Fig. 7

(A) Monoclonal C2C12 populations with stable Arf6 knockout were generated with CRISPR-Cas9 and guides targeting Arf6. Wild-type or Arf6 knockout cells were transfected with pCDNA3.1 empty control or pcDNA3.1 expressing VGLL2-NCOA2 under control of the CMV promoter. Shown is a western blot of generated C2C12 cell lines indicating VGLL2-NCOA2 fusion expression, Arf6 expression, and Tubulin as a loading control.

(B) qRT-PCR for Myh1 expression relative to loading controls, Gapdh and Rpl27, for days 0, 2, and 6 of myogenic fusion. Each data point is a biological replicate. The error bars represent the mean ± SD. The p values were calculated using a one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. This was repeated once.

(C) Representative brightfield images of myotube morphology taken on day 6 of fusion for each treatment group. Scale bar, 100 µm.

(D) Representative brightfield images of colonies in soft agar taken on day 14 post plating for each treatment group. Scale bar, 100 µm.

(E) Quantification of the number of colonies per well on a six-well plate. Each data point is a biological replicate. The error bars represent the mean ± SD. The p values were calculated using a one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. This was repeated twice.

(F) ARF6 expression determined by RNA-seq in human mature skeletal muscle (n = 396; GTEx) and VGLL2-NCOA2 tumors from Watson et al.⁴ (n = 5; one is a primary and recurrent from the same patient). Each data point is an individual tumor or normal tissue sample. The error bars represent the mean ± SD. The p values were calculated using the Mann-Whitney U test.

(G) ARF6 expression from (F) and compared with additional sarcoma tumor samples, including adult sarcoma from The Cancer Genome Atlas (TCGA) (n = 264), clear cell sarcoma of kidney (n = 13), Ewing sarcoma (n = 95), osteosarcoma (n = 87), and rhabdomyosarcoma (n = 42). Each data point is an individual tumor or normal tissue sample. The error bars represent the mean ± SD. The p values were calculated using the Mann-Whitney U test and are a comparison of each tumor cohort with skeletal muscle.