Figure 3

Transcriptomics and their Analyses. (a) Immunostaining of L-NSC and S-NSC cell lines. Left panel in a shows L-NSC cells stained for nestin (green) and Tuj1 (red) (upper row, 20X), Stat 3 (middle row, red) and NFκB (lower row, green), nuclear counterstain blue, DAPI (40X). The right side of the left panel in a shows L-NSC cells immunostained for the cell proliferation marker bromodeoxyuridine, BrdU(upper panel), and propidium, PI (lower panel), nuclear counterstained, DAPI blue (40X). A, right panel, phase microscopic image of S-NSC cells in culture; top panel shows DAPI counterstaining of a nucleus from a cell immunopositive for the cytoskeletal and neural stem/progenitor cell marker proteins nestin (red) and GFAP (green). The panel below shows a single double labeled S-NSC cells double labeled for nestin and GFAP, merged image. The S-NSC double labeled cell interestingly possesses the same morphology and immunostaining pattern of cytoskeletal elements as originally shown in immunocompromised mouse xenografts of the original parental line following intracerebral transplantation and their homing to neurogenic regions (Fig. 3f in Walton et al. Development, 2006⁴⁹). (b) Heat maps showing differentially expressed protein coding and miRNA genes. Left panel, L-NSC microarray gene expression data. Upregulated and downregulated genes in infected cells are shown in red and blue respectively. Middle panel, S-NSC differentially expressed protein-coding genes. Red and green represent genes over- or under-expressed in infected cells respectively. Right panel, microRNA genes over- (red) and under-expressed (green) in infected S-NSC. (c) Functional enrichment analysis of transcriptomics datasets focused on KEGG pathways and GO Biological Processes. Left panels, enrichment analysis on L-NSC transcriptomes; right panels, enrichment analysis on MM6 cells and Steindler’s NSC and NDC cells. Red arrows indicate interesting pathways. DEGs, GO Biological Processes enriched with DAVID software v6.7. GO Biological Processes, p-value < 0.01, number of genes associated with certain GO term >5. (j) KEGG pathway enrichment analysis. Identified DEGs, KEGG pathways enriched with DAVID software v6.7. KEGG pathways, p-value < 0.05. For L-NSC GO and KEGG analysis show neddylation, pathways of Alzheimer’s, Parkinson’s and Huntington’s diseases. For S-NSC there are a variety of interesting pathways marked by red arrows, as in MM6 and NDCs as well involving ribosomes, p53 signaling, cell cycle, TGFβ, purine metabolism, NOD receptor signaling, MAPK signaling, vesicle mediated transport among others. Nominal p values were utilized for KEGG and GO analyses; p-values for pathways that were robust to Benjamini Hochberg correction also are shown in Supplement B: Table S16, 17. Comparison of the nominal and corrected p values indicate the most robust pathways.