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MDA-MB-231 cell lines were purchased from the cell repository of the research institute of biotechnology (Ferdowsi University of Mashhad, Iran). Cells were cultured in RPMI1640 medium with 10% fetal bovine serum (FBS) (Rockville, MD, USA), 100 μg/mL streptomycin, 100 U/mL penicillin (Sigma) and maintained at 37 °C in a humidified atmosphere with 5% CO2 until reaching 80% of confluency. siRNAs The siRNAs targeted human NS (Cat.no: SI04319504, SI04298938, SI04211872 and SI03169593, 20 µM) and scrambled negative siRNA (SI03650325, 20 µM) were purchased from Qiagen company (USA). The lyophilized siRNAs were dissolved in RNase free water to a final concentration of 20 µM. siRNA transfection was performed using HiPerFect transfection reagent (Qiagen, Cat No: 301704, USA), as described in the manufacturer’s instructions. In order to find the optimum concentrations of siRNA, a 0.2 µM siRNA stock was prepared from a 20 µM stock. Briefly, different concentrations of 0.2 µM siRNA stock were spotted in each well of 6-wells plates for qPCR and apoptosis assays (Zhejiang Sorfa Medical Plastic Co., Ltd, Zhejiang, China). Then, 12 μL of HiPerFect transfection reagent (Qiagen, USA) was mixed with 100 μL of RPMI1640 without FBS, and the mixture incubated for 15 min at room temperature. The resulting mixture was added dropwise into each well. Finally, 100,000 cells/well in 2300 μL of 10% FBS-contained RPMI1640 medium were added to each well and were allowed to adhere overnight (reverse transfection. The cells were incubated for 6 to72 h after transfection (in 5% CO2 , 95% humidified air at 37 °C) before additional analyses. |
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| MDA-MB-231 cell lines were purchased from the cell repository of the research institute of biotechnology (Ferdowsi University of Mashhad, Iran). Cells were cultured in RPMI1640 medium with 10% fetal bovine serum (FBS) (Rockville, MD, USA), 100 μg/mL streptomycin, 100 U/mL penicillin (Sigma) and maintained at 37 °C in a humidified atmosphere with 5% CO2 until reaching 80% of confluency. siRNAs The siRNAs targeted human NS (Cat.no: SI04319504, SI04298938, SI04211872 and SI03169593, 20 µM) and scrambled negative siRNA (SI03650325, 20 µM) were purchased from Qiagen company (USA). The lyophilized siRNAs were dissolved in RNase free water to a final concentration of 20 µM. siRNA transfection was performed using HiPerFect transfection reagent (Qiagen, Cat No: 301704, USA), as described in the manufacturer’s instructions. In order to find the optimum concentrations of siRNA, a 0.2 µM siRNA stock was prepared from a 20 µM stock. Briefly, different concentrations of 0.2 µM siRNA stock were spotted in each well of 6-wells plates for qPCR and apoptosis assays (Zhejiang Sorfa Medical Plastic Co., Ltd, Zhejiang, China). Then, 12 μL of HiPerFect transfection reagent (Qiagen, USA) was mixed with 100 μL of RPMI1640 without FBS, and the mixture incubated for 15 min at room temperature. The resulting mixture was added dropwise into each well. Finally, 100,000 cells/well in 2300 μL of 10% FBS-contained RPMI1640 medium were added to each well and were allowed to adhere overnight (reverse transfection. The cells were incubated for 6 to72 h after transfection (in 5% CO2 , 95% humidified air at 37 °C) before additional analyses. |
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