Differential Expression of a Novel Gene in Response to hsp27 and Cell Differentiation in Human Keratinocytes Mojgan Hell-Pourmojib, Peter Neuner, Robert.

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Differential Expression of a Novel Gene in Response to hsp27 and Cell Differentiation in Human Keratinocytes Mojgan Hell-Pourmojib, Peter Neuner, Robert Knobler, Franz Trautinger, M.D. Journal of Investigative Dermatology Volume 119, Issue 1, Pages 154-159 (July 2002) DOI: 10.1046/j.1523-1747.2002.01793.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 mRNA differential display. mRNA was extracted from the epidermal carcinoma cell line A431 (lanes 1, 4), mock-transfected cell lines (lanes 2, 5), and an hsp27-overexpressing clone (lanes 3, 6). mRNA was reverse transcribed and duplicate DDPCR was prepared from each reverse transcription reaction using different combinations of one base anchored oligo-dT and random arbitrary primers. Arrows indicate differentially expressed gene products. Journal of Investigative Dermatology 2002 119, 154-159DOI: (10.1046/j.1523-1747.2002.01793.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reverse northern dot blot analysis. Three randomly picked tetracycline-resistant colonies from the differentially displayed fragments dEST-G24 and dEST-A21 were amplified. The PCR products were blotted on duplicate membranes. The blots were hybridized with 32P-labeled cDNAs from mock-transfected (a) and hsp27-transfected A431 (b). Journal of Investigative Dermatology 2002 119, 154-159DOI: (10.1046/j.1523-1747.2002.01793.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Northern hybridization of clone dEST-G24. Clone dEST-G24 was identified as a gene that is differentially expressed between mock-transfected (lane 1) and hsp27-transfected A431 (lane 2). The blot was hybridized with the labeled dEST-G24 fragment as cDNA probe (a). Ethidium-bromide-stained 18 s and 28 s rRNA were used as control (b). The relative intensity of hsp27-dependent G24 gene expression was determined by gel scanning. Expression in mock-transfected cells was set arbitrarily to 100%. G24 gene expression is 2.5-fold lower in hsp27-overexpressing cells. Bar represents mean ± standard deviation from three independent experiments. Journal of Investigative Dermatology 2002 119, 154-159DOI: (10.1046/j.1523-1747.2002.01793.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Ca2+-dependent expression of hsp27 and G24 in normal human keratinocytes. 20 µg total RNA was loaded onto each lane. RNA was isolated from normal human keratinocytes, cultured in media containing 1 mM (lane 1), 0.15 mM (lane 2), and 0.03 mM Ca2+ (lane 3). The blot was hybridized against hsp27 (a) and G24 (b) complementary DNA. Ethidium-bromide-stained 18 s and 28 s rRNA were used as control (c). The relative intensity of Ca2+-dependent hsp27 and G24 gene expressions was determined by gel scanning. Maximal intensity for hsp27 was observed in the higher Ca2+ concentration (1 mM). In contrast, G24 showed high signals at lower Ca2+ concentrations. Maximum intensity in each hybridization was set to 100%. Values represent percentage of maximum expression and are given as mean ± standard deviation from three independent experiments. Journal of Investigative Dermatology 2002 119, 154-159DOI: (10.1046/j.1523-1747.2002.01793.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 In situ hybridization. Human normal skin was hybridized using a specific G24 riboprobe. G24 is expressed in the basal layer of the epidermis (a). (b) Negative control. Scale bar: 100 μm. Journal of Investigative Dermatology 2002 119, 154-159DOI: (10.1046/j.1523-1747.2002.01793.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Tissue northern hybridization. The blot contains 20 µg total RNA from eight different tissues: tonsil (lane 1), thymus (lane 2), appendix (lane 3), lymph node (lane 4), gall bladder (lane 5), prostate (lane 6), testis (lane 7), and ovary (lane 8). The blot was hybridized using a 32P-adenosine-5′-triphosphate labeled complementary cDNA for G24. Journal of Investigative Dermatology 2002 119, 154-159DOI: (10.1046/j.1523-1747.2002.01793.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Sequence of the isolated dEST-G24. The clone was isolated from the DDPCR gel and amplified using the same primer pair used for DDPCR. DNA was ligated into the pCR-TRAP vector, transformed into GH competent cells, and subjected to sequencing. The sequence is published in the NCBI with the accession number AI207398. Journal of Investigative Dermatology 2002 119, 154-159DOI: (10.1046/j.1523-1747.2002.01793.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Complete sequence of G24. The cDNA clone from GeneBank (accession number AA150222) showed 100% homology to G24and revealed the same northern hybridization pattern as obtained with G24. The full-length sequence of this clone was obtained and is published in GeneBank with the accession number BI173973. Journal of Investigative Dermatology 2002 119, 154-159DOI: (10.1046/j.1523-1747.2002.01793.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions