Further Characterization of the Sho-saiko-to-Mediated Anti-Tumor Effect on Melanoma Developed in RET-Transgenic Mice  Masashi Kato, Ken-ichi Isobe, Yan Dai, Wei Liu, Izumi Nakashima  Journal of Investigative Dermatology  Volume 114, Issue 3, Pages 599-601 (March 2000) DOI: 10.1046/j.1523-1747.2000.02005.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Re-evaluation of Ret protein as a tumor-protective antigen and its reduced expression in tumors at the terminal stage in RET-transgenic mice. (A) Recombinant Ret protein (Kato et al. 1999) in complete Freund’s adjuvant was subcutaneously injected into BCF1 (C57BL/6 × BALB/C) mice twice at 10 d intervals for immunization. Four days after immunization, 2 × 106 Ret lymphoma cells that had been established from an Eμ/RET-transgenic mouse of C57BL/6 ×BALB/C background (Ichihara et al. 1995) were intraperitoneally inoculated to immunized (n=4; •) and non-immunized control mice (n=4; ○). Each point or column shows the mean±SD of four mice. The values marked ** (p<0.01) for • are significantly different from those for ○ by the Mann-Whitney U test. (B–D) Protein expression levels of Ret, MMP-9, and TIMP-2 in lysates of tumor cells were measured by western blot as described previously (Kato et al. 1998a,b). Lane 1, benign stage (tumor volume, 100 mm3); lane 2, malignant stage (tumor volume, 13 750 mm3); lane 3, terminal stage (tumor volume, 100 000 mm3). Journal of Investigative Dermatology 2000 114, 599-601DOI: (10.1046/j.1523-1747.2000.02005.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effect of Sho-saiko-to treatment on Ret protein and heat shock protein expression levels in tumors from RET-transgenic mice. (A) Tumor volumes (mean±SD) are shown for Sho-saiko-to-treated (•: n=60) and untreated control (○´: n=60) transgenic mice on a liner scale. Each point shows the mean±SD of 60 mice. The values marked ** (p<0.01) and * (p<0.05) for • are significantly different from those for ○ by the Mann-Whitney U test. (B–D) The protein expression levels of Ret, HSP-86, and HSP-25 are shown at the benign (lane 1, 100 mm3; lane 2, 96 mm3), malignant (lane 3, 13 750 mm3; lane 4, 13 750 mm3), and terminal stages (lane 5, 100 000 mm3; lane 6, 102 250 mm3) of tumors developed in Sho-saiko-to-treated (+lanes 2, 4, 6) and untreated control (–;lanes 1, 3, 5 ) RET-transgenic mice. Similar results in each stage were also obtained with tumors from other control (n=4) and experimental (n=4) mice. Journal of Investigative Dermatology 2000 114, 599-601DOI: (10.1046/j.1523-1747.2000.02005.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions