Akt1 sirna

Akt1 siRNA and control siRNA were purchased from Cell Signaling Technology. MCF‐7 cells were transfected with siRNA (100 nmol/L) according to the manufacturer's protocol. Cells were harvested at 48 h, and the migration and invasion assays were performed. Efficiencies of Akt1 siRNA were detected by western blot.

Suppression of AKT1 function was performed by knockdown of AKT1 expression by AKT1 small interfering RNA (AKT1-siRNA) (cat# M-004364-00, Cell Signaling Technology). Transient transfections were done with cells at 60% confluence using Dharmafect siRNA transfection reagent according to the manufacturer’s (Thermo Scientific) instructions. Briefly, MLE-12 cells were seeded in six-well plates at 1.5×10⁵ cells/well and incubated at 37°C for 24 h. Each of AKT1-siRNA or non-target control siRNA (100 nmol) was transfected into the cells with 7.5 μl of Dharmafect reagent in serum-free media without antibiotics and incubated for 6 h at 37°C. After removal of siRNA transfection reagent, medium with fetal bovine serum was added and incubated further for 18 h. Cell cultures were subsequently exposed to room air or hyperoxia. The efficacy of AKT1 knockdown and its effects on Nrf2 target gene expression were assessed by real-time RT-PCR analysis.