Trichothiodystrophy Fibroblasts Are Deficient in the Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and (6–4)Photoproducts Yoko Nishiwaki,

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Trichothiodystrophy Fibroblasts Are Deficient in the Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and (6–4)Photoproducts Yoko Nishiwaki, Nobuhiko Kobayashi, Kyoko Imoto, Taka-aki Iwamoto, Aya Yamamoto, Sachiko Katsumi, Toshihiko Shirai, Shigeki Sugiura, Yu Nakamura, Alain Sarasin, Sachiko Miyagawa, Toshio Mori Journal of Investigative Dermatology Volume 122, Issue 2, Pages 526-532 (February 2004) DOI: 10.1046/j.0022-202X.2004.22226.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 TTD cells are deficient in the repair of UV-induced CPD and 6–4PP in genomic DNA. Cells were irradiated with UV (10 J/m2) and incubated to allow for repair. The percentage of the initial number of photoproducts was determined at various times after UV irradiation using an ELISA with monoclonal antibodies specific for each type of lesion. Each point shows the mean (±SD) of four independent experiments. Journal of Investigative Dermatology 2004 122, 526-532DOI: (10.1046/j.0022-202X.2004.22226.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 TTD cells are more sensitive to UV than normal cells. On the fourth day after UV irradiation, cell viability was determined using the MTS assay. Percentage of viability is expressed relative to unirradiated cells. Each point shows the mean (±SD) of three independent experiments. Journal of Investigative Dermatology 2004 122, 526-532DOI: (10.1046/j.0022-202X.2004.22226.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Fluorescent images of localized CPD at various repair times reflect the DNA repair kinetics obtained by ELISA. Cells were cultured in 35-mm glass-bottom dishes for 48 h. Immediately after micropore UV irradiation (100 J/m2) or at various times after UV irradiation, cells were treated with detergent solution and were then fixed. CPD (green) were then visualized with TDM-2 antibody. Nuclear DNA (red) was counterstained with propidium iodide. A filter with 3-μm pores was used. Journal of Investigative Dermatology 2004 122, 526-532DOI: (10.1046/j.0022-202X.2004.22226.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The localization of PCNA at DNA damage sites at early repair times reflects the repair capacity in TTD cell strains. Cells were treated according to the procedure described in Figure 3. Detergent-insoluble PCNA (green) was then visualized with a PCNA antibody. Nuclear DNA (red) was counterstained with propidium iodide. A filter with 3-μm pores was used. Journal of Investigative Dermatology 2004 122, 526-532DOI: (10.1046/j.0022-202X.2004.22226.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The level of XPB at localized CPD sites at early repair times reflects the repair capacity in TTD cell strains. Cells were doubly stained for XPB (green) using a XPB antibody and for CPD (red) with TDM-2 antibody 0.5 h after micropore UV irradiation. A filter with 5-μm pores was used. Journal of Investigative Dermatology 2004 122, 526-532DOI: (10.1046/j.0022-202X.2004.22226.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The level of XPB is significantly lower in TTD cells than in normal cells. Western blot analyses of XPB and actin proteins were performed on cell lysates of normal or TTD cell strains. Protein levels were quantitated by a luminescent image analyzer. The amount of XPB was normalized to the actin content. Journal of Investigative Dermatology 2004 122, 526-532DOI: (10.1046/j.0022-202X.2004.22226.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions