Epidermal Morphogenesis: The Transcriptional Program of Human Keratinocytes during Stratification  Piyush Koria, Stelios T. Andreadis  Journal of Investigative Dermatology  Volume 126, Issue 8, Pages 1834-1841 (August 2006) DOI: 10.1038/sj.jid.5700325 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Culture at the A/L interface promotes complete epidermal stratification. Skin equivalents were cultured (a, c) submerged or at the (b, d) A/L interface for (a, b) 3 or (c, d) 7 days. Paraffin-embedded tissue sections were stained with hematoxylin–eosin (bar=50μm; original magnification × 40). The dashed line denotes basement membrane. (e) Frozen sections were stained for the Ki67 nuclear antigen. Proliferation was measured as the fraction of Ki67+ basal cells. Each value represents the average of six randomly selected sections. The results shown here are representative of two independent experiments each performed with duplicate tissues. Journal of Investigative Dermatology 2006 126, 1834-1841DOI: (10.1038/sj.jid.5700325) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Classification of differentially expressed genes. The differentially expressed genes were annotated and grouped into functional categories. The pie chart shows the proportion of each category among the significantly altered genes. Journal of Investigative Dermatology 2006 126, 1834-1841DOI: (10.1038/sj.jid.5700325) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Fully stratified A/L tissues downregulate expression of integrin α5. Skin equivalents were cultured (a, c) submerged or at (b, d) A/L interface for (a, b) 3 or (c, d) 7 days. Frozen sections were stained for integrin α5 (green) and counterstained with Hoechst (blue). The dashed line denotes basement membrane (bar=50μm; original magnification × 40). The results shown here are representative of two independent experiments each performed with duplicate tissues. Journal of Investigative Dermatology 2006 126, 1834-1841DOI: (10.1038/sj.jid.5700325) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Glycogen accumulates in submerged tissues but not in fully stratified A/L tissues. Skin equivalents were cultured at (a, c) submerged or at the (b, d) A/L interface for (a, b) 3 or (c, d) 7 days. Paraffin-embedded tissue sections were stained for glycogen by PAS stain. Glycogen appeared as red granules in the cell cytoplasm. PAS also stained the glycoprotein-rich basement membrane and dermis of both submerged and A/L tissues (bar=50μm; original magnification × 40). The results shown here are representative of two independent experiments each performed with duplicate tissues. (e) Differential regulation of glycolysis and glycogen synthesis by stratified versus non-stratified epithelium. Bold and italic depict genes that were highly expressed in submerged and A/L tissues, respectively. Journal of Investigative Dermatology 2006 126, 1834-1841DOI: (10.1038/sj.jid.5700325) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Keratin-4 is expressed only in submerged but not in fully stratified A/L tissues. Skin equivalents were cultured (a, c) submerged or at (b, d) A/L interface for (a, b) 3 or (c, d) 7 days. Paraffin sections were stained for K4 (green) after heat-induced antigen unmasking and trypsin digestion. The dashed line denotes basement membrane (bar=50μm; original magnification × 40). The results shown here are representative of two independent experiments each performed with duplicate tissues. Journal of Investigative Dermatology 2006 126, 1834-1841DOI: (10.1038/sj.jid.5700325) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions