BR cells have increased expression and activation of the IGF-1/IGF-1R signaling pathway. (A) The levels of soluble IGF-1 present in cellular supernatants of cells grown in culture overnight were determined using an ELISA. The student's paired t test was used to determine statistical significance (*P < .05 compared with wild-type time points). Data shown are representative from triplicate experiments, and error bars denote ± SD. Basal levels of IGF-1 protein expression were confirmed by Western blotting (inset). (B) IGF-1R expression was determined by Western blotting in cells exposed to vehicle or 10nM bortezomib for 24 hours. (C) Activation of IGF-1R was determined in RPMI 8226 and OPM-2 drug-naive and BR cells using an ELISA. Data shown are representative of 2 experiments, and error bars denote ± SD. Concentrations were determined using a standard curve with activated IGF-1R supplied by the manufacturer. (D) 8226.wt and 8226.BR cells were propagated for 24 hours in media supplemented with 50 ng/mL of human recombinant IGF-1 (right) or IGF-1–free media (left), followed by bortezomib addition for an additional 24 hours, and viable populations were determined using the WST-1 reagent. Data shown are representative from triplicate experiments, and error bars are ± SD.