Downstream targets of GSI in MM cells. (A,B) Pretreatment with cycloheximide (CHX) abrogates the effect of GSI on MM cells. H929 cells were pretreated with 0.5 μg/mL CHX for 2 hours followed by treatment with 5 μM GSI for 24 hours. Apoptosis was measured by flow cytometry using annexin V/7-AAD staining as described in “Flow cytometry.” A typical flow cytometry dot-plot picture (A) and cumulative results from 3 experiments (B) are shown. (C,D) Primary CD138⁺ MM cells were treated in triplicate with GSI or DMSO (vehicle control, vc) for 18 hours. Cells were then collected and RNA was extracted. Expression of indicated genes was evaluated by real-time PCR and normalized to the expression of housekeeping gene 18S. Data are presented as a fold change in GSI treated cells compared with vehicle control-treated cells. In panel C, P < .05 for all genes evaluated. In panel D, actual P values are shown. (E) MM cell lines and primary MM cells were treated with GSI for 24 hours. After that time, cells were collected and the levels of indicated proteins were determined by Western blotting. As a loading control, membranes were reprobed with antibody against β-actin. MM1S cells were treated with 3 different GSI concentrations (4, 5, and 6 μM), and then all of them or only cells treated with the 2 highest doses were used for Western blotting. A portion of the gel (Akt) where cells were treated with 3 doses was repositioned to keep consistency with the 2 highest GSI concentrations used. The reposition is indicated by the vertical white line. (F,G) MM H929 or 8226 cell lines were treated with GSI or VC for 4 hours. Expression of Bcl-2 family and Akt genes (G) or Noxa (F) was evaluated by real-time PCR and normalized to the expression of endogenous control gene 18S. *Statistically significant difference (P < .05) in 2-tailed t test. Error bars represent SD.