RNA interference

Knockdown of JUN expression in BT-549 cells was achieved using Silencer select pre-designed and validated siRNA (Life Technologies). Because endogenous c-Jun levels are so high in this cell line, two rounds of siRNA treatment were required to achieve effective knockdowns. Two siRNA products (manufacturer ID # s7658 and s7659) were used, along with the Silencer select negative control no. 1 siRNA. Transfections were carried out in 10 cm dishes at ∼80% confluency using Lipofectamine 2000 transfection reagent (Life Technologies) and following the manufacturer's instructions. The final siRNA concentration was 15 nM in all cases. Transfection medium was replaced with fresh growth medium 5 h after treatment. At 48 h following the initial transfection, the procedure was repeated, and cells were collected after a further 48 h.