presenilin 1 Mice*

doi: 10.1074/jbc.m110.108100

Figure Lengend Snippet: FIGURE 2. High dose GW3965 is required for elevated apoE levels in tissue. Cortical and hippocampal extracts from APP/PS1 untreated (: white), APP/PS1 treated (: dark gray), APP/PS1 ABCA1/ untreated (: light gray), and APP/PS1 ABCA1/ treated (: black) mice were immunoblotted for apoE. Left panels are representative blots using undiluted samples from individual mice from the following cohorts: APP/PS1 untreated control: n 7 (cortex), n 8 (hippocampus); APP/PS1 prophylactic: n 8 (cortex) n 8 (hippocam- pus); APP/PS1 low dose therapeutic: n 8 (cortex), n 8 (hippocampus); APP/PS1 high dose therapeutic: n 10 (cortex), n 9 (hippocampus); ABCA1/ APP/PS1 untreated control: n 4 (cortex), n 4 (hippocampus); ABCA1/ APP/PS1 prophylactic: n 8 (cortex) n 8 (hippocampus): ABCA1/ APP/PS1 low dose therapeu- tic: n 7 (cortex), n 6 (hippocampus); ABCA1/ APP/PS1 high dose therapeutic: n 4 (cortex), n 4 (hippocampus). Samples from each mouse were blotted at least twice, normalized to actin, and expressed as fold-difference compared with apoE levels in untreated APP/PS1 controls. Data represent mean S.E., where * represents p 0.05 and ** represents p 0.01 by one-way analysis of variance followed by Tukey post test.

Article Snippet: Western Blot Analysis of ABCA1, ApoE, APP, and APP-CTF— For analysis ofABCA1, apoE, andAPP, 50–100 g of carbonate lysate was electrophoresed through 10% SDS-polyacrylamide gels, transferred to PVDFmembrane (Millipore), and immunodetected using a monoclonal anti-ABCA1 antibody, AC10 (1:1000, a kind gift from Dr. M. R. Hayden), a murine-specific apoE antibody (1:1000, Santa Cruz Biotechnology), an antiAPP antibody that recognizes both murine and human APP (clone 22C11, 1:2000, Chemicon), or an anti-actin antibody (1:5000, Chemicon) as a loading control.

Techniques: Control

Journal: Journal of Biological Chemistry

Article Title: ATP-binding Cassette Transporter A1 Mediates the Beneficial Effects of the Liver X Receptor Agonist GW3965 on Object Recognition Memory and Amyloid Burden in Amyloid Precursor Protein/Presenilin 1 Mice*

doi: 10.1074/jbc.m110.108100

Figure Lengend Snippet: FIGURE 3. ABCA1 is required for increased CSF apoE levels in response to GW3965. Equal volumes of CSF from individual mice from untreated control (C), prophylactic (P), low dose therapeutic (T), and high dose therapeutic (TH) groups of APP/PS1 and APP/PS1 ABCA1/ mice were pooled, separated by 6% native PAGE, and immunoblotted for apoE and albumin. Stokes diameter markers are shown on the right.

Article Snippet: Western Blot Analysis of ABCA1, ApoE, APP, and APP-CTF— For analysis ofABCA1, apoE, andAPP, 50–100 g of carbonate lysate was electrophoresed through 10% SDS-polyacrylamide gels, transferred to PVDFmembrane (Millipore), and immunodetected using a monoclonal anti-ABCA1 antibody, AC10 (1:1000, a kind gift from Dr. M. R. Hayden), a murine-specific apoE antibody (1:1000, Santa Cruz Biotechnology), an antiAPP antibody that recognizes both murine and human APP (clone 22C11, 1:2000, Chemicon), or an anti-actin antibody (1:5000, Chemicon) as a loading control.

Techniques: Control, Clear Native PAGE

Journal: Cancer cell

Article Title: Integrative molecular and spatial analysis reveals evolutionary dynamics and tumor-immune interplay of in situ and invasive acral melanoma.

doi: 10.1016/j.ccell.2024.04.012

Figure Lengend Snippet: Figure 6. Spatial transcriptomics analysis (A) Annotated spatial map of AM140 and AM147. Black dashed lines denote tumor regions. Scale bar, 1 mm. (B) Spatial expression levels of selected marker genes. Scale bar, 1 mm. (C) Representative images of single cell segmentation. Scale bar, 100 mm. (D) Proportions of epithelial-mesenchymal transition, EMT, tumor cells (top) and APOE+/CD163+ macrophages (bottom) between C3 and non-C3 tumors. One- sided Mann-Whitney U test. *p < 0.05, **p < 0.01. (E) Comparison of the distance of tumor cells to the nearest APOE+/CD163+ macrophage in C3 patients. Student’s t test. ***p < 0.001. (F) Activities of cells acting as sender cells (x axis) or receiver cells (y axis) in C3 patients based on CellChat. (G) IGF signaling interactions across cell clusters. Data are represented as boxplots in (D and E). The centerline shows the median; boxes indicate the first and third quartiles; whiskers extend 1.5 times the interquartile range. See also Figure S7.

