HAX-1, Identified by Differential Display Reverse Transcription Polymerase Chain Reaction, Is Overexpressed in Lesional Psoriasis Alireza Mirmohammadsadegh,

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HAX-1, Identified by Differential Display Reverse Transcription Polymerase Chain Reaction, Is Overexpressed in Lesional Psoriasis Alireza Mirmohammadsadegh, Ulrike Tartler, Günter Michel, Annett Baer, Markus Walz, Ronald Wolf, Thomas Ruzicka, Ulrich R. Hengge Journal of Investigative Dermatology Volume 120, Issue 6, Pages 1045-1051 (June 2003) DOI: 10.1046/j.1523-1747.2003.12247.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 DDRT-PCR with nonlesional (n) and lesional (l) psoriatic skin. The PCR was performed with arbitrary primer (AP) 25 and different one-base-anchored primers A, G, and C. The radiolabeled PCR products were separated on a denaturing 6% polyacrylamide sequencing gel and visualized by exposing to an X-ray film at –70°C for 12 h. The arrow shows different cDNAs that are dysregulated in lesional versus nonlesional psoriatic skin. Journal of Investigative Dermatology 2003 120, 1045-1051DOI: (10.1046/j.1523-1747.2003.12247.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Northern blot analysis of clone 2/HAX-1 mRNA transcription. Clone 2/HAX-1 mRNA levels were elevated in lesional psoriatic skin. (A) Northern blot analysis of total RNA from cultured HaCaT cells, normal human keratinocytes (KC), and lesional (L) and nonlesional (NL) psoriatic skin. (B) Northern blot analysis of total RNA from nonlesional (NL) and lesional (L) psoriatic skin as well as healthy skin (H) of several volunteers is shown. The signal intensity is compared to 28S RNA. Journal of Investigative Dermatology 2003 120, 1045-1051DOI: (10.1046/j.1523-1747.2003.12247.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Multiple tissue Northern blot analysis. Metabolically active tissues express the highest levels of clone 2/HAX-1. Autoradiograph of a multiple tissue Northern blot of mRNAs from different tissues probed with the insert of clone 2 and exposed overnight. Journal of Investigative Dermatology 2003 120, 1045-1051DOI: (10.1046/j.1523-1747.2003.12247.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Western blot analysis of clone 2/HAX-1 expression. To confirm protein expression Western blot analysis was performed on protein extracts of nonlesional (nl) versus lesional (l) psoriatic skin of three different patients (A). (B) Expression of HAX-1 primary keratinocytes versus HaCaT cells, and in different melanoma cell lines versus primary melanocytes, respectively: 1, primary melanocytes; 2, MV3; 3, BLM; 4, SK-Mel-28; 5, HaCaT; 6, primary keratinocytes. The signal intensity is compared to α-tubulin. Journal of Investigative Dermatology 2003 120, 1045-1051DOI: (10.1046/j.1523-1747.2003.12247.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 In situ hybridization and immunohistochemistry. Clone 2/HAX-1 mRNA and HAX-1 protein localization in normal versus lesional psoriatic skin. (A)–(C) In situ hybridizations of cryosections of healthy (A) and lesional psoriatic skin (B) with antisense cRNA, generated from clone 2. (C) Negative control where a parallel section of lesional skin was hybridized to sense cRNA of clone 2. (D)–(I) Immunohistochemical staining for HAX-1. Healthy skin (D) with HAX-1 antibody and (E) with Ki67 antibody. Lesional psoriatic skin (F) with HAX-1 antibody and (G) with Ki67 antibody. (H)–(I) Close-up view of (F) and (G), respectively. Scale bars: (A), (B), (C), (F), (G) 200 μm; (D), (E) 100 μm; (H), (I) 50 μm. Journal of Investigative Dermatology 2003 120, 1045-1051DOI: (10.1046/j.1523-1747.2003.12247.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 ApoAlert caspase-3 assay in transiently transfected HaCaT cells after irradiation with 280 J per m2 UVB. HAX-1 antisense RNA enhances UVB-induced apoptosis. HaCaT cells were transfected with plasmid constructs expressing HAX-1 sense or antisense mRNA. The negative controls consisted of vector without insert and mock-transfected cells (HaCaT). Caspase-3 activity was determined fluorometrically. A representative experiment is shown. Journal of Investigative Dermatology 2003 120, 1045-1051DOI: (10.1046/j.1523-1747.2003.12247.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions