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. 2017 Aug 7;36(48):6668–6679. doi: 10.1038/onc.2017.278

Figure 4.

Figure 4

cMYC activity is required for optimal MFTF-transforming capacity and cMYC protein is stably overexpressed in transformed cells. (a) JB6 P+ cells were treated with FGF2 (2.5 ng/ml) for indicated time intervals and protein expression was analyzed by western blot. Phosphorylation of Erk1 and mTOR is upregulated and cMyc increases in a time-dependent manner. Actin was used as a loading control. (b) WT JB6 P+ cells and Fgfr-1(−/−) JB6 P+ cells were treated with MFTF (150 μg/ml) for 8 h. Actin was used as a loading control. At this dose and time of MFTF, there was no change in ERK and mTOR activity. cMYC was induced in WT cells but not KO cells. (c) Inhibition of cMyc, mTOR and Erk1 activity by pharmacological inhibitors significantly attenuates MFTF-stimulated JB6 P+ colony formation in agar. Data are presented as mean±s.d. of values from triplicates and statistical significance was determined using a one-way ANOVA (*P<0.05, **P<0.001, ***P<0.0001). (d) cMYC immunofluorescence (red) in JB6 P+ cells and JB6 P+ cells transformed with MFTF. MFTF-transformed JB6 P+ cells have a higher expression of cMYC compared with non-transformed cells. Transformed JB6 P+ cells are obtained by isolating colonies from soft agar and growing the cells in liquid culture. These cells were passaged several times for 28 days and maintained a high expression of cMYC. Images were taken at × 40 magnification.