Article Snippet: Primary antibodies used in this study include CD163 (clone-10D6, Abcam, Cat#ab74604, ready-to-use), APOE (clone-D17N, Cell Signaling, Cat#13366S, 1:750), pERK1/2 (Thr202/Tyr204, Cell Signaling, Cat#9101S, 1:200), pAKT (Ser473, Cell Signaling, Cat#9271S, 1:100), E-Cadherin (clone-4A2, Cell Signaling, Cat#14472S, 1:100), N-Cadherin (clone-D4R1H, Cell Signaling, Cat#13116S, 1:100), and FN1 (clone-E5H6X, Cell Signaling, Cat#26836S, 1:200).

Techniques: Expressing, Marker, MANN-WHITNEY, Comparison

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Single‐Cell RNA Sequencing Reveals Granzyme K + CD8 + T Cells as a Target for Mitigating Plaque Instability Following Radiation

doi: 10.1161/JAHA.124.040632

Figure Lengend Snippet: A , Schematic diagram of the research, including participant groups and scRNA‐seq technology. B , UMAP plots for the entire cell population, initially categorized into 19 clusters labeled 1 to 19 (left), with each cell color‐coded to represent its cell type (middle) and group of origin (right). C , Classic marker genes were used to delineate all cell types. D , Distribution of each cell type across various stages. E , Top enriched KEGG pathway bubble charts in ES. F , Top enriched KEGG pathway bubble charts in the AS. APOE indicates apolipoprotein E; AS, advanced stage; ES, early stage; HFD, high‐fat diet; IL, interleukin; ILC, innate lymphoid cells; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen‐activated protein kinase; NC, nonradiation control; NK, natural killer; NOD, nucleotide‐binding oligomerization domain; RT, radiotherapy; scRNA‐seq, single‐cell RNA sequencing; SMC, smooth muscle cell; STAT, signal transducers and activators of transcription. TNF, tumor necrosis factor; and UMAP, Uniform Manifold Approximation and Projection.

Article Snippet: Ninety‐six male ApoE −/− (apolipoprotein E −/− ), 20 GZMB −/− ApoE −/− , and 16 recombination‐activating gene 2 ( Rag2 ) −/− apoE −/− mice were purchased from the Cyagen Laboratory, all on a C57BL/6 background, aged 8 to 10 weeks, and weighing an average of 33.2±1.1 g. They were randomly divided into groups for radiation or sham treatment (nonradiation control and early stage) 4 days after radiation and advanced stage (2 weeks after radiation) and housed in ventilated cages with free access to a diet containing 3.7% fat and water.

Techniques: Labeling, Marker, Control, Binding Assay, RNA Sequencing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Single‐Cell RNA Sequencing Reveals Granzyme K + CD8 + T Cells as a Target for Mitigating Plaque Instability Following Radiation

doi: 10.1161/JAHA.124.040632

Figure Lengend Snippet: A , Flow cytometry plots of Gzmb + CD8 + T cells in different groups from atherosclerotic lesions in Gzmb −/− ApoE −/− mice. B , Frequencies of Gzmb + CD8 + T cells in atherosclerotic lesions were analyzed in different groups (n=5). C , Typical H&E‐stained aortic roots images are presented for various groups. D , Plaque area was determined and quantified relative to the aortic area (n=5). E , Immunofluorescence staining of atherosclerotic plaques showed SMA‐α (green), CD68 (red) in the fibrous caps among different groups. F , Quantification of SMA‐α + cell ratio per image (n=6). G , Quantification of CD68 + cell ratio per image (n=6). HFD represents the high‐fat diet group; HFD+RT, high‐fat diet group with radiotherapy; HFD+ Gzmb −/− , high‐fat diet group in Gzmb −/− A poE −/− mice; HFD+RT+ Gzmb −/− , high‐fat diet group with radiotherapy in Gzmb −/− ApoE −/− mice. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.001. ApoE indicates apolipoprotein E; CD8, cluster of differentiation 8; CD68, cluster of differentiation 68; DAPI, 4′,6‐diamidino‐2‐phenylindole; GZMB, granzyme B; H&E, hematoxylin and eosin; HFD, high‐fat diet; ns, not significant; RT, radiotherapy; and SMA‐α, smooth muscle actin‐α.

Article Snippet: Ninety‐six male ApoE −/− (apolipoprotein E −/− ), 20 GZMB −/− ApoE −/− , and 16 recombination‐activating gene 2 ( Rag2 ) −/− apoE −/− mice were purchased from the Cyagen Laboratory, all on a C57BL/6 background, aged 8 to 10 weeks, and weighing an average of 33.2±1.1 g. They were randomly divided into groups for radiation or sham treatment (nonradiation control and early stage) 4 days after radiation and advanced stage (2 weeks after radiation) and housed in ventilated cages with free access to a diet containing 3.7% fat and water.

Techniques: Flow Cytometry, Staining, Immunofluorescence