Figure 3.

KDM3A is upregulated in Ewing Sarcoma, and promotes clonogenic and anchorage-independent growth. (a) KDM3A protein levels in the indicated Ewing Sarcoma cell lines, and mesenchymal stem cells (hMSCs), were determined by immunoblotting, with tubulin as loading control, as in Figure 2. MSCs were obtained from Lonza (L) and SciCell (SC), as previously,⁶ and grown in Dulbecco’s modified Eagle’s media (D) or proprietary stem cell media (M). (b) KDM3A protein levels in A673, SK-ES-1 and TC71 cells stably transduced with two different KDM3A-targeting lentiviral small hairpin RNAs (shRNAs) and non-targeting control, as determined by immunoblotting. ShRNA-mediated gene expression silencing via lentiviral delivery was performed as previously described.⁶ The control non-targeting shRNA consisted of a scrambled sequence (Addgene plasmid 1864⁴⁵). ShRNAs 1 and 2 for KDM3A correspond to TRCN0000021150 and TRCN0000021152 (all Sigma Mission shRNAs, distributed via the University of Colorado Cancer Center Functional Genomics Core Facility). (c) Clonogenic and soft agar assays were performed and analyzed as in Figure 1. Quantifications represent the mean and s.e.m. of two independent experiments, each performed in at least triplicate; representative images from one experiment are also shown. *P<0.05, determined using a two-way Student’s t-test with unequal variance.