Figure 5. FGF8 is involved in the oncogenicity of primary tumors from the myoblast system.

A. Western blot analysis of FGF8 expression in doxycycline-treated primary TD cells (Prim 1) after transduction with lentiviruses containing CRISPR constructs targeting FGF8 (#1, 2 or 3) or a non-targeting CRISPR construct (control). P3F induction was confirmed in these cells and GAPDH was used as a loading control. B. Cell growth of doxycycline-treated primary TD cells transduced with CR-FGF8 or CR-control constructs was monitored using the IncuCyte system. The data are displayed as the mean +/− SE of 4 different wells. Statistical significance was determined using unpaired Student t-test. ** P<0.01. C. Representative images of focus formation and clonogenicity that were assayed in doxycycline-treated primary TD cells transduced with CR-FGF8 or CR-control constructs; two replicates are shown for each assay. D. Foci counting from focus formation assay shown in part C. Focus counting was performed in triplicate for the CRISPR groups indicated in part C. The two-sided unpaired Student’s t-test was used to determine significant differences between the control and each test group. ** P<0.01. *** P<0.001. E. Tumor formation using FGF8 knockout and control cells. Tumor growth curves in NOD-SCID mice (5 mice per group) injected intramuscularly with cells from transduction of CR-FGF8 or CR-control constructs into Prim 1 cells. For statistical significance, CR-FGF8 #1 and #3 were compared to the CR-control, for the four last time points. Unpaired two-sided Student’s t-test was used, and significance is indicated for the two comparisons. * P<0.05 ** P<0.01, Ns: not significant. F. Focus assay of Dbt/MYCN cells grown in CM collected from cultures of primary TD cells (Primary #1 or Primary #2) or parental Dbt/MYCN/iP3F cells with (dox+) or without (dox-) doxycycline induction of P3F expression.