RhoC Promotes Human Melanoma Invasion in a PI3K/Akt-Dependent Pathway

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RhoC Promotes Human Melanoma Invasion in a PI3K/Akt-Dependent Pathway Mariah C. Ruth, Yisheng Xu, Ian H. Maxwell, Natalie G. Ahn, David A. Norris, Yiqun G. Shellman Journal of Investigative Dermatology Volume 126, Issue 4, Pages 862-868 (April 2006) DOI: 10.1038/sj.jid.5700211 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Invasion assays of WM35-derived cells. Matrigel invasion chambers were used to compare invasive ability. (a) Assays were performed with 72hours incubation. Pictures of the bottom membranes were taken at original magnification × 400. (b) Quantification of invasion ability. Assays were performed with 48hours incubation. (c) The effects of Rho inhibitor, C3 transferase treatment (30μg/ml for 48hours) on invasiveness of WM35RhoC and control cells. Pictures of the top and bottom membranes were taken at original magnification × 100. Experiments have been repeated twice. Journal of Investigative Dermatology 2006 126, 862-868DOI: (10.1038/sj.jid.5700211) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Western blot analysis of pAkt expression. (a) Samples from WM35, WM35GFP, and WM35RhoC cells were probed with antibodies to the indicated proteins. (b) Samples from WM35RhoC cells were analyzed after treatment with or without C3 transferase (Rho inhibitor) for the indicated time. (c) Samples were analyzed after treatment with or without LY294002 (PI3K inhibitor) for 18hours. Experiments have been repeated three times. Journal of Investigative Dermatology 2006 126, 862-868DOI: (10.1038/sj.jid.5700211) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The effects of Akt inhibitor III treatment on invasiveness of WM35RhoC and control cells. Invasion assays were performed with or without Akt inhibitor III (5μM for 48hours). (a) Quantification of invasion efficiency. (b) Pictures of top and bottom membranes were taken at original magnification × 100. Data summarize four experiments. Journal of Investigative Dermatology 2006 126, 862-868DOI: (10.1038/sj.jid.5700211) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The effects of PI3K inhibitor (LY294002) treatment on invasiveness of WM35RhoC and control cells. Invasion assays were performed with or without LY294002 (10μM for 48hours). (a) Quantification of invasion efficiency of WM35RhoC cells. (b) Pictures of top and bottom membranes were taken at original magnification × 100. Data summarize three experiments. Journal of Investigative Dermatology 2006 126, 862-868DOI: (10.1038/sj.jid.5700211) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The effects of ROCK inhibitor (Y27632) treatment on invasion and pAkt expression in WM35RhoC cells. (a) Invasion assays were performed with or without Y27632 (40μM for 48hours). Data summarize three experiments. (b) WM35RhoC cells were treated with or without Y27632 at the indicated concentration for 24hours before being harvested for Western blot. Data represent four different experiments. Journal of Investigative Dermatology 2006 126, 862-868DOI: (10.1038/sj.jid.5700211) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The effects of treatment with both Akt and ROCK inhibitors combined on invasion. Invasion assays were performed as in Figures 3 and 5. WM35RhoC cells were treated with Akt inhibitor III (5μM) and ROCK inhibitor (Y27632, 40μM) alone or combined for 48hours. The results represent two experiments. P-values were calculated as indicated for each column versus treatment with Akt+ROCK inhibitor. Journal of Investigative Dermatology 2006 126, 862-868DOI: (10.1038/sj.jid.5700211) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Diagram of proposed pathways. Journal of Investigative Dermatology 2006 126, 862-868DOI: (10.1038/sj.jid.5700211) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions