Dynamic Bcl-2 family binding patterns are involved in S63845 resistance. BIM (A), Bcl-xL (B), and BAK (C) coimmunoprecipitation experiments were performed 90 minutes (A) or 24 hours (B-C) after treatment with 100 nM S63845. Eluates were analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis for the presence of BIM:Bcl-xL, BIM:Bcl-2, BAK:Bcl-xL, and BAK:MCL-1 complexes, respectively; GAPDH served as the loading control. Numbers are representative of 2 biological replicates. *Immunoglobulin light chain. (D) Doxycycline inducible KMS12BM-EGFP (control) and -Bcl-xL cells were treated for 24 hours with 1000 ng/mL doxycycline. Subsequently, cells were treated for 2 hours with 100 nM S63845 and used for BIM or BAK coimmunoprecipitation experiments, which confirmed the dynamic formation of BAK:Bcl-xL and BIM:Bcl-xL complexes. (E) Inducible Bcl-xL expression protects MM cells from S63845. Cells were treated in the presence of doxycycline and S63845 for 72 hours (n = 3 biological replicates). Cells with doxycycline-inducible EGFP expression served as the control. Graphs represent viability relative to control at the end of the incubation period. Mean ± standard deviation of 3 independent experiments. ***P < .001.