SiRNA-mediated knockdown of YB-1 induces apoptosis in the MM cell line INA-6. (A) Western blot analysis of YB-1 protein expression in INA-6 cells 72 hours after transient transfection of siRNA expression constructs against YB-1. Staining of α-tubulin served as a loading control. (B) Viability of INA-6 cells in the absence of BMSCs was assayed with annexin V–FITC/PI staining 96 hours after transfection. (C,D) Coexpression of an YB-1 protein derived from an expression construct that cannot be targeted by the YB-1 siRNA (pcDNA6/YB-1_mut) served as a control for the specificity of the siRNA-mediated effects. Transfection of 20 μg/mL of pcDNA6/YB-1_mut into INA-6 cells resulted in normal levels of YB-1 despite siRNA treatment as detected by Western blot analysis (C) and largely protected cells from YB-1 siRNA–mediated apoptosis as assessed by annexin V–FITC/PI staining (D). (E) INA-6 cells were either kept with or without 10 ng/mL of recombinant IL-6, or kept in the presence or absence of BMSCs for 24 hours, before YB-1 expression levels were analyzed by Western blot. Staining with β-actin served as loading control. (F) Viability of INA-6 cells in the presence of BMSCs was assayed with annexin V–FITC/PI staining 96 hours after transfection with YB-1 siRNA. (G) Western blot analysis of cyclin D1 expression 72 hours after transient transfection of siRNA expression constructs against YB-1. Staining of α-tubulin served as a loading control. The error bars denote the range of values derived from 3 independent experiments.