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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Cancer Res. 2019 May 2;79(11):2892–2908. doi: 10.1158/0008-5472.CAN-18-3026

Figure 4. ARF6-GTP is sufficient for PI3K and AKT activation.

Figure 4.

(a) Catalytic activity of endogenous PI3K in mouse tumor cell lines, one–way ANOVA with Tukey’s multiple comparisons test (a). (b) Mouse melanoma cells BrafCA;Cdkn2af/f control (5588), BrafCA;Cdkn2af/f + Arf6Q67L (6431 and 6455). (c) Adenoviral delivery of MYC-tagged ARF6Q67L to serum-starved NIH3T3 cells. (b-c) Graphs show individual data points normalized to control along with geometric means, 95% C.I., ratio paired t test, two tailed. Phosphorylated AKTS473 detected by immunohistochemistry in (d) primary tumors from Arf6Q67L mice but not BrafCA;Cdkn2af/f controls. Scale bars = 20μm, 400x magnification. (e) pAKT stains tended to highlight tumor cells at the invasive front (arrows). Mouse tumors 8845, 6807 and 6808 scale bars = 20 μm, 400x magnification. In 6807 pAKT highlights tumor cells invading edematous subcutaneous tissue (top right of image). In 6808, pAKT highlights tumor cells invading adipose (left portion of image). Mouse 7760 shows pAKT highlighting tumor invading skeletal muscle, scale bar = 50 μm, 100x magnification. (f) Heterogeneous pAKT staining of metastatic tumors. Pulmonary metastasis (met), scale bars = 20 μm, 400x magnification. Lymph node met scale bars = 50 μm, 200x magnification. BrafCA;Cdkn2af/f mice are known to develop benign pulmonary adenomas (unrelated to melanoma) and these fail to stain with pAKT, providing a negative internal control (right panel, scale bar 100μm, 100x magnification).