Figure 6.
Simultaneously targeting V600EB-Raf and Akt3 inhibits in vitro melanoma growth in an additively cooperating manner. A, In vitro anchorage independent growth of UACC 903 cells nucleofected with siRNA targeting V600EB-Raf and Akt3 in cell culture media containing 0% serum. UACC 903 cells nucleofected with siRNA were plated onto PolyHEMA coated 96 well plates, allowed to grow for 3 days in DMEM containing no serum and cell viability quantified by MTS assay. UACC 903 cells nucleofected with no siRNA (lane 1) was set as 100% and others compared to this value. Targeting both B-Raf and Akt3 resulted in statistically significant inhibition of growth compared to cells in which B-Raf or Akt3 had been targeted individually. B, Western blots showing effects of targeting V600EB-Raf and Akt3 on cyclin D1, cleaved caspase-3 and p27Kip1 protein levels. siRNA-mediated inhibition of V600EB-Raf and Akt3 led to significant reductions in cyclin D1 and corresponding increases in cleaved caspase3 and p27Kip1 protein levels compared to cells in which proteins had been targeted individually.