Soluble Adenylyl Cyclase Defines a Nuclear cAMP Microdomain in Keratinocyte Hyperproliferative Skin Diseases Jonathan H. Zippin, Paul A. Chadwick, Lonny.

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Soluble Adenylyl Cyclase Defines a Nuclear cAMP Microdomain in Keratinocyte Hyperproliferative Skin Diseases Jonathan H. Zippin, Paul A. Chadwick, Lonny R. Levin, Jochen Buck, Cynthia M. Magro Journal of Investigative Dermatology Volume 130, Issue 5, Pages 1279-1287 (May 2010) DOI: 10.1038/jid.2009.440 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunostaining of sAC in normal skin. (a) Hematoxylin (blue) and eosin (red) staining of normal human skin. (b) Normal human skin immunostained with R21 (red) and hematoxylin (blue). (c) Normal human skin immunostained with R21+ blocking peptide (red) and hematoxylin (blue). (d) Normal epidermis immunostained with R21 (red), Melan-A (brown), and hematoxylin (blue). (e) Normal human skin eccrine duct immunostained with R21 (red) and hematoxylin (blue). (f) Normal human skin cutaneous nerve immunostained with R21 (red), PGP9.5 (brown), and hematoxylin (blue). Bars=50μm (a–c) and 10μm (d–f). Journal of Investigative Dermatology 2010 130, 1279-1287DOI: (10.1038/jid.2009.440) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunostaining of sAC in viral infections of the epidermis. (a) Hematoxylin (blue) and eosin (red) staining of a verruca vulgaris skin lesion. (b) Verruca vulgaris skin lesion immunostained with R21 (red) and hematoxylin (blue). (c) Verruca vulgaris skin lesion immunostained with R21 (red) and hematoxylin (blue). The black arrow demonstrates a nucleus negative for sAC. The gray arrow demonstrates a nucleus postive for sAC. (d) Hematoxylin (blue) and eosin (red) staining of a molluscum contagiosum skin lesion. (e) Molluscum contagiosum skin lesion immunostained with R21 (red) and hematoxylin (blue). (f) Molluscum contagiosum skin lesion immunostained with R21 (red) and hematoxylin (blue). Bars=100μm (a, b, d, e) and 10μm (c, f). Journal of Investigative Dermatology 2010 130, 1279-1287DOI: (10.1038/jid.2009.440) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 sAC is present in the nucleus when epithelial cells are proliferating and not when epithelial cells are differentiating. (a) Differentiated MDCK cells stained with DAPI (left), anti-N-term sAC antibody (center), and overlay of DAPI and anti-N-term (right). (b) Proliferating undifferentiated MDCK cells stained with DAPI (left), anti-N-term sAC antibody (center), and overlay of DAPI and anti-N-term (right). Bar=10μm. Journal of Investigative Dermatology 2010 130, 1279-1287DOI: (10.1038/jid.2009.440) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunostaining of sAC in AK and SCC. (a) Hematoxylin (red) and eosin (blue) staining of AK. (b) AK immunostained with R21 (red) and hematoxylin (blue); inset, magnified view of the area in panel b. (c) Hematoxylin (red) and eosin (blue) staining of SCCIS. (d) SCCIS immunostained with R21 (red) and hematoxylin (blue); inset, magnified view of the area in panel d. (e) Hematoxylin (red) and eosin (blue) staining of SCC. (f) SCC immunostained with R21 (red) and hematoxylin (blue). (g) Hematoxylin (red) and eosin (blue) staining of SCC. (h) SCC immunostained with R21 (red) and hematoxylin (blue). Bars=50μm. Journal of Investigative Dermatology 2010 130, 1279-1287DOI: (10.1038/jid.2009.440) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Nuclear sAC is enhanced in psoriatic skin and is associated with phosphorylated CREB. (a) Hematoxylin (red) and eosin (blue) staining of psoriatic lesional skin. (b) Psoriatic lesional skin immunostained with R21 (red) and hematoxylin (blue). The inset is a magnified view of a portion of the epidermis demonstrating that some nuclei are positive for sAC (white arrow) and some are negative for sAC (black arrow). (c) Psoriatic lesional skin immunostained with R21 (red) and an antibody against phosphorylated CREB (light blue). Panel c and inset: Nuclei positive for both sAC and phosphorylated CREB appear dark blue to purple (gray arrows). White arrows indicate nuclei positive for phosphorylated CREB only (light blue). Black arrows indicate nuclei positive for sAC only (red). Bars=50μm. Journal of Investigative Dermatology 2010 130, 1279-1287DOI: (10.1038/jid.2009.440) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions