Osteopontin Expression Correlates with Melanoma Invasion Youwen Zhou, Derek L. Dai, Magdalena Martinka, Mingwan Su, Yi Zhang, Eric I. Campos, Irene Dorocicz, Liren Tang, David Huntsman, Colleen Nelson, Vincent Ho, Gang Li  Journal of Investigative Dermatology  Volume 124, Issue 5, Pages 1044-1052 (May 2005) DOI: 10.1111/j.0022-202X.2005.23680.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Osteopontin (OPN) mRNA is more abundantly expressed in metastatic melanoma nodules as compared with benign melanocytic nevi. Confirmation of OPN expression using real-time RT-PCR in melanoma cell lines (n=14), cultured normal melanocytes (n=4), metastatic melanoma nodules (n=4), and normal nevi (n=11). The OPN expression was normalized to the level of glyceraldehyde phosphate dehydrogenase (GAPDH) from the same sample and the average copy numbers per million GAPDH were plotted. The bars indicate standard errors. Journal of Investigative Dermatology 2005 124, 1044-1052DOI: (10.1111/j.0022-202X.2005.23680.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of osteopontin (OPN) protein in cultured human epidermal melanocytes and melanoma cell lines by western blot analysis. Lane 1, epidermal melanocytes; lanes 2–11, melanoma cell lines (Sk-mel-3, Sk-mel-93, Sk-mel-24, Sk-mel-5, Sk-mel-2, PEDI, MMAN, KZ-28, KZ-13, and KZ-2, respectively). Journal of Investigative Dermatology 2005 124, 1044-1052DOI: (10.1111/j.0022-202X.2005.23680.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Osteopontin (OPN) protein expression in melanocytic lesions by immunohistochemistry. Sections from benign, dysplastic, and malignant melanocytic lesions were stained as described in Materials and Methods. (A) primary melanoma tissue incubated with pre-immune serum. (B–F), various stages of melanocytic lesions stained with OPN antibody. (B), benign nevus. (C), dysplastic nevus. (D), melanoma in situ. (E), primary melanoma. (F), metastatic melanoma. Scale bars=50 μm. Journal of Investigative Dermatology 2005 124, 1044-1052DOI: (10.1111/j.0022-202X.2005.23680.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Osteopontin (OPN) protein expression in benign, dysplastic, primary invasive, and metastatic melanocytic lesions by immunohistochemistry. (A) Staining intensity of OPN in various melanocytic lesions. NN, normal nevi (n=15); DN, dysplastic nevi (n=20); MIS, melanoma in situ (n=17); PM, invasive primary melanoma (n=68); MM, metastatic melanomas (n=18). (B) Correlation of OPN expression with tumor thickness. The sample size for the melanoma of various tumor thickness: <0.75 mm (n=11); 0.75–1.50 mm (n=40); 1.51–3.0 mm (n=9); and >3.0 mm (n=8). Journal of Investigative Dermatology 2005 124, 1044-1052DOI: (10.1111/j.0022-202X.2005.23680.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Inhibition of osteopontin (OPN) mRNA expression by RNA interference reduces the cell growth rate of melanoma cells. (A) Inhibition of OPN mRNA expression in KZ-28 cells by OPN small interference RNA (siRNA). The cells were also transfected by glyceraldehyde phosphate dehydrogenase (GAPDH) siRNA. The data were obtained using real-time PCR and presented as fold change in expression of both OPN and GAPDH normalized in the non-siRNA control sample. No OPN reduction was observed in GAPDH siRNA-transfected cells and vice versa. (B) Reduction in cell growth rate by MTT assay. The viabilities of cells transfected with various concentrations of OPN siRNA were represented as percentage over the control cells. Each datum represents the average of four replicates. Bars indicate the standard errors. Significant reduction was observed in cells transfected with 150 nM siRNA as compared with control cells (*p=0.0134). Journal of Investigative Dermatology 2005 124, 1044-1052DOI: (10.1111/j.0022-202X.2005.23680.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions