GSI induces apoptosis of MM cells by up-regulation of Noxa. Introduction of Noxa siRNA into MM cells cancels GSI-induced apoptosis. Human 8226 cells were transfected with Noxa siRNA or siCONTROL nontargeting pool siRNA using Amaxa nucleofector device. (A) Cells were incubated for the indicated period of time, and the levels of Noxa were determined by Western blotting. As a loading control, the membrane was reprobed with antibody against β-actin. (B) Transfected cells were treated in triplicate with either vehicle control (DMSO) or the indicated concentrations of GSI. After 12 hours, cells were collected, and apoptosis was measured by annexin V/7-AAD staining using FACSCalibur flow cytometer. Mean plus or minus SD values are shown. The differences were statistically significant for all GSI concentrations (P < .05). (C) Bax and Bak were activated after Noxa up-regulation. MM cells were treated with vehicle control (filled histogram) or GSI (thick line) for 16 hours, then collected and fixed. Staining with antibodies against the conformationally active forms of Bak and Bax was performed as described in “Flow cytometry.” As a control, cells were stained with secondary antibody only (thin line). Cells were analyzed by flow cytometry, and typical histograms from 3 experiments are shown. (D) 8226 MM cells were treated with 7 μM GSI for 24 hours. After that time, cells were collected, lysed, and immunoprecipitated with an antibody against Bad or normal rabbit IgG. The membrane was probed with anti-Noxa and anti-Bad antibody. Three experiments yielded similar results.