The 10058-F4 induced apoptosis in INA-6 cells is not prevented by BMSCs and is associated with down-regulation of MYC and antiapoptotic Bcl-2 proteins. (A) INA-6 cells were grown in 2% human serum in RPMI, with or without IL-6 (1 ng/mL) or 10058-F4, either alone or in the presence of a monolayer of BMSCs derived from an MM patient. The stromal cells were labeled with CFDA-SE (1μM) before INA-6 cells were added to the wells. After 72 hours, cells were detached by Accutase treatment, labeled with annexin-V Alexa-647, and analyzed by flow cytometry. INA-6 cells that were both CFDA-SE and annexin-V negative were considered viable. Error bars represent SD of triplicate measurements. (B) INA-6 cells were treated for 48 hours with the indicated concentrations of 10058-F4, DHA, ART, or PS-341 before measurement of cell viability. Cells were analyzed by flow cytometry, and cells that were both annexin-V FITC- and PI-negative were considered viable. Error bars represent SD of duplicate measurements. (C) Expression of c-MYC or GAPDH protein levels in INA-6 cells treated for 20 hours with 10058-F4 (50μM), DHA (10μM), ART (10μM), or PS-341 (8nM). (D) Immunoblot showing INA-6 and U266 after treatment for 20 hours with 10058-F4 as indicated. Protein levels of Bcl-xL, Mcl-1, Bcl-2, and GAPDH were measured using specific antibodies. (E) Myeloma cell lines were treated with the indicated lentiviral short hairpin RNA particles, and cell viability was evaluated after 3 days. (F) Relative MYC mRNA levels in myeloma cell lines treated for 2 days with the indicated lentiviral short hairpin RNA particles. The relative quantification (RQ) of mRNA levels was calculated using the δ δ C_t method with GAPDH as housekeeping gene. The MYC mRNA values were set to 1 in the LKO.1-transduced control cells.