Isolation of Extracellular Vesicles From Mouse Brown Adipose Tissue Secretome Using Size‐Exclusion Chromatography

Figures

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  FIGURE 1. Isolation of brown adipose tissue and organ culture. (A) Incision and exposed interscapular region; (B) Located and lifted interscapular BAT; (C) excision of BAT; (D) Excised BAT; (E) BAT explant culture; and (F) BAT‐secretome.

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  FIGURE 2. Size‐exclusion Chromatography using qEV1 Column. (A) and (B) qEV Column set‐up; (C) flushing of the qEV column; (D) BAT‐secretome loaded at the frit; (E) Elution of BAT‐EVs; (F) BAT‐EV Fractions.

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  FIGURE 3. Characterization of BAT‐EVs from Male and Female Mice. (A) Concentration of BAT‐EVs from C57BL/6 male mice showing the greatest BAT‐EV concentration among the tested collagenase conditions at 0.50 mg/mL collagenase type 1 concentration after incubation for 48 h post 4 h equilibration. (B) Concentration of BAT‐EVs from C57BL/6 female mice showing the highest concentration of BAT‐EVs at 0.50 mg/mL collagenase type 1 concentration after incubation for 48 h post 4 h equilibration. (C) The mean size of BAT‐EVs from male mice was significantly lower with 0.25 mg/mL and 0.50 mg/mL collagenase type 1 concentration. (D) The mean size of BAT‐EVs from female mice was lower with 0.50 mg/mL collagenase concentration. (E) Size distribution of the BAT‐EVs obtained from male mice. (F) show the size distribution of the BAT‐EVs obtained from female mice. N = 3–4 per group; Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001.

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  FIGURE 4. Step‐by‐step schematics of the BAT‐EVs isolation protocol.

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  FIGURE 5. EV marker and biogenesis‐associated protein signature in BATEVs. Heatmap of log2 protein intensities (no row scaling) for a curated panel of proteins commonly associated with EVs and EV biogenesis/trafficking pathways, measured across BAT‐EV replicates. Proteins are grouped by functional panel annotation, including tetraspanins (e.g., Cd9, Cd82), ESCRT components (e.g., Pdcd6ip/ALIX, Chmp2a, Chmp4b), trafficking regulators (e.g., Rab5a, Rab7a), annexins (e.g., Anxa1/2/5/6), chaperones (e.g., Hspa8, Hsp90aa1, Hsp90ab1), and cytoskeletal proteins (e.g., Actb, Tuba1b, Vim). This panel supports EV identity and reflects expected biogenesis‐related cargo within the BAT‐EV proteome. Bar annotations indicate sample group and protein‐panel classification.

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  FIGURE 6. Reactome BAT‐associated proteins and BAT marker proteins identified in BATEVs. (A) Heatmap of log2 protein intensities (no row scaling) for the top 30 BAT‐associated Reactome proteins detected in mouse BAT‐derived EVs isolated using the experimental workflow. Values represent within‐dataset EV abundance profiles (clipped to reduce saturation). (B) Heatmap of log2 protein intensities (no row scaling) for key BAT/mitochondrial‐associated marker proteins detected in BAT‐derived EVs. Marker selection was based on frequent reporting in BAT proteomic studies and their established association with BAT identity and metabolic function. These include the canonical thermogenic marker Ucp1, as well as proteins linked to mitochondrial/energy metabolism and stress‐response pathways such as Vdac1, Atp2a1, Ephx1/Ephx2, and Hspa5, confirming their presence within the EV proteome.

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  FIGURE 7. Functional enrichment analysis of the BATEV proteome. (A) Gene Ontology (GO) Biological Process enrichment for proteins identified in the BAT‐EV dataset. Bar length represents the number of genes/proteins mapped to each GO term, and bar color indicates the Benjamini‐Hochberg adjusted p‐value (p.adjust). Terms are ordered by count from highest to lowest. (B) KEGG pathway enrichment for the same BAT‐EV protein set, visualized as gene/protein count (bar length) and p.adjust (color), also ordered by count. Enriched pathways highlight metabolic and bioenergetic programs characteristic of BAT biology, including carbon metabolism, glycolysis/TCA cycle, oxidative phosphorylation, and lipid and amino‐acid metabolism.

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  FIGURE 8. BAT‐EVs increased respiration in human induced pluripotent stem cells cardiomyocytes (hiPSC‐CM). (A) Bioenergetics profile of IPSC‐CM incubated with 3000/cell scWAT‐EVs or BAT‐EVs for 24 h. Bar graphs for (B) Maximal Respiration, (C) Spare Respiratory Capacity (D) Non‐mitochondrial Respiration in iPSC‐CMs incubated with BAT‐EVs and scWAT‐EVs. Data are mean ± SEM, n = 4 per group; *p < 0.05.