Characterization of Acquired Drug Resistance

SUM149 and SUM190 cells (obtained from Asterand, Inc., Detroit, MI, USA) were cultured as previously described.^{16 (link), 59 (link)} Asterand characterizes cell lines using short-tandem repeat polymorphism analysis. Cells were banked upon receipt and cultured for no more than 6 months before use in this study. rSUM149 and rSUM190 are isogenic, acquired resistance cell lines established in the laboratory.^{16 (link)}Transient cell transfections were performed using the Mirus TransIT 2020 transfection reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer's instructions. rSUM149 and rSUM190 cells were transfected with a plasmid containing XIAP-targeting short hairpin RNA and 48 h post transfection, viable cells were used in the ADCC assay. Effective knockdown was confirmed by western immunoblot analysis.
For caspase activity and cell viability, cells were treated with indicated doses of recombinant human TRAIL (Enzo Life Sciences, Farmingdale, NY, USA) for 24 h. Cell viability was determined by trypan blue exclusion as previously described.^{59 (link)} For proliferation measurement, cells were seeded in a 96-well plate and treated with indicated doses of antibodies for 72 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich, St. Louis, MO, USA) assay reagent was added and cellular proliferation measured as previously described.^{60 (link), 61 (link)}