Jens M. Baron, Ruth Heise, William S

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Retinoic Acid and its 4-Oxo Metabolites are Functionally Active in Human Skin Cells In Vitro Jens M. Baron, Ruth Heise, William S. Blaner, Mark Neis, Sylvia Joussen, Alexandra Dreuw, Yvonne Marquardt, Jean-Hilaire Saurat, Hans F. Merk, David R. Bickers, Frank K. Jugert Journal of Investigative Dermatology Volume 125, Issue 1, Pages 143-153 (July 2005) DOI: 10.1111/j.0022-202X.2005.23791.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Analysis of differentially expressed genes in NHEK (a–d) and dermal fibroblasts (e–g) after treatment with 13-cis- and 4-oxo-13-cis-retinoic acid (RA) for 24 h using the DNA-Chip technology. Total RNA was isolated using peqGOLD RNA Pure. Two micrograms of aRNA from each probe was labeled by reverse transcription with Cy5 and Cy3 fluorescence, respectively, and subjected to a PIQOR skin microarray. Processing of microarrays was performed using a fully automated hybridization station. Image capture and signal quantification of hybridized cDNA arrays were carried out with the ScanArray Lite and ImaGene software version 4.1. Array images and Scatter blot analysis of keratinocytes induced by 10–6 M 13-cis-RA (a, c) and 4-oxo-13-cis-RA (b, d) compared with untreated control. For analysis of gene expression in dermal fibroblast, generation of 33P-labeled cDNA probe was carried out by reverse transcription of 10 μg total RNA with 33P dCTP for 90 min at 37°C following the Mamalian GeneFilters (Research Genetics) protocol. Purified radiolabeled probe was applied to the array for hybridization at 42°C overnight. Afterwards, filters were exposed to a Kodak X-OMAT AR-5 film at -70°C with intensifying screens. Scatter blot analysis of fibroblasts induced by 10–5 M 13-cis-RA (e, f) and 4-oxo-13-cis-RA (e, g) compared with untreated control. Journal of Investigative Dermatology 2005 125, 143-153DOI: (10.1111/j.0022-202X.2005.23791.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Confirmation of cDNA microarray and 2D-gel electrophoresis data using real-time PCR. (a–f) TaqMan real-time PCR analyses: normal human epidermal keratinocytes (NHEK) were stimulated with different RA isomers and metabolites at a concentration of 10–6 M for 24 h. Total RNA was isolated and untreated NHEK were used as control. The relative keratin 1, 10, 13, 19, matrix metalloproteinase-3, and lamin B1 RNA levels were normalized to 18S RNA. Journal of Investigative Dermatology 2005 125, 143-153DOI: (10.1111/j.0022-202X.2005.23791.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Confirmation of cDNA microarray and 2D gel electrophoresis data using immunofluorescence. For immunofluorescent examination, normal human epidermal keratinocytes were seeded into chamber slides and induced with 10–6 M 13-cis-, all-trans-, 9-cis-, 4-oxo-13-cis-, 4-oxo-all-trans-, or 4-oxo-9-cis-RA for 24 h. The chamber slides were then incubated with specific primary antibodies against cytokeratin 7 (CK7) or cytokeratin 19 (CK19) for 60 min and subsequently with a secondary FITC-conjugated rabbit anti-mouse antibody for 30 min. Journal of Investigative Dermatology 2005 125, 143-153DOI: (10.1111/j.0022-202X.2005.23791.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions