Fig. 1: Copy number amplification of MCL1 occurs at a high frequency in OS, representing a potential vulnerability.

A The copy number variations (CNVs) of MCL1 and BCL2L1 in OS cells measured by qPCR. Each copy number is quantified relative to LINE1 levels and represented as fold change. The human genomic DNA is used as a normal reference DNA. Data are shown as mean ± SD (n = 3). B Protein expression levels of Bcl-2 family proteins in OS cells. Each protein expression is detected using immunoblot with the indicated antibodies. C Detection of copy number gains of MCL1 in OS cells by Fluorescence in situ hybridization (FISH) analysis. The ChromaTide TexasRed-12-dUTP targeting the human MCL1 gene in 1q21.2 (red), and Fluorescein-12-dUTP targeting sequences in the chromosomal region 1p13.1 (green) are hybridized to cell blocks. Scale bars: 20 μm. D–L Effects of RNAi mediated gene knockdown of Mcl-1, Bcl-xL, or Bcl-2 in NOS-10, Sa-xeno-147, and G-292 cells. The knockdown efficacy is confirmed 24–48 h after siRNA treatment by the immunoblotting (D–F) and qPCR (Supplementary Fig. S2). Each protein expression is detected using immunoblot with the indicated antibodies. G–I Effects of siRNAs on the caspase-3/7 activity of OS cells. Caspase activities are measured using a Caspase-Glo 3/7 Assay reagent after 48 h of siRNA treatment. Data are shown as mean ± SD (n = 4). **p < 0.01 as determined by Student’s t-test. J–L Effects of siRNAs on OS cell viability. Cell viability is measured using a CellTiter-Glo Assay reagent after 48 h of siRNA treatment. Data are shown as mean ± SD (n = 4). ^**p < 0.01 as determined by Student’s t-test.