Effects of Dicer1 silencing on bortezomib-induced apoptosis in MM cells. (A) Representative dose-response curve of MMS1 and L363 cells after incubation for 24 hours with various bortezomib concentrations. Cell viability (%) was measured by annexin V/propidium iodide staining (AnnV/PI) (Invitrogen, Paisley, United Kingdom), and viable cells are defined as AnnV^–/PI^–. Results are shown as the mean of duplicates (n = 2). (B) Lysates were generated from Dicer1 knockdown with 100 nM short interfering RNA (siRNA) or nontarget siRNA control MMS1 and L363 cells treated with 5 to 10 nM bortezomib (Cell Signaling Technology, Danvers, MA) or vehicle for 24 hours, and the expression of Dicer1 (Cell Signaling Technology) and p21 (BD Transduction Laboratories, Heidelberg, Germany) proteins were analyzed by western blot. Tubulin served as a loading control. The image shows a representative of 3 independent experiments run on the same gel. Briefly, 4.5 × 10⁵ MM cells were transiently transfected with Dicer1 siRNA (Cell Signaling Technology) or nontarget smart pool siRNA (Dharmacon, Lafayette, CO) using the TransIT-siQUEST transfection reagent (Mirus Bio, Madison, WI). MM cells were transfected with a solution containing 250 µL Opti-Mem medium (Invitrogen), 3.5 µL TransIT-siQUEST transfection reagent, and 100 nM Dicer1 siRNA or nontarget siRNA. Transfection efficiency was assessed by western blot. (C) Dicer1 knockdown increased apoptosis upon bortezomib treatment in MM cells. Dicer1 knockdown with 100 nM siRNA or nontarget siRNA control MMS1 and L363 cells was treated with 5 to 10 nM bortezomib or vehicle for 24 hours before an assessment of the percentage of AnnV⁺/PI⁺ cells by flow cytometry. Dicer1 knockdown potentiates the effect of bortezomib in L363 cells. The data presented are means ± SD of 3 experiments.