Proteomic Analysis Identifies Molecular Players and Biological Processes Specific to SARS-CoV-2 Exposure in Endothelial Cells

Figures

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  Figure 1. Shematic showing an experimental workflow for studying the response of HUVECs to SARS-CoV-2 L strain.

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  Figure 2. Absence of SARS-CoV-2 productive infection in primary HUVECs. HUVECs were infected with SARS-CoV-2 at MOI of 1 or mock-infected for 1 h and were cultured for up to 48 h. (A) Representative confocal microscopy images of HUVECs at 48 h after exposure show the integrity of the actin cytoskeleton without alterations. (B) Viral titer measurement by TCID50 assay of HUVECs culture after the indicated infection time showed a decrease in titer over time. Values shown are the means ± standard deviation (SD) of three independent biological samples. (C) RT- qPCR analysis shows no mRNA expression of ACE2, TMPRSS2, or CD147 in primary HUVECs. Lung cells (HK2) and water were used as positive and negative controls, respectively. Bar charts show the delta CT values of the three RT- qPCR measurements. (D) Representative confocal microscopy images showing the negative immunostaining profile of the viral nucleoprotein in HUVECs infected with SARS- CoV-2 at MOI of 1 or with mock and the positive immunostaining profile in Vero-E6 cells infected with SARS-CoV-2 at MOI of 0.001 at 48 hpi. In confocal images, red depicts actin stained with Alexa Fluor 647 phalloindin, blue depicts nuclei stained with…

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  Figure 3. Functional clustering and enrichment analysis of differentially abundant proteins in HUVECs in response to SARS-CoV-2 exposure. (A,B) Volcano plots of the relative quantified protein groups showing a significantly high abundance of proteins in red (log2Fc ≥ 0.58 and −log10 p-value ≥ 1.12 = p-value ≤ 0.05) and low abundance proteins in blue (log2Fc ≤ −0.58 and −log10 p-value ≥ 1.12 = p-value ≤ 0.05). The gene names depicted in each plot were from proteins that presented the highest fold change or the lowest p values (p-value ≤ 0.001). Network representation of the enriched reactome pathways of the high (red) or low abundant (blue) protein clusters of SARS-CoV-2-exposed HUVEC samples after 48 h (C) or 120 h (E). Network representation of the enriched GO terms for biological processes of high (red) or low abundant (blue) protein clusters of SARS-CoV-2 exposed samples at 48 h (D) or 120 h (F). Nodes depict the enriched terms for pathways or biological processes. The connections between the nodes represent the shared proteins among them. Dots represent the main proteins associated with each biological process or pathway. The size of the nodes was directly proportional to the…

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  Figure 4. Increase in SUMO2 abundance upon SARS-CoV-2 exposure in HUVECs. (A,B) Bar chart and Venn diagram showing common differentially abundant proteins at both time points. The dotted blue box highlights the decrease in NTPL/NCP2 abundance over time and the dotted red box highlights the increase in SUMO2 abundance over time. Network of protein–protein interaction analysis using the K-means cluster of the highly abundant proteins at 48 h (C) and 120 h (D). Blue circles and arrows highlight the link between SUMO2 and PTMA. The red circles and arrows highlight that SUMO2 and FABP4 are clustered together. Green and red dots represent the cluster divisions based on K-means performed using STRING functional protein association networks. (E) Representative confocal microscopy images showing increased immunostaining for SUMO2/3 in virus-exposed HUVECs compared with mock-exposed cells after 48 h. Zoomed-in panels highlight the stronger staining of SUMO2/3 in SARS-CoV-2 exposed cells and (F) bar chart showing SUMO2/3 clusters in virus- and mock-exposed cells (p-value < 0.05, U-test Mann–Whitney. ** p ≤ 0.01) Red depicts actin stained with Alexa Fluor 647 phalloindin, blue depicts nuclei stained…