Fig. 11

Effect of exposing NSCs to a Dose Range of Ethanol on the Transcriptome of Secreted EVs and Cells. A–D Scatter plots of effect size (Hedges’ g with nonzero-containing 95% Confidence Interval) vs. normalized RNA expression level, document that statistical significance (p < 0.05, red symbols) is independent of RNA expression (‘Y’ axis), but generally associated with decreased effect size. Relationship between effect size and statistical significance in EVs from 120 mg/dL-treated NSCs (A) or 320 mg/dL-treated NSCs (B) relative to control (0 mg/dL) NSCs. These analyses show that more differentially regulated EV transcripts from 320 mg/dL-treated NSCs also exhibited a negative effect size, indicative of downregulation in EVs. Relationship between effect size and statistical significance in parental NSCs treated with 120 mg/dL (C) or 320 mg/dL (D) relative to control (0 mg/dL) NSCs. Similar to EVs, these analyses show that more differentially regulated parental NSC transcripts also exhibited a negative effect size, indicative of downregulation. Horizontal green-dotted line represents the average transcript expression level of all transcripts, while horizontal blue-dotted line represents the average expression level of only transcripts for which the effect size had a non-zero containing 95% confidence estimate in EV (A, B) or parental cell (C, D) samples. Vertical purple-dotted lines denote effect size values of − 0.8, − 0.4, − 0.2, + 0.2, + 0.4, + 0.8, respectively. Positive effect size signifies increased expression of a transcript, and negative effect size signifies decreased expression of a transcript, in ethanol treatment group vs. control group. Each data point represents a transcript, with red color for significant p-value; n = 6 samples per group; paired t-test, p < 0.05. E A bar graph of the number of unique genes whose expression was significantly increased or decreased by ethanol. F, G Volcano plots of the relationship between effect size due to treatment and p-value. Each data point represents transcript enrichment in EVs relative to parental cells (EV/Cell) between moderate ethanol-treated (120 mg/dL, F) or heavy ethanol-treated (320 mg/dL, G) groups compared to the control group (0 mg/dL). These data show that transcripts that were significantly enriched in EVs due to heavy ethanol exposure were also depleted in parental NSCs. Blue triangles above the horizontal dotted line represent transcripts for which the effect size had a non-zero containing 95% confidence estimate that were also significantly affected by ethanol exposure by paired t-test.; n = 6 samples per group; paired t-test, p < 0.05. H A bar graph of the number of unique genes that are significant DEGs by sex (sex-variant) or not (sex-invariant) in EVs whose expression were significantly increased in EVs relative to cells by ethanol. I Pathway overrepresentation plots for transcripts that met the criteria for an effect size, >  + 0.4 for heavy ethanol exposure (320 mg/dL), a non-zero containing 95% confidence estimate and a significance of p < 0.05 by paired t-test. The size of each data point represents the number of genes/transcripts in a pathway that were within this subset of transcripts while color of data point denotes the FDR-corrected p-value for pathway overrepresentation. J Plot of key enriched pathways and constituent transcripts involved in these pathways, for heavy ethanol exposure. The size of each pathway element denotes the number of transcripts in a pathway that were within that subset of pathway transcripts, while the color of each element represents the effect size for that pathway component