A, Western blot analysis of Rh41 cells stably expressing doxycycline-inducible empty vector control or CDK4. B, qPCR analysis indicates that CDC25A and CCNE2 mRNA expression is unaffected by doxycycline-inducible CDK4 overexpression. Data represent the mean ± SD of quadruplicate samples from three independent experiments. * p < 0.0005 by Student’s t-test. C, The number of upregulated genes harboring E2F-binding motifs per sample is comparable between fusion-positive RMS tumors with and without 12q13-14 amplification. p = 0.32 by Student’s t-test. Doxycycline-inducible CDK4 overexpression has no significant effect on (D) cell cycle distribution, (E) cell proliferation, or (F) focus formation. G, Co-IP analysis of CDK4 and cyclin D1 in Rh41 cells stably expressing doxycycline-inducible empty vector control or CDK4.