Fig 1.
Graphical overview of methods for cellular treatment.
1. HepG2 cells, representing parent cells, were treated as either a control or with a per and polyfluoroalkyl substance mixture. 2. EVs were isolated from parent HepG2 cells (EVHepG2/CT or EVHepG2/PFAS) and 3. used as a treatment on separate HepG2 cells, representing target cells. The six 6 treatment types included a control, individual EVHepG2/CT, individual EVHepG2/PFAS, and a PFAS mixture, and PFAS mixture with and without EVHepG2/CT or EVHepG2/PFAS. 4. Target HepG2 cells were subsequently evaluated for changes in cell viability and protein expression. Key findings are summarized.
Fig 2.
Cellular cytotoxicity in response to HepG2-derived EV exposure conditions.
This bar plot depicts average cell viability per condition with individual datapoints (conditions 1-4, n = 6; conditions 5-6, n = 3). Statistical comparisons to the control are highlighted via multiple t-tests (* indicated p < 0.05). Interpretation of results for every condition are described on the right. Conditions additionally align with those outlined in the graphical overview of methods in Fig 1.
Fig 3.
Heatmap of differentially expressed protein signatures following exposure to EVs.
This heatmap highlights differentially expressed proteins (log2 FC > |0.58|, p-adjust <0.1) with their corresponding gene symbols following individual exposure to EVHepG2/CT and EVHepG2/PFAS compared to their respective control. Average Log2 FCs for proteins with differential expression are listed where applicable. Log2 FC is also indicated by color where FC > 0 (increased protein expression compared to control) is purple, and FC < 0 (decreased protein expression compared to control) is green. All conditions were evaluated with n = 6. NS indicates not significant.
Fig 4.
Summary of differentially expressed proteins following exposure to single EV exposure and EV co-exposure with PFAS.
A) Proteomic expression changes (log2 FC > |0.58|, p-adjust <0.1) following EVHepG2/CT or EVHepG2/PFAS exposure are summarized in the bar plot. Bars are organized by number of proteins with increased expression and number of proteins with decreased expression compared to their respective control. B) Venn diagram displaying number of unique or overlapping proteins differentially expressed following EVHepG2/CT or EVHepG2/PFAS. C) Bar plot summarizing protein expression changes for PFAS co-exposures with EVHepG2/CT or EVHepG2/PFAS. D) Venn diagram displaying the number of differentially expressed proteins for each condition including PFAS exposure and co-exposures with EVHepG2/CT or EVHepG2/PFAS. All conditions were evaluated with n = 6.
Table 1.
Canonical pathways with predicted enrichment following individual exposure to EVHepG2/CT or EVHepG2/PFAS. Top pathways are presented with corresponding -log p-values. Differentially expressed proteins predicted to be involved in these pathways are additionally listed.
Table 2.
Literature-supported roles of proteins with predicted pathway involvement on liver health outcomes. For all enriched pathways previously presented, the roles of implicated differentially expressed proteins are further defined in the specific context of liver health with associated references.
Fig 5.
Predicted biological pathway enrichment and predicted upstream regulators following HepG2 cell exposure to PFAS + EVHepG2/CT or PFAS + EVHepG2/PFAS.
A) Most significantly enriched biological pathway alterations following exposure to PFAS + EVHepG2/CT. B) Most significantly enriched biological pathway alterations following exposure to PFAS + EVHepG2/PFAS. C) Predicted decrease in activity of upstream regulator TP53 following PFAS + EVHepG2/CT exposure. D) Predicted increase in activity of upstream regulator TP53 following PFAS + EVHepG2/PFAS exposure.
Fig 6.
Theoretical mechanism of EV-mediated proteomic expression alterations.
1. PFAS exposure to PFOS, PFOA, and PFHxA reach the cell and induce the release of EVs. 2. EVs released from PFAS exposed HepG2 cells contain altered miRNA expression profiles, including decreased Let-7e. 3. EVs reach separate HepG2 cells and, in theory, target proteins of Let-7e are increased including S100A8.