Myc expression correlates with BZ/SAHA-induced apoptosis. (A) Myc expression in MM and immortalized B cells. Levels of c-Myc, L-Myc, and β-actin were determined by immunoblotting. N-Myc was not expressed in these cells (data not shown). (B) Translation rates of MM and EBV-immortalized B cells. Equal numbers of cells were incubated with [³⁵S]cysteine/[³⁵S]methionine for 30 minutes. Protein was collected as described in “Metabolic labeling,” and [³⁵S]cysteine/[³⁵S]methionine incorporation was measured using a scintillation counter. n = 3 plus or minus SD. * indicates a significant difference compared with both MM cell lines, P < .05. (C) Determination of ER content. Cells were collected, fixed, and imaged using an electron microscope. (D-F) SAHA enhances bortezomib (BZ)–induced apoptosis. Cells were treated with 5 nM BZ, 2 μM SAHA, or this combination for 24 hours. Apoptosis was measured by (D) immunoblotting for active caspases-8, -9, and -3, (E) propidium iodide (PI) staining followed by FACS analysis, and (F) annexin V staining followed by FACS analysis as described in “Quantification of drug-induced apoptosis.” Mean (n = 3), SD. (G) SAHA augments bortezomib's ability to impair clonogenic survival. Cells were incubated with 2 nM BZ, 2 μM SAHA, or this combination for 24 hours. Clonogenic survival was determined as described in “Colony assays.” Mean (n = 3), SD. * indicates a significant difference from control and ** represents a significant difference from single-agent treatments, P < .05.