Journal: iScience

Article Title: APOE4 drives widespread changes to the hepatic proteome and alters metabolic function

doi: 10.1016/j.isci.2026.115035

Figure Lengend Snippet: Whole liver proteomics in female and male APOE3 and APOE4 mice (A) Study schematic showing primary outcomes in APOE -targeted replacement (TR) mice. n = 3 mice per group for all mouse proteomic outcomes. (B) APOE protein expression from whole liver measured via ELISA in male and female APOE3 and APOE4 mice. Statistical significance was determined by two-way ANOVA. Data are presented as mean ± SD. n = 8 mice per genotype and sex. (C) The number of differentially expressed (DE) proteins in male and female APOE3 and APOE4 mice. (D) Volcano plot showing upregulated and downregulated proteins in female APOE4 vs. APOE3 mice. (E) IPA pathway showing top 5 upregulated and downregulated pathways in female APOE4 vs. APOE3 mice. (F) Volcano plot showing upregulated and downregulated proteins in male APOE4 vs. APOE3 mice. (G) IPA pathway analysis showing top 5 upregulated and downregulated pathways in male APOE4 vs. APOE3 mice. (H) Heatmap of proteins involved in fatty acid metabolism, cholesterol and bile acid metabolism, lipid transport, and lipid storage showing upregulated and downregulated proteins between APOE4 and APOE3 mice. (I) Venn diagram showing the number of shared upregulated proteins between comparisons of female APOE4 vs. APOE3 mice and male APOE4 vs. APOE3 mice. (J) Venn diagram showing the number of downregulated proteins shared between comparisons of female APOE4 vs. APOE3 mice and male APOE4 vs. APOE3 mice. (K) Violin plot showing normalized Pgam1 protein expression from liver proteomics in male and female APOE3 and APOE4 mice. Statistical significance was determined by two-way ANOVA with Fisher’s LSD post hoc. ∗ p < 0.05. G, genotype. n = 3 mice per genotype and sex. See also .

Article Snippet: These APOE TR mice were previously generated by removing and replacing the endogenous mouse Apoe gene with the human APOE gene., Mice were maintained on a standard chow diet (Teklad Global Rodent Diet, 8604) for the entire study and housed at ∼25°C with a standard 12-h light/dark cycle.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: APOE4 drives widespread changes to the hepatic proteome and alters metabolic function

doi: 10.1016/j.isci.2026.115035

Figure Lengend Snippet: Proteomics analysis of APOE isogenic iHLCs (A) iHLC study schematic. Two pairs of isogenic iPSCs (A and B) homozygous for either APOE3 or APOE4 were used to generate iHLCs and examine proteomic and bioenergetic outcomes. n = 5 per group for all iHLC proteomic outcomes. (B) Violin plots of APOE protein expression between two sample batches in pair A isogenics. Statistical significance was determined by unpaired t test. ns, not significant, ∗∗∗ p < 0.001. (C) Volcano plot showing upregulated and downregulated proteins between APOE4 and APOE3 iHLCs from batch 1. (D) Volcano plot showing upregulated and downregulated proteins between APOE4 and APOE3 iHLCs from batch 2. (E) Heatmap of the top 20 most significant upregulated and top 20 most significant downregulated proteins ( p < 0.05) shared between batch 1 and batch 2. (F) IPA pathway analysis showing top 12 upregulated and top 12 downregulated pathways shared in both batch 1 and 2 between APOE4 and APOE3 iHLCs. (G) STRING network analysis of the most significant upregulated and downregulated proteins shared between batch 1 and batch 2 in APOE4 vs. APOE3 iHLCs. (H) Cellular component Gene Ontology (GO) analysis of the top 20 upregulated and top 20 downregulated proteins in APOE4 vs. APOE3 iHLCs. See also .

Article Snippet: These APOE TR mice were previously generated by removing and replacing the endogenous mouse Apoe gene with the human APOE gene., Mice were maintained on a standard chow diet (Teklad Global Rodent Diet, 8604) for the entire study and housed at ∼25°C with a standard 12-h light/dark cycle.

Techniques: Expressing

Journal: iScience

Article Title: APOE4 drives widespread changes to the hepatic proteome and alters metabolic function

doi: 10.1016/j.isci.2026.115035

Figure Lengend Snippet: Mitochondrial function in APOE isogenic iHLCs (A) Mitochondrial stress test (MST) tracing from isogenic pair A. (B) Quantification of basal respiration, maximal respiration, proton leak, and ATP-production linked respiration from isogenic pair an MST. n = 13–14 per group. (C) MST tracing from isogenic pair B. (D) Quantification of basal respiration, maximal respiration, proton leak, and ATP-production-linked respiration from isogenic pair B MST. n = 12–15 per group. (E) Electron transport chain (ETC) flux through complex I, II, III, and IV in isogenic pair A. n = 7–22 per group. (F) ETC flux through complex I, II, III, and IV in isogenic pair B. n = 14–22 per group. (G) Representative tetramethyl rhodamine, ethyl ester, perchlorate (TMRE) images from isogenic pair A. Scale bars, 50 μm. (H–K) Quantification of TMRE (H), MitoSOX (I), Amplex Red (J), and Rhod-2 AM (K) fluorescence intensities from isogenic pair A. n = 8–16 per group. (L) Representative TMRE images from isogenic pair B. Scale bars, 50 μm. (M–P) Quantification of TMRE (M), MitoSOX (N), Amplex Red (O), and Rhod-2 AM (P) fluorescence intensities from isogenic pair B. n = 8–16 per group. (B, D, E–F, H–K, and M–P) Statistical significance was determined by unpaired t test. Data are shown as mean ± SD. ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: These APOE TR mice were previously generated by removing and replacing the endogenous mouse Apoe gene with the human APOE gene., Mice were maintained on a standard chow diet (Teklad Global Rodent Diet, 8604) for the entire study and housed at ∼25°C with a standard 12-h light/dark cycle.

Techniques: Fluorescence

Journal: iScience

Article Title: APOE4 drives widespread changes to the hepatic proteome and alters metabolic function

doi: 10.1016/j.isci.2026.115035

Figure Lengend Snippet: Glycolytic function and glucose/pyruvate oxidation in APOE isogenic iHLCs (A) Glycolytic stress test (GST) tracing from isogenic pair A. (B) Quantification of basal glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification from isogenic pair A GST. n = 11 per group. (C) GST tracing from isogenic pair B. (D) Quantification of basal glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification from isogenic pair B GST. n = 10–16 per group. (E) Glucose/pyruvate oxidation stress test from isogenic pair A. (F) Quantification of basal respiration, acute response, and maximal respiration from isogenic pair A glucose/pyruvate oxidation stress test. n = 7–10 per group. (G) Glucose/pyruvate oxidation stress test from isogenic pair B. (H) Quantification of basal respiration, acute response, and maximal respiration from isogenic pair B glucose/pyruvate oxidation stress test. n = 7–11 per group. (I) Significant DE proteins involved in glucose metabolism pathway from IPA. (J) Glucose metabolism protein network from STRING analysis. (B and D) Statistical significance was determined by unpaired t test and (F and H) two-way ANOVA with Fisher’s LSD post hoc. Data are shown as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. D, drug, G, genotype.

Article Snippet: These APOE TR mice were previously generated by removing and replacing the endogenous mouse Apoe gene with the human APOE gene., Mice were maintained on a standard chow diet (Teklad Global Rodent Diet, 8604) for the entire study and housed at ∼25°C with a standard 12-h light/dark cycle.

Techniques:

Journal: iScience

Article Title: APOE4 drives widespread changes to the hepatic proteome and alters metabolic function

doi: 10.1016/j.isci.2026.115035

Figure Lengend Snippet: Fatty acid oxidation and LipidTOX staining in APOE isogenic iHLCs (A) Long chain fatty acid (LCFA) oxidation stress test from isogenic pair A. (B) Quantification of basal respiration, acute response, and maximal respiration from isogenic pair An LCFA oxidation stress test. n = 6–8 per group. (C) LCFA oxidation stress test from isogenic pair B. (D) Quantification of basal respiration, acute response, and maximal respiration from isogenic pair B LCFA oxidation stress test. n = 7–10 per group. (E) Representative LipidTOX images from isogenic pair A. Scale bars, 50 μm. (F) Quantification of lipid droplet (LD) numbers per cell and diameter (μm) from isogenic pair A. n = 24 per group. (G) Representative LipidTOX images from isogenic pair B. Scale bars, 50 μm. (H) Quantification of LD numbers per cell and diameter (μm) from isogenic pair B. n = 24 per group. (I) Comparison analysis showing significantly altered proteins in SREBF2 network from proteomics. (B and D) Statistical significance was determined by a two-way ANOVA with Fisher’s LSD post hoc and (F and H) unpaired t test. Data are shown as mean ± SD. ns = not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. D, drug, G, genotype.

Article Snippet: These APOE TR mice were previously generated by removing and replacing the endogenous mouse Apoe gene with the human APOE gene., Mice were maintained on a standard chow diet (Teklad Global Rodent Diet, 8604) for the entire study and housed at ∼25°C with a standard 12-h light/dark cycle.

