Figure 2

Sensitivity to CDK4/6 inhibitors is not determined by Rb1 status or Pax3:Foxo1a expression. (A) Median inhibitory concentration (IC₅₀) determinations by cell viability assays for Pax3:Foxo1a,p53 (n = 3) and Pax3:Foxo1a,p53,Rb1 (n = 3) RMS primary cell cultures treated with Panobinostat, PD0332991, SAHA and SNS-032. P values based on a linear model of cell viability in terms of genotype, concentration and the genotype by concentration interaction with Bonferroni multiple testing correction. IC₅₀ of PD0332991 was approximately 3 μM for both groups. Error bars, mean ± 1 standard error of the mean. (B) Western blotting for Rb1 wildtype primary tumor cell cultures (n = 3) and Rb1 null RMS primary tumor cell cultures (n = 3). C2, C2C12 mouse myoblasts; pro, proliferating culture conditions; dif, differentiation culture conditions. pRb and phospho-pRb (p-Rb) are distinguished by mobility on a 5% gel using a single antibody. Whereas pRb expression is diminished in RMS cell cultures relative to C2C12 proliferating myoblasts, p107 expression is comparable. (C) Reduced relative expression levels of Rb1 by qRT-PCR in Rb1 wildtype aRMS primary cell cultures relative to C2 pro or dif. 3 independent aRMS primary cultures. All measurements performed in triplicate. P ≤0.035 for comparisons of aRMS cultures with either C2 pro or dif. (D) Two independent shRNA clones for eYFP knockdown (shY08, shC09; also achieves Pax3:Foxo1a knockdown, see Results) were compared with shRNA controls (shC01, shC05) for expression of pRb, which was unaltered. P3F, Pax3:Foxo1a. (E) Insensitivity to PD0332991 was not improved in Pax3:Foxo1a knockdown tumor cell culture clones (IC₅₀ of all clones ~2.75 μM). Specifically, shC01 and shC05 independent clones did not differ significantly, shY08 and shY09 independent clones did not differ significantly, nor did the shC versus shY clones differ significantly with regard to mean cell viability.