Fig. 2: Cluster analysis of differentially expressed genes in EBOV-infected Leydig cells.
A Counts of significantly upregulated (log2 FC ≥ 2; P ≤ 0.05) and downregulated (log2 FC ≤ −2; P ≤ 0.05) genes for Leydig cells. Values were calculated from three replicates for each set of variables, using normalized feature counts determined by RNAseq analysis. For each time point, separate queries of upregulated and downregulated genes were performed against the Gene Ontology Biological Processes (https://geneontology.org; accessed October 5, 2023) and Reactome Gene Sets (https://reactome.org; accessed October 5, 2023) to identify associated ontological terms31,32,33,34. Significance of associated terms was determined by calculating accumulative hypergeometric p-values (cutoff: 0.01) and enrichment factors (minimum 1.5). Samples were clustered based by significant terms using Kappa-statistical similarities (0.3 kappa threshold) among upregulated (B) and downregulated (C) member genes. A heatmap was generated and colored by p-values, where white cells indicate the lack of enrichment for that term in the corresponding gene list. The term with the best p-value for each term cluster was used as a representative on the heatmap. Protein-protein interaction networks were generated based on upregulated (D) and downregulated (E) genes, and the MCODE algorithm was applied to these networks to identify neighborhoods where proteins are densely connected. Enrichment analysis was applied to each MCODE network to extract “biological meanings” from the network component, where top three best p-value terms were retained and represented based by one term per cluster as a label. A breakdown of all MCODE interpretations for each time point is available as supplementary data. Nodes represent individual proteins, node pie sectors indicate which samples differentially expressed each protein, and edges represent interactions between proteins. Analysis was performed using the Metascape online tool (https://metascape.org; accessed October 5, 2023), which incorporates network visualization by Cytoscape30,71. L01, Leydig + infectious EBOV 1 dpi; L02, Leydig + infectious EBOV 2 dpi; L04, Leydig + infectious EBOV 4 dpi; L07, Leydig + infectious EBOV 7 dpi; L14, Leydig + infectious EBOV 14 dpi.
