HuR affects PDAC EV cargoes relating to endothelial cell functions. A, Schematic of EV isolation from PANC-1 WT vs. HuR-KO cells for EV validation, RNA-seq, and proteomics. B, Immunoblot of cell lysates (CL) and SEC fractions 3–6 (pre-EV), 7–10 (EV), and 11–14 (post-EV) from PANC-1 WT (HuR status +) and HuR-KO (HuR status −) cells. Blot probed for total protein (Ponceau), HuR, EV markers (TSG101 and CD81), and cell lysate control (cytochrome c). C, Electron microscopy validation of SEC fractions 7–10 (scale bar, 50 nm). D, Mean particle size and concentration normalized to final cell number measurements via fluorescent nanoparticle tracking analysis. P values were calculated using an unpaired two-tailed Student t test (n = 4). E, RNA-seq volcano plot of differentially abundant genes in WT (blue, left) vs. HuR-KO (orange, right) EVs (n = 4). F, RNA-seq pathway enrichment analysis highlighting the top nine enriched pathways in WT EVs (blue) and the top four enriched pathways in HuR-KO EV mRNA (orange). G, Volcano plot isobaric-labeling quantitative proteomics illustrating the differentially abundant proteins in WT (blue, left) vs. HuR-KO (orange, right) EVs (n = 3). H, Proteomics pathway enrichment analysis highlighting the top nine enriched pathways in WT EVs (blue) and the top four enriched pathways in HuR-KO EV proteins (orange). ER, endoplasmic reticulum; ns, not significant; PDT, photodynamic therapy; OvCa, ovarian cancer; UPS, unfolded protein response.
Finan et al. (Wed,) studied this question.