Fig 2: Protein levels of the OSM receptors LIF-R/GP130 and IL-31RA are increased after HCF-V stimulation in vitro. (a) Representative Western blots for GP130, IL-31RA, and IL-31RB and ß-tubulin (Tub) expression in HCF-V cells were measured under basal conditions (Ctrl), after starvation (Starved) and after starvation and stimulation with a pro-fibrotic cocktail containing L-ascorbic acid 2-phosphate, dextran sulphate, and recombinant TGFß-1. Samples were collected both after starvation and after 0.5, 1, 2, 4, 24, and 48 h of starvation and fibrosis stimulation. Untreated cells in basal conditions were used as control. (b) GP130 protein expression quantification. (c) IL-31RB protein expression quantification. (d) IL-31RA protein expression quantification. Protein content was measured by densitometry in ImageJ 1.53t, using Tub as a loading control for each condition. Results were obtained from three independent experiments and were analyzed with ANOVA and Tukey’s post hoc test, using values from ctrl cells as a reference and represented as mean ± SEM (* p < 0.05; *** p < 0.001).

Fig 3: SEVs derived from genetically modified MSC-T cells carry the recombinant proteins mat/mutOSM-CD81 on their surface but maintain the same physical characteristics in terms of morphology and size distribution. (a) OSM detection in protein samples of cell lysates or SEVs from MSC-T (Ctrl) (lane 1), MSC-T-CD81 (CD81) (lane 2), MSC-T-matureOSM-CD81 (matOSM-CD81) (lane 3), and MSC-T-mutantOSM-CD81 (mutOSM-CD81) (lane 4). OSM was identified at both native molecular weight (22–24 kDa) and its expected molecular weight after fusion to CD81 (45–47 kDa). SEV protein extracts, 10 µg/lane; cell lysate protein extracts, 30 µg/lane. (b) Representative images of SEVs obtained by transmission electron microscopy (scale bar = 100 nm). (c,d) Nanoparticle tracking analysis, showing average particles size (nm) and particles size distribution histograms. (e) ExoView analysis showing percentage of OSM-, CD81-, or CD9-positive SEVs in samples after capture with a CD63 antibody. (f) Both the proportion of SEVs double-positive for CD63 and other proteins (OSM, CD81 and CD9) and triple-positive for these markers was analyzed in all SEVs experimental groups and plotted as pie charts. Each experiment was performed three times, and data were analyzed by ANOVA and Tukey’s post hoc test, using values from control cells or SEVs as the reference and represented as mean ± SEM (ns: non-significant).