Overexpression of the Oncofetal Fn Variant Containing the EDA Splice-in Segment in the Dermal–Epidermal Junction of Psoriatic Uninvolved Skin  Kathleen M. Ting, Dinah Rothaupt, Thomas S. McCormick, Craig Hammerberg, Guofen Chen, Anita C. Gilliam, Seth Stevens, Lloyd Culp, Kevin D. Cooper  Journal of Investigative Dermatology  Volume 114, Issue 4, Pages 706-711 (April 2000) DOI: 10.1046/j.1523-1747.2000.00871.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cellular Fn staining is localized to the dermal–epidermal junction in uninvolved psoriasis. Immunohistochemical staining was performed for cellular Fn in control and uninvolved psoriatic tissue. Tissue sections from control (A), uninvolved psoriatic tissue (B) and psoriasform atopic dermatitis (C) were stained with a monoclonal antibody specific for the cellular Fn splice variant EDA+ (monoclonal antibody DH1, ICN), and detected using goat anti-mouse IgG1 horseradish peroxidase conjugated secondary antibody. The horseradish peroxidase conjugated secondary antibody was visualized using diaminobenzidine as the substrate for the horseradish peroxidase in the normal and psoriasis tissues, and AEC as the substrate for horseradish peroxidase in the atopic dermatitis sample. Uninvolved tissue stained positively for cellular Fn compared with both control and isotype staining. Scale bar: 50μm. Journal of Investigative Dermatology 2000 114, 706-711DOI: (10.1046/j.1523-1747.2000.00871.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Cellular Fn penetrates the basal epidermis and surrounds basal keratinocytes in psoriasis involved tissue. Psoriasis involved (A, B) and uninvolved (C, D) tissue was stained with anti-cellular EDA+ specific antibody (clone DH1). The primary antibody was detected using secondary specific antibodies conjugated to either rhodamine (A, C) or fluorescein isothiocyanate (B, D). Localized staining at the dermal–epidermal junction is visible in the sections presented. Positive staining of endothelial cells (vessel) is also evident (A), as shown by the striped arrows. Transient amplifying cells (PCNA positive staining, green) are shown in contact with the basal layer of the dermal–epidermal junction/EDA Fn positive staining (A), indicated with white arrows. Fn staining also appears to penetrate the basal epidermis from the dermal papillary area and surround basal keratinocytes, as shown with white arrows in (B). Sections (B) and (D) were counterstained with Evan's blue, as described for single staining under Materials and methods. Journal of Investigative Dermatology 2000 114, 706-711DOI: (10.1046/j.1523-1747.2000.00871.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Several splicing variants are prominent in Fn. RT-PCR primers and their location are depicted. Splice in variants of cellular Fn (EDA, EDB, and IIICS are depicted). The control sequence III-9 is also shown as a positive control. Journal of Investigative Dermatology 2000 114, 706-711DOI: (10.1046/j.1523-1747.2000.00871.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 EDA+mRNA expression appears restricted to the epidermal fraction in both involved and uninvolved psoriatic skin. RT-PCR amplification of the splice variant EDA+ Fn from separated epidermal (E)/dermal(D) skin fractions from control (N), psoriasis uninvolved (PU) or involved (PI) skin. RNA that had been homogenized in Trizol was reverse transcribed and the cDNA was then amplified with primer sets specific for the EDA+ Fn splice in the region (as detailed in Materials and methods). Prominent EDA+ Fn message is demonstrated in the epidermal fraction of both psoriasis involved and uninvolved skin. Journal of Investigative Dermatology 2000 114, 706-711DOI: (10.1046/j.1523-1747.2000.00871.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Other cellular specific variants of Fn (EDB) do not appear to be changed in a psoriasis-specific manner. RT-PCR amplification of other cellular Fn markers, including EDB, III9, and IIICS, were amplified from mRNA obtained from either normal individuals (N) or individuals with psoriatic involved (PI) or uninvolved (PU) areas. The tissue was homogenized in Trizol buffer and RNA was extracted and amplified using primers specific for the indicated splice variants of Fn. Amplification of the IIICS region appears to be restricted to the dermal fraction of skin. Journal of Investigative Dermatology 2000 114, 706-711DOI: (10.1046/j.1523-1747.2000.00871.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions