ASK1 triggers apoptosis in human MM cell lines. (A) Ectopic expression of ASK1 increases death of H929 and U266 cells. Retroviral vector producing FLAG-tagged WT, CA, or dominant-negative (DN) ASK1 or the control vector, pGC, was transduced into H929 (left) and U266 (right) cells. The transduced cells were sorted according to the expression of yfp at 1 day after transduction and cultured for the days indicated. The percentage of cell death was determined by trypan blue exclusion. Immunoblotting with anti-FLAG shows the expression of transduced proteins. Tubulin was used as an internal control. (B) Tet-on-inducible stable H929 and IM9 transfectants were treated with Dox (1 μg/mL) for 3 days to induce ASK1 expression. Percentage of cell death was determined by trypan blue exclusion with the use of cells harvested on various days after Dox treatment, and the expression of inducible ASK1 was evaluated by immunoblotting using anti-FLAG. Results represent mean ± SD (n = 3). (C) Ectopic expression of ASK1 causes apoptosis of H929 cells. H929 cells were transduced with retroviral vector to produce WT ASK1 (black line), CA ASK1 (gray line), or pGC control vector (gray fill) for 3 days. The apoptotic cells in the yfp⁺ gate were determined by annexin V staining. Results are from 1 experiment and are representative of 3 independent experiments. *P < .05, **P < .01.