Macrophage Inhibitory Cytokine-1 Is Overexpressed in Malignant Melanoma and Is Associated with Tumorigenicity  Glen M. Boyle, Julie Pedley, Adam C. Martyn, Kelly J. Banducci, Geoffrey M. Strutton, David A. Brown, Samuel N. Breit, Peter G. Parsons  Journal of Investigative Dermatology  Volume 129, Issue 2, Pages 383-391 (February 2009) DOI: 10.1038/jid.2008.270 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of MIC-1 in melanocytic cells. (a) Semi-quantitative RT-PCR analysis of primary human melanocytes, the melanoma cell line MM96L, primary human keratinocytes, or immortalized keratinocytes (HaCaT) following either solar-simulated (SS)UV or acute UVB exposure. Data shown are representative from separate isolations of mRNA from two independent pools of melanocytes, keratinocytes, HaCaT, and MM96L cells. (b) Quantitative real-time reverse transcription-PCR detection of MIC-1 mRNA in normal human melanocytes and 23 melanoma cell lines. All data was normalized to GAPD expression and expressed relative to melanocyte MIC-1 levels. Data represent duplicate assays in duplicate experiments. (c) Expression of MIC-1 protein in melanoma cell lines. Representative western blot analysis of MIC-1 precursor expression in primary melanocytes and 35 melanoma cell lines. Western blots were probed with MIC-1 antibody followed by GAPD antibody. Journal of Investigative Dermatology 2009 129, 383-391DOI: (10.1038/jid.2008.270) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunohistochemical detection of MIC-1 in melanoma biopsies. (a) Stage IB primary melanoma (Clark level IV) showing no staining for MIC-1. (b) Primary melanoma biopsies showing cells staining positive for MIC-1 expression (red staining, examples of positive cells indicated by black arrows). Samples are from left to right: Stage IA (Clark level II), Stage IB (Clark level III), and Stage IB (Clark level IV). (c) Metastatic melanoma biopsies showing strong staining for MIC-1 expression. Top and bottom panels are separate parts of the same tumor. (d) Metastatic melanoma biopsies showing very strong staining for MIC-1 expression. Top and bottom panels are separate parts of the same tumor. Melanin (brown pigment) is also visible in some sections. Scale bar, 100μm. Journal of Investigative Dermatology 2009 129, 383-391DOI: (10.1038/jid.2008.270) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of kinase inhibitors on MIC-1 expression in melanoma cell lines. (a) A2058 or MM96L cells were treated with either vehicle alone (DMSO, mock), 2μM staurosporine, 10μM U0126, 10μM SB203580, 50μM LY294002 or 5μM bisindolylmaleimide-1 (BIS-1) for 16hours. The cells were harvested, and 30μg of total cell lysate was subjected to western blot analysis. The blots were probed with MIC-1 antibody followed by GAPD antibody. Results from two representative cell lines are shown of the five examined. (b) Melanoma cell lines were treated with either vehicle alone (DMSO, mock), 10μM U0126 or 20μM PD098059 for 16hours. The blots were probed with phosphorylated-ERK1/2 antibody followed by total ERK1/2 and MIC-1 antibody, and GAPD separately. Representative western blots from two independent treatments, protein lysate extractions, and western blot analyses of five melanoma cell lines are shown. Journal of Investigative Dermatology 2009 129, 383-391DOI: (10.1038/jid.2008.270) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 MIC-1 is a transcriptional target of MITF in melanoma cells. (a) MM96L cells were serum starved for 12hours and then treated with 20ng/ml SCF for periods between 15minutes and 24hours before generation of protein lysates and examining by western blot analysis. The blots were probed with MITF and MIC-1 antibodies followed by TYRP-1 and actin. A representative western blot from two independent treatments of two cell lines (A2058 and MM96L) is shown. (b) Chromatin immunoprecipitation (ChIP) assays from A2058 or MM96L cells were performed as described in the Materials and Methods. PCR products were resolved on 1.5% agarose by electrophoresis. DNA from lysates prior to immunoprecipitation (1% of total) was used as positive input control. (c) Protein lysates from melanoma cell lines were examined by western blot analysis. The blots were probed with MITF antibody followed by MIC-1 and GAPD antibodies. Journal of Investigative Dermatology 2009 129, 383-391DOI: (10.1038/jid.2008.270) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Reduction of MIC-1 reduces melanoma cell tumorigenicity. (a) Western blot analysis of stably-expressing short hairpin (sh)RNA MIC-1 clones from D04, A2058, and C32 melanoma cells. (b–d) Tumorigenicity of MIC-1-targeted shRNA melanoma cells in athymic mice. Two million cells expressing either GFP-targeted shRNA (squares, control) or MIC-1-targeted shRNA (circles, MIC-1 shRNA) were injected subcutaneously into each of four sites of three 4- to 6-week-old male BALB/c nu/nu mice. Three-dimensional measurement was performed two times per week, and the tumor volume expressed as mm3; data are mean±SE. Curves differ at P<0.0001 by permutation test. Journal of Investigative Dermatology 2009 129, 383-391DOI: (10.1038/jid.2008.270) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions