Establishment of drug-resistance cell lines

The parental RPMI8266 cells (5x10⁵) were seeded into 6-well plates containing 10 nM bortezomib (Sigma-Aldrich; Merck KGaA) when they reached the exponential growth phase, and were incubated in 37˚C with 5% CO₂ condition. The cell culture medium was replaced every 2-3 days until the cells return to the normal growth state. After 1-2 weeks, bortezomib (10 nM) was added to the culture medium again; when this process has been repeated for three times, the final concentration of bortezomib was increased to 20 nM. The aforementioned steps were repeated until the bortezomib concentration was increased to 200 nM. After 8-10 months, the bortezomib-resistant RPMI8266 cells were obtained. A similar approach was also used to establish adriamycin- and dexamethasone-resistant cell lines. Finally, ADR-resistant RPMI8226 cells (160 nM of ADR), BOR-resistant RPMI8226 cells (200 nM of BOR), and DEX-resistant RPMI8226 cells (180 nM of DEX) were successfully established.