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Oxidative Medicine and Cellular Longevity
Volume 2012, Article ID 850684, 13 pages
Research Article

Preventive Effects of Epigallocatechin-3-O-Gallate against Replicative Senescence Associated with p53 Acetylation in Human Dermal Fibroblasts

1Department of Applied Nanoscience and BK 21 Nano Fusion Technology Division, College of Nanoscience & Nanotechnology, Pusan National University, San 30 Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea
2Cellbiocontrol Laboratory, Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Republic of Korea
3Center for Innovation in Immunoregulative Technology and Therapeutics, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
4Department of Biobased Materials Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

Received 19 July 2012; Revised 17 September 2012; Accepted 8 October 2012

Academic Editor: Gabriele Saretzki

Copyright © 2012 Dong-Wook Han et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Materials and Methods. Fluorescence Microscopic Observation for Cellular Uptake of FITC-EGCG. To compare the cellular uptake patterns of EGCG into human dermal fibroblasts (HDFs) (primary cells at 5 ~ 7 passages) and L-929 cells (murine fibroblastic cell line), fluorescence microscopy was performed in the cells either suspended or cultured with FITC-EGCG treatment. After 30 min of preincubation in phosphate-buffered saline (PBS, pH 7.4), suspended cells were treated with FITC-EGCG (50 µM) in PBS for 4 h, washed three times with PBS and then centrifuged at 250 × g for 5 min at 4°C. After centrifugation, the cell pellets were reuspended in PBS and fixed with 3.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 5 min at room temperature. The incorporation of FITC-EGCG into the cytoplasm of the cells and its subsequent nuclear translocation were immediately observed under a fluorescence microscope (Biozero – 8000, Keyence, Osaka, Japan). For studies in cultured cells, 2 × 105 cells in growth medium were plated into each well of a 24-well plate. When reached 80% confluence, the cells were treated with FITC-EGCG (100 µM) for 8 h and then fixed with 3.5% paraformaldehyde as described above. After incubation, the cellular uptake of FITC-EGCG was observed under a fluorescence microscope.

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