Tumor Necrosis Factor α Increases and α-Melanocyte-Stimulating Hormone Reduces Uveal Melanoma Invasion Through Fibronectin  Irene Cantón, Paula C. Eves, Sheila MacNeil  Journal of Investigative Dermatology  Volume 121, Issue 3, Pages 557-563 (September 2003) DOI: 10.1046/j.1523-1747.2003.12417.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Effect of TNF-α on invasion of three uveal melanoma cell lines through human fibronectin. Triplicate wells of cells were treated with TNF-α (100–300 units per ml) for 20 h during the fibronectin invasion experiment. TNF-α significantly increased the invasion in all three cell lines at 200 and 300 units per ml. Results are expressed as a mean percentage of the control±SEM. |, SOM 196b (n=4 experiments);?, 177w7B7 (n=4 experiments);?, VUP (n=5 experiments). *p<0.05; **p<0.01. Journal of Investigative Dermatology 2003 121, 557-563DOI: (10.1046/j.1523-1747.2003.12417.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Western immunoblotting for the MC-1R. 12.5 μg total cellular protein was loaded per gel track from three uveal melanoma cell lines (196b, VUP, and 177w7B7, as indicated at the top of each track). M is the SDS-7B molecular weight marker. A single prominent immunoreactive band was identified migrating at approximately 37 kDa (indicated) for each uveal melanoma cell type. Journal of Investigative Dermatology 2003 121, 557-563DOI: (10.1046/j.1523-1747.2003.12417.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of α-MSH on invasion of three uveal melanoma cell lines through human fibronectin. Triplicate wells of cells were incubated with α-MSH (10-11 M-10-7 M) for 20 h during the fibronectin invasion experiment. α-MSH significantly decreased uveal melanoma invasion levels in all three lines, with maximum inhibition at 10-9 M for all lines. Results are expressed as a mean percentage of the control±SEM. ¦, SOM 196b (n=5 experiments);?, 177w7B7 (n=4 experiments);?, VUP (n=6 experiments). *p<0.05; **p<0.01. Journal of Investigative Dermatology 2003 121, 557-563DOI: (10.1046/j.1523-1747.2003.12417.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Comparison between α-MSH and MSH 11–13 KP-D-V on 196b uveal melanoma invasion through fibronectin. Both peptides significantly inhibited invasion (triplicate wells of cells were examined in all cases), but no significant differences between α-MSH (n=5 experiments) and KP-D-V (n=4 experiments) were observed at the concentrations studied. Results are expressed as a mean percentage of the control±SEM. *p<0.05; **p<0.01. Journal of Investigative Dermatology 2003 121, 557-563DOI: (10.1046/j.1523-1747.2003.12417.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of TNF-α, α-MSH, and KP-D-V on uveal melanoma invasion. TNF-α (200 units per ml) increased invasion of all three uveal melanoma cell lines, whereas α-MSH and KP-D-V (at 10-9 M) decreased invasion. The presence of α-MSH together with TNF-α significantly opposed the ability of the TNF-α to increase invasion in two out of three cell lines. KP-D-V opposed the effect of TNF-α in all three cell lines. All experiments were conducted using triplicate wells of cells. Mean values from these wells were compared to control untreated wells using Student's paired t test based on a minimum of three experiments. Values differing significantly from control values are indicated by *p<0.05, **p<0.01, ***p<0.001. Values differing significantly from TNF-α-stimulated values are indicated by #p<0.05, ##p<0.01. The total number of experiments is illustrated in each histogram and all data shown are normalized with respect to the control value for ease of presentation. Journal of Investigative Dermatology 2003 121, 557-563DOI: (10.1046/j.1523-1747.2003.12417.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions