Hyperthermia Induces Endoplasmic Reticulum-Mediated Apoptosis in Melanoma and Non-Melanoma Skin Cancer Cells Yiqun G. Shellman, William R. Howe, Leslie.

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Hyperthermia Induces Endoplasmic Reticulum-Mediated Apoptosis in Melanoma and Non-Melanoma Skin Cancer Cells Yiqun G. Shellman, William R. Howe, Leslie A. Miller, Nathaniel B. Goldstein, Theresa R. Pacheco, Roop L. Mahajan, Susan M. LaRue, David A. Norris Journal of Investigative Dermatology Volume 128, Issue 4, Pages 949-956 (April 2008) DOI: 10.1038/sj.jid.5701114 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Hyperthermia induced cytotoxicity in multiple cutaneous cell lines. Cells were seeded in 96-well plates overnight before being treated at the indicated temperature (°C) for the indicated amount of time. MTS assays were performed 24hours after treatment. Viability at 37°C of each cell line was set as 100%. Percentages of cell viability at other temperatures were compared to the cell viability of 37°C. (a) Viability at 41°C. (b) Viability at 45°C. (c) Viability at 48°C. Error bars were defined as “mean±SEM,” and there were 6–8 samples for each point. Journal of Investigative Dermatology 2008 128, 949-956DOI: (10.1038/sj.jid.5701114) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Hyperthermia stimulated Caspase 3/7 activity under certain conditions. Cells were seeded in 96-well plates overnight before being treated at the indicated temperature (°C) for the indicated amount of time (minutes). Caspase 3/7 Glo activity assays were performed 24hours after treatment. Caspase 3/7 activity of 37°C of each cell line was set as 1. Activities of other temperatures were compared to that of 37°C. Error bars were defined as “mean±SEM,” and n=3. Journal of Investigative Dermatology 2008 128, 949-956DOI: (10.1038/sj.jid.5701114) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Time-course experiments with hyperthermia treatment of ASZ001 cells. Cells were seeded in 96-well plates overnight before being treated at the indicated temperature for indicated amount of time. MTS cell viability assays and Caspase 3/7 activity Glo assays were performed according to the manufacturer's instructions at the indicated amount of post-treatment time. (a) Cell viability assays. Viabilities of cells in the 37°C incubator at each time point were set at 100%. Error bars were defined as “mean±SEM,” and there were 6–8 samples for each point. (b) Caspase 3/7 activity assay. Error bars were defined as “mean±SEM,” and n=3. Journal of Investigative Dermatology 2008 128, 949-956DOI: (10.1038/sj.jid.5701114) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Representative pictures of EB/AO staining at indicated time points. The same experimental settings were used as in Figure 3. The EB/AO staining method was used to assess the cell status at the indicated amount of post-treatment time (hours). Arrows point to the cells representing certain cell viable status: L, is the live cells; A, the apoptotic cells; and N, the necrotic cells. Bar=200μm. Journal of Investigative Dermatology 2008 128, 949-956DOI: (10.1038/sj.jid.5701114) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Hyperthermia induced activation of Caspase 3, but not Caspase 8 or 9 in ASZ001 cells. Cell lysates from Jurkat treated with PBS or 100ngml−1 of anti-Fas antibody for 5hours were used as positive controls for (a) Caspases 8 and 9 activity assays. The same experimental settings were used as in Figure 3 for (b) Caspase 8, (c) 9, or (d) 3/7 activity. Glo assays were performed according to the manufacturer's instructions at the indicated post-treatment time. Error bars were defined as “mean±SEM,” and n=3. Journal of Investigative Dermatology 2008 128, 949-956DOI: (10.1038/sj.jid.5701114) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Hyperthermia induced activation of Caspase 3, but not Caspase 8 or 9 in A375 cells. The same experimental settings were used as in Figure 5, except that A375 cells were used here instead of ASZ001. Caspase G10 activity assays were performed for (a) Caspase 8, (b) Caspase 9, and (c) Caspase 3/7 at the indicated post-treatment time. Error bars were defined as “mean±SEM,” and n=3. Journal of Investigative Dermatology 2008 128, 949-956DOI: (10.1038/sj.jid.5701114) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Hyperthermia induced ER stress and ER-mediated apoptosis. Cells were grown overnight in 10cm dishes before being subjected to hyperthermia treatment. The cell lysates were prepared at indicated time points post-treatment before being subjected to western blot. (a) Grp78 and full-length Caspase 4 expression in A375 cells. (b) Grp78 and full-length Caspase 12 expression in ASZ001 cells. Journal of Investigative Dermatology 2008 128, 949-956DOI: (10.1038/sj.jid.5701114) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Simplified apoptotic pathways. Journal of Investigative Dermatology 2008 128, 949-956DOI: (10.1038/sj.jid.5701114) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions