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

Journal: Journal of Lipid Research

Article Title: Apolipoprotein A5 reduces clearance of VLDL by altering apolipoprotein E content

doi: 10.1016/j.jlr.2025.100917

Figure Lengend Snippet: Verification of key protein abundance differences assessed by Western blot. Ultracentrifugally isolated VLDL from WT or Apo a 5 KO mice was prepared, assayed for APOB content by ELISA, separated by SDS-PAGE, and probed with antibodies for APOA5, APOE, SAA (shown to be reduced in the KO by MS in ), and APOA1 (increased in KO VLDL). Diluted unfractionated WT mouse plasma (P) was used to show the correct size of each protein. Each numeral represents a single mouse.

Article Snippet: The primary antibodies used were rabbit-anti-mouse ApoB100 (GTX135994, 1:1,000 dilution; GeneTex), rabbit-anti-mouse Apoa5 (#OACD01724, 1:1,000 dilution; Aviva Systems Biology), rabbit-anti-mouse Apoa1 (#PA5-29557, 1:1,000 dilution; Thermo Fisher), rabbit-anti-mouse Apoe (#68587, 1:2000 dilution; Cell Signaling Technology), goat serum amyloid A1/A2 (SAA1/SAA2) (#AF2948, 1:500 dilution; R&D Systems), and rabbit-anti-mouse Apod (# Ab187513 , 1:500 dilution; Abcam).

Techniques: Quantitative Proteomics, Western Blot, Isolation, Enzyme-linked Immunosorbent Assay, SDS Page, Clinical Proteomics

Journal: Journal of Lipid Research

Article Title: Impaired ApoB secretion triggers enhanced secretion of ApoE to maintain triglyceride homeostasis in hepatoma cells

doi: 10.1016/j.jlr.2025.100795

Figure Lengend Snippet: TG biosynthesis regulates the secretion of both ApoE and ApoB. A: Immunoblots showing depletion of SOAT1 and SOAT2 in Huh-7.5 cells expressing indicated sgRNAs. B: RT-qPCR determination of DGAT1 and DGAT2 mRNA levels in control and SOAT1 / 2 sgRNA-expressing Huh-7.5 cells transfected with indicated siRNAs. ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). C: BODIPY 493/503 staining of LDs (green) in Huh-7.5 cells transfected with indicated siRNAs and expressing indicated sgRNAs. BODIPY values were normalized to the cell number as measured by DAPI staining (blue). ∗∗∗ P < 0.001 versus control (n = 4, one-way ANOVA with Dunnett’s multiple comparisons test). Scale bar, 100 μm. D: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) from Huh-7.5 cells expressing indicated sgRNAs and transfected with indicated siRNAs. Immunoblots are shown below. ∗∗ P < 0.01, ∗∗∗ P < 0.001 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test). E: Relative fluorescence intensity of Huh-7.5 cells expressing indicated sgRNAs stained with BODIPY 493/503. ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). F: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) from Huh-7.5 cells expressing indicated sgRNAs. Immunoblots are shown below. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). G: (top) Incorporation of 13 C-OA into TG in Huh-7.5 cells expressing indicated sgRNAs. TG abundance was quantified by GC-MS. (bottom) TG abundance in the same set of cells was determined by a colorimetric assay. ∗ P < 0.05, ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). H: (top panel) Immunoblots showing expression of FLAG-tagged DGAT proteins in Huh-7.5 cells. (bottom panels) RT-qPCR determination of DGAT1 and DGAT2 mRNA levels. ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). I: TG abundance in Huh-7.5 cells expressing DGAT1-FLAG, DGAT2-FLAG or empty vector determined by a colorimetric assay. ∗∗ P < 0.01, ∗∗∗ P < 0.001 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test). J: Relative abundances of ApoE and ApoB in supernatants (upper panels) and lysates (lower panels) from Huh-7.5 cells expressing DGAT1-FLAG or DGAT2-FLAG. Immunoblots are shown below. ∗ P < 0.05, ∗∗ P < 0.01 versus control (n = 3, one-way ANOVA with Dunnett’s multiple comparisons test).

Article Snippet: Primary antibodies to ApoB (1:200 dilution, sc-13538) and SOAT1 (1:200 dilution, sc-137013, non-reducing condition) were from Santa Cruz Biotechnology; ApoE (1:500 dilution, MCA5639GA) was from Bio-Rad; SOAT2 (1:1,000 dilution, 21852-1-AP, non-reducing condition), Albumin (1:50,000 dilution, 16475-1-AP), and LDLR (1:5,000 dilution, 10785-1-AP) were from Proteintech; Actin (1:10,000 dilution, A2066) and FLAG (1:1,000 dilution, F7425) were from Sigma; GAPDH (Clone 5A12; 1:5,000 dilution, 016–25523) was from Wako; IRDye 680 or 800 secondary antibodies including #926–68072, #926–32213, and #926–32212 (1:20,000 dilution) were from LI-COR.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Control, Transfection, Staining, Fluorescence, Gas Chromatography-Mass Spectrometry, Colorimetric Assay, Plasmid Preparation

Journal: Journal of Lipid Research

Article Title: Impaired ApoB secretion triggers enhanced secretion of ApoE to maintain triglyceride homeostasis in hepatoma cells

doi: 10.1016/j.jlr.2025.100795

Figure Lengend Snippet: MTP inhibition promotes ApoE secretion by enhancing its expression. A: Immunoblots of ApoE and ApoB in supernatants and cell lysates of Huh-7.5 cells treated with EtOH (Vehicle) or 200 μM oleic acid (OA) in the presence or absence of 2.5 μM MTPi. Relative abundances of ApoE (left panels) and ApoB (right panels) are shown below. ∗∗ P < 0.01, ∗∗∗ P < 0.001 (n = 3, two-tailed Student's t test). B: Results of MTPi treatment as in (A) in serum-free medium. Loading controls (Actin and human albumin) are shown at the bottom. ∗∗∗ P < 0.001 (n = 3, two-tailed Student's t test). C: Immunoblots of LDLR in lysates from Huh-7.5 cells treated with MTPi. ∗∗∗ P < 0.001 versus control (n = 3, two-tailed Student's t test). D: Confocal microscopic images of Huh-7.5 cells treated with MTPi or DMSO control. Cells were stained with antibodies against ApoE (red) and the Golgi marker GM130 (magenta), and DAPI to stain nuclei (blue). Scale bar, 10 μm. E: Stability of ApoE protein in MTPi-treated Huh-7.5 cells after treatment with 50 μg/ml puromycin and 100 ng/ml brefeldin A. Data were fit to a one-phase decay model (n = 3, R 2 = 0.9279–0.9726). F: Effect of MTPi on APOE and APOB mRNA levels in cells treated with EtOH (Vehicle) and OA. ∗∗ P < 0.01 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test). G: Inhibition of de novo transcription of APOE by treating with different concentrations of actinomycin D (ActD). ∗∗∗ P < 0.001 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test).

Article Snippet: Cells were then washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, washed twice with PBS, and permeabilized with 0.2% Triton X-100 for 10 min. After washing with PBS, cells were blocked with Intercept Blocking Buffer for 1 h. Cells were then stained with primary antibodies to ApoE (1:100 dilution, MCA5639, Bio-Rad) and washed three times with PBS containing 0.5% Tween-20, followed by overnight incubation with Anti-PDI (1:100 dilution, ADI-SPA-890, Enzo Life Sciences) or Anti-GM130 (1:500 dilution, PM061, MBL) antibodies.

Techniques: Inhibition, Expressing, Western Blot, Two Tailed Test, Control, Staining, Marker

Journal: Journal of Lipid Research

Article Title: Impaired ApoB secretion triggers enhanced secretion of ApoE to maintain triglyceride homeostasis in hepatoma cells

doi: 10.1016/j.jlr.2025.100795

Figure Lengend Snippet: ApoB and ApoE redundantly contribute to TG turnover. A: Subcellular localization of ApoE and TG in Huh-7.5 cells. Cells were stained with antibodies against ApoE (red), the ER marker PDI (magenta), and the Golgi marker GM130 (magenta). TG and nuclei were stained with BODIPY 493/503 (green) and DAPI (blue), respectively. Scale bar, 10 μm. B: Three-dimensional (3D) view of a merged image shown in A (lower panel). Dashed lines indicate the position of the Z-stack images shown below. Scale bar, 10 μm. C: Flow cytometric analysis of BODIPY 493/503 staining in control or APOE -KO1 Huh-7.5 cells treated with MTPi (2.5 μM) or DMSO. ∗∗ P < 0.01 (n = 3, two-way ANOVA with Sidak’s multiple comparisons test).

Article Snippet: Cells were then washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, washed twice with PBS, and permeabilized with 0.2% Triton X-100 for 10 min. After washing with PBS, cells were blocked with Intercept Blocking Buffer for 1 h. Cells were then stained with primary antibodies to ApoE (1:100 dilution, MCA5639, Bio-Rad) and washed three times with PBS containing 0.5% Tween-20, followed by overnight incubation with Anti-PDI (1:100 dilution, ADI-SPA-890, Enzo Life Sciences) or Anti-GM130 (1:500 dilution, PM061, MBL) antibodies.

Techniques: Staining, Marker, Control