1. SOX9 and SOX10 but Not BRN2 Are Required for Nestin Expression in Human Melanoma Cells 
Anna Flammiger, Robert Besch, Anthony L. Cook, Tanja Maier, Richard A. Sturm, Carola Berking 
Journal of Investigative Dermatology 
Volume 129, Issue 4, Pages (April 2009)
DOI: /jid
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
mRNA and protein expression of nestin and BRN2 in melanoma cell lines. (a) Expression levels of nestin and BRN2 mRNA were assessed by RT–PCR. They are presented as mean of three independent experiments±standard deviation normalized relative to HPRT mRNA levels. Standard deviation of the cell line WM278 was (b) Protein expression of nestin and BRN2 in melanocytes, keratinocytes (Keratin.), fibroblasts (Fibrobl.), and melanoma cell lines was assessed by western blot analysis. One representative of three independent experiments is shown. Detection of β-actin served as loading control. (c) Cytoplasmatic localization of nestin protein is demonstrated by immunofluorescence in the metastatic cell line 1205Lu. Scale bar=100μm (left panel) and 10μm (right panel).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
BRN2 does not affect nestin expression in melanoma cells. Efficacy of BRN2-targeting siRNA was assessed by Western blot analysis in 1205Lu (a) and WM9 (b) cell lines 2 and 3 days after transfection. The effect of BRN2 downregulation on nestin mRNA expression was investigated by quantitative RT-PCR 2, 3, and 6 days after transfection with re-transfection of 1205Lu cells after 3 days (c) and 2 and 3 days after transfection in WM9 cells (d). mRNA levels in (c) and (d) were normalized to hypoxanthine-phosphoribosyl-transferase (HPRT) levels. The effect of BRN2 downregulation on nestin protein expression was demonstrated by Western blot analysis 6 days after the first transfection with re-transfection after 3 days in 1205Lu cells (e) and 2 days after transfection in A2058 melanoma cells (f). In (a–f) cells were either treated with BRN2-specific siRNA (BRN2), control siRNA (Neg Ctrl), transfection reagent without siRNA (no siRNA), or with nestin-specific siRNA (f). Detection of β-actin or IFA served as a loading control.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Correlation of BRN2 and SOX10 expression in melanoma cell lines. The levels of SOX10 (top panel) and BRN2 (middle panel) were determined by Western blot analysis relative to β-actin (bottom panel) in 11 melanoma cell lines.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
SOX10, but not BRN2, alters nestin expression in A2058 melanoma cells. (a) Real-time RT-PCR analysis of BRN2, SOX10, and nestin mRNA in A2058 melanoma cells transiently transfected with siRNA molecules targeting either negative control, BRN2, or SOX10. (b) Western immunoblot of A2058 cells transfected as in (a). The siRNA transfected is shown above each lane, with the protein detected shown to the left of each gel panel. Detection of IFA served as loading control.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
SOX9 and SOX10 are required for nestin expression in 1205Lu and WM793 melanoma cells. Quantitative RT-PCR analysis of SOX9, SOX10, and nestin mRNA in metastatic melanoma cell line 1205Lu (a) and in VGP melanoma cell line WM793 (c) 48hours after transfection with siRNA molecules targeting either negative control, SOX9, or SOX10 or after cotransfection with siRNA molecules targeting SOX9 and SOX10. The mean of three independent experiments±standard deviation is shown. Western immunoblot of 1205Lu (b) and WM793 (d) cells transfected as in (a) and (c), respectively. Detection of β-actin served as loading control.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Expression of nestin, SOX9, SOX10 and BRN2 in melanoma tissues in situ. Immunohistochemical analysis (red stain) of nestin (a, e), SOX10 (b, f), SOX9 (c, g), and BRN2 (d, h) expression in two different primary melanomas (arrows point to the tumor) of the skin (a–d and e–h). Nonspecific background staining was seen in the epidermis of SOX9-stained sections (c). Scale bar=100μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions