Akti-1/2 inhibits Akt phosphorylation in primary MM cells and induces apoptosis in the presence or absence of BMSCs. Shown are 2 examples of biochemical analysis of the effects of Akti-1/2 on primary MM cells. (A) Western analysis of freshly purified CD138⁺ MM cells (200 000 per lane) treated overnight with either DMSO or Akti-1/2 (10 μM). At 15 minutes before harvest, cells were stimulated with a combination of IL-6 and IGF-1 to show inducibility of activated Akt and lack of this effect in cells treated with the Akt inhibitor. Phospho-ERK1/2, which was always unchanged by Akti-1/2 treatment in primary MM cells or in MM cell lines, serves as surrogate loading control for the blot for patient 14. (B) Phospho-Akt–specific flow cytometry for the same primary samples as in panel A. A total of 50 000 cells per well were kept for a total of 24 hours in medium alone or in coculture with BMSCs. They were then treated overnight with Akti-1/2 (10 μM) or DMSO. Primary MM cells stimulated with IL-6 and IGF-1 for 15 minutes served as positive control. (C) Cytotoxic effect of Akti-1/2 treatment on the same primary MM cells as shown in panels A and B. Freshly purified MM cells were kept overnight either in medium supplemented with 2 ng/mL IL-6 or in the presence of BMSCs. They were then exposed to different concentrations of Akti-1/2 and their viability was measured after 5 days by annexin V–FITC/PI staining and flow cytometry.