Techniques: Staining, Comparison

Journal: bioRxiv

Article Title: Human breast milk extracellular vesicles from donors with asthma differentially the modulate release of inflammatory cytokines by primary human airway smooth muscle cells in a recipient-cell specific manner

doi: 10.64898/2026.03.02.709065

Figure Lengend Snippet: (A) Schematic representation of BM-EVs isolation and characterization. BM was collected from 3-4 months post-partum mothers with and without asthma diagnosis. BM-EVs were isolated by SEC (qEV original columns, Izon Science Ltd) and analyzed for size, concentration and zeta potential through TRPS using the qNano Gold instrument (Izon Science Ltd.). BM-EVs were concentrated for protein yield quantification through MicroBCA Protein Assay kit (Thermo Scientific), Western blot analysis for exosome and microvesicles markers and hASM cells treatment for cytokines release analysis. (B) TEM showing exosomal morphology and approximate size of BM-EVs (scale bar 100[nm). (C) Western blotting was performed (12% SDS-PAGE) and Ponceau S staining used as a loading control. Proteins enriched in exosomes (CD9, CD81, CD63, TSG101, HSP70 and Flotillin-1), in microvesicles (MMP2 and ARF6), and lipoproteins (APO-A1) were analysed. F7 to 9 were considered exosome-rich while microvesicles and lipoprotein-poor. CL: cell lysate from breast tissue, M: marker lane.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-TSG101 (T5701, Sigma-Aldrich Co, 1:200), rabbit polyclonal anti-CD63 (SAB4301607, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-CD81 (sc-166029, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-CD9 (CBL162, Sigma-Aldrich Co, 1:100), rabbit polyclonal anti-Flotillin-1 (F1180, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-heat shock protein 70 (HSP70) (H5147, Sigma-Aldrich Co, 1:500), mouse monoclonal anti-ARF6 (sc-7971, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-MMP2 (sc-13595, Santa Cruz Biotechnology, 1:200) and mouse monoclonal anti-Apolipoprotein A1 (Apo-A1) (0650-0050, Bio-Rad Laboratories, 1:200).

Techniques: Isolation, Biomarker Discovery, Concentration Assay, Zeta Potential Analyzer, Western Blot, SDS Page, Staining, Control, Marker

Journal: bioRxiv

Article Title: Human breast milk extracellular vesicles from donors with asthma differentially the modulate release of inflammatory cytokines by primary human airway smooth muscle cells in a recipient-cell specific manner

doi: 10.64898/2026.03.02.709065

Figure Lengend Snippet: (A) Equal amounts of protein (5 µg/mL) of BM-EVs from mothers with and without asthma were subjected to SDS-PAGE (12% SDS) and the expression of proteins enriched in exosomes [CD81 (22 kDa), CD63 (28 kDa), CD9 (25 kDa), Flotillin-1 (48 kDa), TSG101 (46 kDa), HSP70 (70 kDa)], in microvesicles [MMP2 (63kDa)], and in lipoproteins [APO-A1 (25kDa)] were quantified. Ponceau or Coomassie staining was used as a loading control to normalize protein content for all markers. (B) CD81 levels remained unchanged between the groups, (C) while asthma BM-EVs expressed ∼86% lower CD63 (p=0.0224) and (D) ∼ 24% lower CD9 (p=0.0646). (E) Flotillin-1 expression was lower by ∼40% in asthma BM-EVs compared to control (p=0.0196). (F) TSG101 levels remained unchanged, (G) while HSP70 was ∼69% lower in the asthma BM-EV (p=0.08). (H) APO-A1 was used as a purity control to investigate the presence of lipoproteins in the samples and did not show difference between groups. BM-EVs did not show any expression for MMP2 protein. Data were analyzed using an unpaired Student’s t-test with p<0.05 considered as significant and expressed as mean ± standard error (N=5).

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-TSG101 (T5701, Sigma-Aldrich Co, 1:200), rabbit polyclonal anti-CD63 (SAB4301607, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-CD81 (sc-166029, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-CD9 (CBL162, Sigma-Aldrich Co, 1:100), rabbit polyclonal anti-Flotillin-1 (F1180, Sigma-Aldrich Co, 1:200), mouse monoclonal anti-heat shock protein 70 (HSP70) (H5147, Sigma-Aldrich Co, 1:500), mouse monoclonal anti-ARF6 (sc-7971, Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-MMP2 (sc-13595, Santa Cruz Biotechnology, 1:200) and mouse monoclonal anti-Apolipoprotein A1 (Apo-A1) (0650-0050, Bio-Rad Laboratories, 1:200).

Techniques: SDS Page, Expressing, Staining, Control

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