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Figure S1. Schematic of the DCT-TVA melanoma mouse model and associated procedures. DCT-TVA mice were crossed with Ink4a/Arflox/lox mice to generate DCT-TVA; Ink4a/Arflox/lox mice. TVA negative mice were used as controls. All mice were injected subcutaneously with the indicated viruses at birth. Melanocytes and tumors were isolated and established in culture. Further analysis of tumorigenicity was performed in nude mice and the DCT-TVA; Ink4a/Arflox/lox mouse strain.

Figure S2. Fluorescent intravital imaging of GFP in RCASBP(A)GFP injected DCT-TVA mice. (A–D) Confocal/multi-photon intravital imaging. (A) RCASBP(A)GFP infected DF-1 cells imaged prior to injection. (B) Image of a DCT-TVA mouse 5 min post-injection. (C) RCASBP(A)GFP injected TVA-negative mouse 3 weeks post-injection. (D) TVA-negative mouse 3 weeks post injection with RCASBP(A)GFP infected viral producing cells. (E–J) Whole body fluorescent imaging using the FluorVivo system. Each image represents a different time point post injection, indicated in hours (h).

Figure S3. Characterization of tumors in DCT-TVA/Ink4a/Arflox/lox mice induced by expression of NRAS-IRES-Cre. Mice were injected with NRASQ61R-IRES-Cre viral producing cells at birth. (A and C) H&E stained tumor sections from a representative mouse (age 39 days). (B) IHC for the HA epitope tag on NRASQ61R. (D) IHC for HMB-45 and MART-1 (pan-MEL). All sections were counterstained with hematoxylin. Scale bars represent 200 μm for all sections.

Figure S4. Characterization of a tumor with brain involvement in an injected DCT-TVA/Ink4a/Arflox/lox mouse. Mice were injected with NRASQ61R-HA and Cre viruses at birth. (A, B) H&E stained tumor sections from a representative mouse (age 44 days). (C, D) IHC for the HA epitope tag on NRASQ61R. (E, F) IHC for Ki67. (G, H) IHC for GFAP. (I, J) IHC for Nestin. All sections were counterstained with hematoxylin. Scale bar represents 200 μm for all sections.

Figure S5. NRas expression in explanted melanoma cells. Expression of endogenous NRas and virally delivered NRASQ61R-HA from D6-MEL immortal melanocytes (control) and three different tumor samples (336, 335 and 333). The cells were lysed in SDS lysis buffer and separated on 4–20% gradient polyacrylamide gels. Expression from the virus was compared to endogenous NRas expression using an NRAS specific antibody. NRASQ61R-HA can be distinguished by its slower migration through the gel resulting from the addition of the HA tag. The blots were re-probed with α-tubulin as a loading control. The levels of virally delivered NRASQ61R-HA expression from three separate samples were not significantly different than the level of endogenous NRas expression in the control cells. Band density was quantitated using Image J, normalized to tubulin levels, and compared with the control. Sample 336 NRASQ61R-HA (top band) was 99 ± 6.3%, sample 335 was 109 ± 6.7% and sample 332 was 86 ± 10.0% compared with control endogenous NRas expression.

Figure S6. Growth of melanoma cells in soft agar. Cells were seeded at a density of 1.5 × 105 per well in a 6-well dish. D6-MEL cells are previously described immortal Ink4a/Arf-deficient melanocytes that served as a negative control (Whitwam et al., 2007). D6-NRAS are D6-MEL cells expressing NRASQ61R that served as a positive control. Each cell line was assayed in triplicate. Data are expressed as the mean ± SEM.

Figure S7. Characterization of a syngeneic tumor with kidney invasion in a DCT-TVA/Ink4a/Arflox/lox mouse. Mice were injected subcutaneously with explanted tumor cells at birth. (A, B) H&E stained tumor sections from a representative mouse (age 78 days). (C, D) IHC for the HA epitope tag on NRASQ61R. (E, F) IHC for phosphorylated Erk (p-Erk). (G, H) IHC for phosphorylated p70 S6 kinase (p-S6K). (I, J) IHC for Ki67. (K, L) IHC for Nestin. All sections were counterstained with hematoxylin. Scale bar represents 200 μm for all sections.

Figure S8. Peritoneal metastases detected in DCT-TVA/Ink4a/Arf lox/lox mice injected subcutaneously with syngeneic tumor cells. Mice were injected subcutaneously with explanted tumor cells at birth. (A–I) Representative H&E stained tumor sections from three different mice. Tumor cells were detected within the (A) kidney, (B–E) pancreas, (F–G) intestine and (I) esophagus. (H) lymph nodes were frequently involved by primary tumors and metastases. Scale bars represent 200 μm for all sections.

Figure S9. Schematic representation of the viral vectors. These vectors are all Gateway compatible to allow for the easy insertion of experimental sequences. LTR, long terminal repeat; Ψ, packaging signal; SD, splice donor; SA, splice acceptor.

Table S1. Tumorigenicity of primary tumor cells following subcutaneous injection into Nude or DCT-TVA/Ink4a/Arflox/lox mice.

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