1. Functional Modulation of IGF-Binding Protein-3 Expression in Melanoma
Altaf A. Dar, Shahana Majid, Mehdi Nosrati, David de Semir, Scot Federman, Mohammed Kashani-Sabet 
Journal of Investigative Dermatology 
Volume 130, Issue 8, Pages (August 2010)
DOI: /jid
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2. Figure 1
Expression of IGF-binding protein-3 (IGFBP3) mRNA and protein in cell lines and human specimens. mRNA and protein expression levels of IGFBP3 in a normal melanocyte cell line and six melanoma cell lines as determined by qRT-PCR (a) and western blot analysis (b). (c) Secreted IGFBP3 levels in different melanoma cell lines as determined by enzyme-linked immunosorbent assay. (d) Expression of IGFBP3 at the mRNA level quantified by qRT-PCR in nevus (n=26) and melanoma (n=36) samples. *P<0.001.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Effect of 5-AZA-2′deoxycytidine treatment (5AZA) on IGF-binding protein-3 (IGFBP3) expression. Five melanoma cell lines (C8161.9, DO4, WM3211, MaMel71, and MaMel144a1) were treated with 5AZA (5μM), and IGFBP3 mRNA (a) and protein (b) expression levels were determined by qRT-PCR and western blot analysis. (c) Effects of 5AZA on histone modifications of the IGFBP3 promoter. Chromatin immunoprecipitation assay was performed on C cells after treatment with 5AZA (5μM). Acetylated H3 and H4 and methyl-2H3K4 and 3H3K4 levels in C cells treated with 5AZA showed significant enrichment compared with untreated cells. (d) Bar graph showing quantification of histone enrichment. Enrichment was calculated as the ratio between the net intensity of each bound sample normalized to its input sample and the vehicle control sample normalized to vehicle control input samples.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
DNA methylation analysis of the IGF-binding protein-3 (IGFBP3) promoter. (a) Schematic representation of the genomic region of IGFBP3 analyzed for methylation: CpG island -550 to +350; CpG sites are represented by vertical lines. F1 and F2 indicate the region for forward primers, and R1 indicates the region for reverse primer. Methylation status of C and DO4 melanoma cell lines is depicted. (b) 5AZA (5μM) treatment of C and DO4 cells completely demethylated the IGFBP3 promoter region. (c) DNA sequence of the promoter region of IGFBP3 from C cells untreated or treated with 5AZA. Cytosine residues in untreated cells were converted to thymines after 5AZA treatment. CpG sites are underlined. (d) PCR amplicons from C cells treated or untreated with 5AZA were subcloned, and DNA from five individual clones was sequenced. The color reproduction of the figure is available on the HTML full-text version of the paper.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Methylation pattern of IGF-binding protein-3 (IGFBP3) in tumor samples. (a, b) The promoter region of IGFBP3 in nevus (n=10) and tumor (n=15) samples was amplified by a nested PCR approach using methylation-specific primers and analyzed for DNA methylation. Bisulfite modification of DNA was carried out before sequencing. (c, d) PCR amplicons from nevus and tumor samples were subcloned into TOPO cloning vector and DNA was extracted from five individual clones and subsequently sequenced for DNA methylation.
Journal of Investigative Dermatology  , DOI: ( /jid )
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6. Figure 5
Overexpression of IGF-binding protein-3 (IGFBP3) suppresses melanoma cell survival. (a) Western blot analysis was performed to determine the expression of IGFBP3 in transfected C cells. (b) Cell cycle analysis of C performed using a fluorescence-activated cell sorter to determine the percentage of cells in different phases of the cell cycle (the bar graph represents three independent analyses). (c) Cell survival assay of C cells transfected with IGFBP3. (d) TUNEL assay showing rates of apoptosis after IGFBP3 transfection. (e) Western blot analysis showing expression of different proteins following IGFBP3 overexpression in C melanoma cells. *P<0.001 and **P<0.01.
Journal of Investigative Dermatology  , DOI: ( /jid )
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7. Figure 6
Stable overexpression of IGF-binding protein-3 (IGFBP3) suppresses colony formation, invasiveness, and tumor growth, and induces apoptosis. (a) Western blot analysis of IGFBP3 expression in C cells stably expressing IGFBP3 versus vector control. (b) Cell survival analysis of C stably overexpressing IGFBP3 clones as compared with control vector-expressing clone. (c) Colony formation capability of IGFBP3 clones. The bar graph shows the mean number of colonies. (d) TUNEL assay of IGFBP3-overexpressing clones versus vector control. (e) Tumor cell invasion into Matrigel of C cells overexpressing IGFBP3 versus those expressing vector control. (f) Tumor volume following subcutaneous injection of 1 × 106 C cells expressing IGFBP3 or C cells expressing vector control. *P<0.001 and **P<0.01
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions