Akt2 ablation resulted in a substantial decrease in cell proliferation and cell survival.
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(A) The isoform-specific siRNA oligos used in this study effectively downregulated protein synthesis in an isoform-specific manner. Western blotting was carried out with samples isolated from MDA-MB231 cells transfected with isoform-specific siRNA alone or in combination with other siRNA oligos as indicated. “R” denotes cells transfected with reagent alone (i.e., a mock transfection control). Akt1–3 at the left of the panel denotes antibodies against Akt1–3 proteins, respectively. γ-tubulin was used as a loading control. Data shown is representative of four independent experiments using siRNA set #1. (B) Among three Akt isoforms, Akt2 ablation caused the greatest decrease in the colony forming ability. Results shown have employed siRNA set #1. Data shown is an average of three independent experiments. Error bars are standard errors. (C) Data from an MTT assay showed that ablation of Akt2 downregulated cell proliferation. MDA-MB231 cell proliferation was analyzed at 48–120 h post-transfection using siRNA set #2. Data shown is representative of four independent experiments. (D) DNA replication is dramatically downregulated in MDA-MB231 cells transfected with Akt2 siRNA. Cells were pulse-labelled with BrdU for 45 min, followed by analysis of DNA replication at 0–48 h post-transfection with an anti-BrdU antibody. Both siRNA oligos to Akt2 (#1 and #2) show reduced cell proliferation relative to the control (p<0.05). Data is expressed as mean BrdU incorporation, and error bars are standard errors. Results shown are an average of three independent trials.