Murine myeloma cell line MPC-11 and bone marrow stromal HS-5 cells were purchased from Cobioer Biosciences (Nanjing, Jiangsu, China). The cell lines were authenticated by the supplier using the STR profiling method. The cells were maintained in McCoy’s medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham) and 1% penicillin/streptomycin (Hyclone, Logan, UT, USA) under the condition of 37°C and 5% CO2. To establish the carfilzomib-resistant MPC-11 cells, the cells were exposed to the following increasing dose of carfilzomib (MedChemExpress, Shanghai, China): 4 nM carfilzomib for 4 weeks, 6 nM for the next 6 weeks, and 12 nM for further 8 weeks. The experiments were then performed between the resistant cells and the parental cell line. The following experiment was performed by treating both cells with 4 nM carfilzomib.
For the cell co-culture experiment, HS-5 cells were pre-labeled with CellTrace™ Violet dye (Thermo Fisher Scientific) to differentiate them from MPC-11 cells during flow cytometry analysis. MPC-11 and HS-5 cells were seeded at a 1:1 ratio (2 × 105 cells/well in a 6-well plate) and co-cultured for 48 h in the presence of 4 nM carfilzomib. Tumor necrosis factor (TNF)-α protein was purchased from Genscript Biotech company (Nanjing, Jiangsu, China), and added at 10 ng/mL for 48 h.
For the cell co-culture experiment, HS-5 cells were pre-labeled with CellTrace™ Violet dye (Thermo Fisher Scientific) to differentiate them from MPC-11 cells during flow cytometry analysis. MPC-11 and HS-5 cells were seeded at a 1:1 ratio (2 × 105 cells/well in a 6-well plate) and co-cultured for 48 h in the presence of 4 nM carfilzomib. Tumor necrosis factor (TNF)-α protein was purchased from Genscript Biotech company (Nanjing, Jiangsu, China), and added at 10 ng/mL for 48 h.