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Figure 3. Effects of TMZ on the protein cargo of GBM-derived EVs and their internalisation in recipient GBM cells. (A) Venn diagrams show the quantified proteins in EVs derived from the four GBM cell lines, comparing TMZ-treated and untreated conditions (EVs +TMZ vs. EVs -TMZ); (B) Volcano plots showing DEPs identified in comparisons between TMZ-treated and untreated conditions (EVs +TMZ vs. EVs -TMZ). Data are graphed as ratios and -log(P-value): grey dots represent proteins not differentially expressed in the comparison; red dots represent significantly upregulated proteins, and green dots indicate proteins that were significantly downregulated in EVs isolated from TMZ-treated cells. Data are presented as mean ± SD of three independent biological replicates (n = 3). Statistical significance was determined using Student’s t-test, with P-value < 0.05 considered statistically significant; (C) Heatmap of the main downstream effects predicted by the IPA tool in the performed comparisons. Data are graphed by z-score values, as shown in the legend above (red for activation and blue for inhibition). Biofunctions are grouped into larger biological categories; (D) Schematic representation of the treatments of recipient GBM cells with EVs from untreated or TMZ-treated U87MG and U251MG cells performed in this study; (E) Microscopy fluorescence images showing the uptake of PKH26-labelled EVs from untreated and TMZ-treated U87MG and U251MG cells in recipient GBM cells after 24 h of incubation (representative images at 1 and 3 h, together with PKH26-only controls are provided in Supplementary Figure 3); (F) Quantitative analysis of the MFI of internalised PKH26-labelled EVs in recipient U87MG and U251MG cells after 1, 3, and 24 h of incubation, measured using ImageJ Software version 1.54p^[35]. Statistically significant differences between groups were determined using one-way ANOVA followed by Tukey’s post hoc test ^***P < 0.001. Data are presented as mean ± SD of three independent biological replicates (n = 3). ANOVA: Analysis of variance; DEPs: differentially expressed proteins; EVs: extracellular vesicles; GBM: glioblastoma; IPA: Ingenuity Pathway Analysis; MFI: mean fluorescence intensity; PKH26: red fluorescent cell membrane dye; TMZ: temozolomide.