Scrambled sirna

Cells were grown in six‐well plates (Costar, Cambridge, MA, USA) and transfected with a mixture of 30 nmol/ml MXRA5 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Opti‐MEM I Reduced Serum Medium and Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Life technologies, Paisley, UK). After 18 hr, cells were washed and cultured for 48 h in complete medium and serum‐depleted for 24 hr before stimulation. A scrambled siRNA (Ambion, Applied Biosystems, Foster City, CA, USA) was used as control.

Small interfering RNA (siRNA) directed against mouse visfatin was designed and purchased from Ambion Cenix (Austin, TX, USA). The sequence specific for mouse visfatin was forward 5’-GGCACCACUAAUCAUCAGAtt-3’, reverse 5’-UCUGAUGAUUAGUGGUGCCtc-3’.
Mouse chondrocytes were cultured as described above. Confluent cells were removed with trypsin, and 6 × 10⁵ chondrocytes were seeded in 6-cm tissue culture plates and grown for 24 hours, to 70 to 80% confluence. Normal growth medium containing 10% fetal bovine serum was changed prior to siRNA transfection. Transfections were performed as described for the RNAi Starter Kit (Qiagen). Cells were incubated for 18 hours with siRNA and transfection reagent, rinsed twice with phosphate-buffered saline (PBS), and placed in DMEM (1 mg/L glucose) supplemented with penicillin, streptomycin, and L-glutamine containing 1% BSA, with or without IL-1β (10 ng/ml) for 24 hours. Transfection of siRNA against MAPK-1, a ubiquitously produced mouse cell protein, was used as a positive control. A nonsilencing siRNA that has no homology with any known mammalian gene (RNAi Starter Kit) and scrambled siRNA (Ambion) were used as negative controls.

The knockdown of BUD31 was performed using small interfering RNAs. A pre-designed siRNA silencer and a scrambled siRNA (as negative control) were obtained from Ambion, Grand Island, NY, USA. Briefly, PC3 and LNCaP cells were plated in six well plates until they reached about 70–80% confluency. Next, the reaction mix for the transfection of siRNA was prepared using Opti-MEM (GIBCO life technology, Grand Island, NY, USA) and Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A Western blot analysis was performed to confirm the knockdown of BUD31, while also assessing the efficiency and duration of the transient BUD31 knockdown.

Knockdown of PRKCZ, IGF1R, and ITGB3 expression in ovarian cancer cell lines was achieved by transfection of siRNAs (Ambion). siRNAs targeting of these genes was performed with Dharmafect-4 transfection reagent (Dharmacon). In brief, cells were seeded in 12-well or 6-well plates at densities of 1 x 10⁵ or 2 x 10⁵ cells/well, respectively. Cells were then treated with siRNA transfection mixtures following the manufacturer’s protocol. Scrambled siRNA (Ambion) was used as a control. Additional controls included mock-treated cells that received transfection reagent without siRNA, as well as untreated cells that received only fresh media. Cells were harvested after 48 or 72 hours for RNA and protein extraction, respectively.

Huh7 cells were seeded into 24-well plates and transfected with ON-TARGET plus SMARTpool siRNA against NF-YA (Thermo Scientific) or scrambled siRNA (Ambion) using DharmaFECT4 (Dharmacon). U87 cells were seeded into 24-well plates and transfected with FlexiTube siRNA against HDAC1 (Qiagen) or scrambled siRNA using DharmaFECT1. RNA was extracted from transfected cells 72 h later, and gene expression was determined by RT-qPCR.