(A) Gene expression versus gene copy number is shown for each of the 4 ubiquitin-encoding genes across all 1,053 cell lines from the CCLE. Each dot represents a single cell line. Note that the range of y-axis values reflects an expression range specific for each gene. Cell lines highlighted in red and black were selected for subsequent studies to validate levels of ubiquitin expression. The x axis shows the number of gene copies per diploid genome. (B) Box plots of data from A directly comparing expression of ubiquitin genes. Expression is a unimodal distribution for all ubiquitin genes except for UBB, which exhibits a substantial outlier population. GAPDH is included as a control. (C) Selected cell lines were cultured and RNA was analyzed by RT-qPCR using ubiquitin-gene-specific primers and expression levels represented as log₂ fold-change mRNA relative to OC316. Data bars are colored red or black corresponding to the scheme in A. Biological quadruplicates with mean ± SEM are shown. This study was repeated twice. (D) UBB^LO cell lines (red) are sensitive to UBC knockdown. Cell lines were transfected with 5 nM siRNAs targeting PLK1 or UBC. Mock transfections performed without siRNA are shown and results from each cell line are normalized to 1.0 based on viability of cells treated with nontargeting siRNA (data not included). Dashed lines indicate 10% viability level. CellTiter-Glo was added 72 hours after transfection. Results shown were singlicates and this experiment was performed 3 times independently.