SiRNA knockdown of Akt isoforms in MM cell lines. (A) Specificity of Akt isoform knockdown in MM cell lines AMO-1 and MM.1S as determined by Western analysis 3 days after electroporation with pSUPER-derived siRNA expression vectors (siAkt1, siAkt2, and siAkt3; pSU = empty vector) and subsequent enrichment of strongly transfected cells. MM.1S cells display a constitutive phospho-Akt signal, whereas AMO-1 cells do not. AMO-1 cells are devoid of Akt3 protein. Staining of α-tubulin was used as loading control. (B) Western analysis of the molecular effects of combined knockdown of Akt1 and Akt2 or of all 3 isoforms in AMO-1 and MM.1S cells. Shown is the down-regulation of single Akt isoforms, its effect on the total level of Akt protein (pan-Akt), on the level of activated Akt protein (phospho-Akt [Ser473]), and on the amount of phosphorylated Akt substrate FoxO1/3a. (C) Viability of MM cells transfected with (combinations of) Akt knockdown constructs. Purified populations of transfected cells were cocultured with BMSCs and their survival determined by annexin V–ATTO 647/PI staining 6 days after electroporation. The graph comprises the results from 3 independent experiments. The bars display the percentage of viable cells for the respective Akt knockdown constructs compared with identically treated empty vector controls. Error bars mark standard deviations. P values were determined by 1-tailed unpaired Student t tests (*P < .01; **P = .04).