Figure 7. Modulating FOXR2 expression significantly alters MPNST tumorigenic properties.

(a) Immunofluorescent imaging of FOXR2 expression in HSC1λ targeted with either a Luciferase expression construct (left) or a FOXR2 expression construct (right). (b) FOXR2 western blot on cells from (a). (c) Bar graph depicts results from a soft-agar colony-forming assay performed in triplicate. Statistical analysis was done using a two-tailed student t-test, *** p<0.0001 (d) Western blot analysis of FOXR2 expression on STS26T and S462-TY cell lines targeted with FOXR2 TALENs. WT = wildtype, KO = knockout, MD = mutation detected. (e) Bar graph depicts results from a soft agar colony-forming assay performed in triplicate with biological replicate cell lines. STS26T wildtype (n= 3), STS26T mutation detected (n=4), STS26T knockout (n=4), S462-TY wildtype (n=2), S462-TY mutation detected (n=4 ), and S462-TY knockout (n=4). Statistics were done using a two-tailed student t-test comparing to the respective wildtype control. **p-=0.0032, ***p<0.0001. (f) One million STS26T wildtype (n=8, left flank) and STS26T FOXR2 KO (n=8, right flank) cells were injected into Nu/Nu mice. Tumors were measured over a 1 month period. Wildtype STS26T tumors grew significantly larger than paired KO (two-tailed student t-test ***p=0.0009). Images were captured at time of necropsy of the tumors from WT (n=5, left) or regions were injections occurred (n=5, right). H&E staining of tissue sections from the masses indicate the wildtype masses were tumors while the masses from the KO cells were predominantly fat pad tissue that contained the injected cells.