Fig. 5. Gene expression and gene set enrichment analysis of 2-D attached spheroids and corresponding 2-D monolayer mass cultures.

(A) Unsupervised hierarchical clustering of log-transformed mean-centered mRNA expression levels for all transcripts differentially expressed greater ± 2-fold in 3-D-to-2-D attached spheroids compared to corresponding 2-D monolayer NHKc mass cultures. (B) Volcano plot of differentially regulated genes between 2-D attached spheroids and corresponding monolayer NHKc mass cultures. Vertical dashed lines denote greater ± 2-fold change and horizontal dashed lines denote ANOVA p-value < 0.05. Colored dots indicate top enriched (Red) or depleted (Green) genes with a GO annotation relating to hair formation (note marked depletion of cell death genes such as Death Associated Protein Like 1 (DAPL1). (C) Dot plot of differentially regulated genes with ≥ 2-fold change and = 0.05 ANOVA p-value in 2-D attached spheroids and corresponding monolayer cultures. Colored dots indicate top enriched (Red) or depleted (Green) genes with a GO annotation relating to skin barrier formation (D) Venn diagram depicting global numbers of deregulated and upregulated mRNAs expressed by 2D attached spheroids. (E) Table charting major gene changed in 2-D attached spheroids compared to corresponding monolayer cultures. (F) Heatmap depicting select genes changes implicated in skin formation (G) Pie chart of upstream regulators targeted for activation in 2-D attached spheroids. Pie chart segments are proportional to the -log of the overlap p-value. Respective activation z-score are indicated in red; * indicate undetermined activation pattern. (H) Downstream functional effects of gene transcripts upregulated and downregulated in 2-D attached spheroids (2-fold change FDR < 0.05). Gene Ontology analysis indicates activation of pathways related to cell proliferation and epidermis formation.