Modulation of ASK1 activity affects MM cell survival. (A) Schematic representation of the mechanism by which PP5 regulates ASK1 activity. (B) ASK1-inducible H929 cells simultaneously treated with the indicated doses of AA and Dox (1 μg/mL) for 48 hours were harvested for lysate preparation, followed by immunoblotting with the indicated antibodies. Tubulin was used as an internal control. Phosphorylated ASK1 is denoted as p-ASK1. (C) ASK1-inducible H929 cells treated without Dox (−Dox), with Dox (+Dox; 1 μg/mL), or with Dox plus AA (+Dox at 1 μg/mL +AA at 10 μg/mL) for 72 hours were subjected to annexin V staining. Results show the percentage of annexin V⁺ cells and are the mean ± SD (n = 3). *P < .05. (D) H929 cells treated with OA (10 ng/mL) for 2 or 3 days or treated with the solvent (DMSO, control) for 3 days were subjected to immunoblotting with the indicated antibodies. (E) Human primary plasma cells from patients with MM isolated with CD138⁺ beads were treated with OA at the indicated doses or with DMSO (control) and were harvested 8 or 16 hours later for annexin V staining. Data are for 1 patient and are representative of data from 3 patient samples. (F) H929 cells were transfected by electroporation with an siRNA against ASK1 mRNA (ASK1i) or a control siRNA (ctrli). After 24 hours, a portion of cells were harvested for immunoblotting to ensure the knockdown of ASK1. Cells were then treated with 10, 50, or 100 ng/mL OA or with DMSO (0 ng/mL of OA) for an additional 24 hours, followed by annexin V staining. Results show the percentage of annexin V⁺ cells and are the mean ± SD (n = 2). **P < .01.