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Figure 2. STn-positive gastric cancer-derived EVs and secretome promote browning and lipolysis of 3T3-L1 adipocytes. (A) Schematic representation of the experimental workflow, in which differentiated 3T3-L1 adipocytes were treated with the secretome/CM or isolated EVs from Mock and MST6-I gastric cancer cells. Created in BioRender. Duarte, H. (2026) https://BioRender.com/k39obbd; (B) RT-qPCR analysis of Ucp1 and Prdm16 gene expression in 3T3-L1 adipocytes after 24 h of treatment with either the secretome/CM (left panel) or EVs (right panel - 5μ or 10µg) from Mock and MST6-I cells. Results are shown as mean ± SEM, with statistical significance determined by One-way ANOVA; (C) Immunofluorescence images of 3T3-L1 adipocytes (white adipocytes), untreated or treated with the secretome/CM from Mock or MST6-I cells. Lipid droplets were stained for perilipin (red), and nuclei were counterstained with DAPI (blue). Images were acquired at 200× and 630× magnification. Scale bar: 50 µm; (D and E) Quantification of the lipolytic activity by measuring the free glycerol (D) and fatty acid (E) release by 3T3-L1 adipocytes after 24 h of treatment with the secretome/CM or EVs (5μg) from Mock and MST6-I gastric cancer cells. Results are shown as mean ± SEM. One-way ANOVA was used for statistical analysis; (F) Western blot analysis of ACC and phosphorylated HSL expression in untreated 3T3-L1 adipocytes or after being exposed to either the secretome/CM or 5µg of EVs from Mock and MST6-I cells. ACC and HSL expressions were normalized to Actin, and phosphorylated HSL levels were normalized to both Actin and total HSL. Results are shown as mean ± SEM, with statistical significance determined by One-way ANOVA. Biological replicates are represented as individual data points. ns: P-value > 0.05; *: P-value ≤ 0.05; **: P-value ≤ 0.01; ***: P-value ≤ 0.001; ****: P-value ≤ 0.0001. CM: Conditioned medium; EV: extracellular vesicle; ACC: acetyl-CoA carboxylase; DAPI: 4',6-diamidino-2-phenylindole; RT-qPCR: real-time quantitative polymerase chain reaction; ANOVA: analysis of variance; HSL: hormone-sensitive lipase; SEM: standard error of the mean.