fig1

Figure 1. (A) Schematic representation of the biosynthetic pathway of the short O-glycan, STn. The MKN45 ST6GalNAc-I (MST6-I) gastric cancer cell line exhibits overexpression of the ST6GalNAc-I gene, which codifies the enzyme responsible for STn synthesis. Created in BioRender. Duarte, H. (2026) https://BioRender.com/6xht3c; (B) Immunofluorescence detection of STn antigen in Mock and MST6-I cells (green). Nuclei were counterstained with DAPI (blue). Images were captured at 200× and 630× magnification. Scale bar: 50 µm; (C) Western blot analysis of parental cells and derived EVs for EV markers (HSP70, Syntenin-1, Alix, Actin, CD9, and CD81), mitochondrial marker cytochrome C, and STn antigen. At least two biological replicates were performed for EV markers and three for STn evaluation, each with one technical replicate; (D) Representative Nanoparticle tracking analysis (NTA) histogram image showing the particle-size distribution of Mock and MST6-I-derived EVs; (E) Total particle number and size of EVs released from Mock and MST6-I cells. Results are shown as mean ± SEM. An unpaired t-test was used for statistical analysis; (F) TEM images, acquired at 100,000× magnification, of EVs showing their morphology and integrity. Scale bar: 100 nm; (G) Quantification of the uptake of DiO-labeled EVs (5µg) by 3T3-L1 adipocytes after 24 h of incubation. Internalization was assessed by flow cytometry, and results were normalized to control samples (DiO without EVs - Ctr- + DiO). Results are represented as mean ± SEM. One-way ANOVA was used for statistical analysis; unless otherwise indicated, differences were not significant (ns). Biological replicates are represented as individual data points. ns: P-value > 0.05; *: P-value ≤ 0.05; ***: P-value ≤ 0.001; ****: P-value ≤ 0.0001. STn: Sialyl-Tn; DAPI: 4',6-diamidino-2-phenylindole; EV: extracellular vesicle; TEM: transmission electron microscopy; SEM: standard error of the mean.