Inhibitory Effect of β-Thujaplicin on Ultraviolet B-Induced Apoptosis in Mouse Keratinocytes Takako Baba, Hajime Nakano, Katsuto Tamai, Daisuke Sawamura,

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Inhibitory Effect of β-Thujaplicin on Ultraviolet B-Induced Apoptosis in Mouse Keratinocytes Takako Baba, Hajime Nakano, Katsuto Tamai, Daisuke Sawamura, Katsumi Hanada Journal of Investigative Dermatology Volume 110, Issue 1, Pages 24-28 (January 1998) DOI: 10.1046/j.1523-1747.1998.00078.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 β-Thujaplicin suppression of SBC formation in UVB-irradiated mouse skin. Mouse ear skin was biopsied, stained with hematoxylin and eosin, and observed by light microscopy. About 40% reduction in the number of SBC was observed in the β-thujaplicin-treated mouse ear skin as compared with nontreated skin (p < 0.01). β-Thujaplicin treatment did not affect SBC formation in nonirradiated ear skin. Error bars represent the mean of eight separate experiments ± SD. Journal of Investigative Dermatology 1998 110, 24-28DOI: (10.1046/j.1523-1747.1998.00078.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 β-Thujaplicin suppression of TUNEL-positive cell induction in UVB-irradiated mouse skin. Mouse ear skin was biopsied, stained by the TUNEL technique, and observed by light microscopy. The number of TUNEL-positive cells was reduced by 43% (p < 0.001) as compared with nontreated skin. β-Thujaplicin treatment did not affect positive cell formation in nonirradiated ear skin. Error bars represent the mean of eight separate experiments ± SD. Journal of Investigative Dermatology 1998 110, 24-28DOI: (10.1046/j.1523-1747.1998.00078.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 β-Thujaplicin and Cd2+ inhibition of UVB-induced DNA ladder formation in cultured mouse keratinocytes. DNA extracted from the cells was electrophoresed on 2% agarose gel. DNA ladder formation was observed 24h after UVB irradiation at 50 mJ per cm2 (lane 2). Treatment with β-thujaplicin (lane 3, 10 μM; lane 4, 25 μM; lane 5, 50 μM) and 10 μM Cd2+ (lane 6) prior to UVB irradiation suppressed ladder formation in a concentration-dependent manner (lanes 3, 4, and 5) (lane 1, size marker). Journal of Investigative Dermatology 1998 110, 24-28DOI: (10.1046/j.1523-1747.1998.00078.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Increase in expression of MT mRNA induced by β-thujaplicin.Thirty micrograms of total RNA per lane was fractionated on 1.5% agarose gel, transferred to nitrocellulose filters, and northern hybridization was performed with a 0.5 kb human MT-II cDNA (A). The filters were hybridized to GAPDH cDNA, and the bands were scanned with a densitometer (B). The induction level of MT mRNA with 50 μM β-thujaplicin was double that in the presence of 10 μM Cd2+ (B). Journal of Investigative Dermatology 1998 110, 24-28DOI: (10.1046/j.1523-1747.1998.00078.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Increase in expression of MT protein induced by β-thujaplicin in mouse skin. Twenty-four hours after treatment with β-thujaplicin, the skin was biopsied and stained immunohistochemically using a monoclonal mouse anti-MT antibody. In the treated skin (A), MT expression was increased in comparison with nontreated skin (B). Scale bar, 25 mm. Journal of Investigative Dermatology 1998 110, 24-28DOI: (10.1046/j.1523-1747.1998.00078.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Increase in expression of MT protein induced by β-thujaplicin in cultured mouse keratinocytes. Epidermal proteins were separated on 15% sodium dodecyl sulfate-polyacrylamide gel, transferred electrophoretically to nitrocellulose, and reacted with a monoclonal mouse anti-MT antibody. Shown are the cells treated with Cd2+ (lane 2), 10mM β-thujaplicin (lane 3), 50mM β-thujaplicin (lane 4), or 500 pg MT-II protein (lane 5) and nontreated cells (lane 1, control). Immunoblot analysis demonstrated an increase of MT protein expression in the cells incubated with 10mM and 50mM β-thujaplicin (lanes 3 and 4). Journal of Investigative Dermatology 1998 110, 24-28DOI: (10.1046/j.1523-1747.1998.00078.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions