CD151 siRNA (h)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Incubate the FuGENE HD Transfection Reagent/medium mixture for 5 minutes at room temperature  Incubate transfection mixture for 30 min at room temperature and add to cells and shake for 10-30 seconds.  Replace medium after 6h and re-incubate cells for 72 h

Protocol tips
Incubate the FuGENE HD Transfection Reagent/medium mixture for 5 minutes at room temperature  Incubate transfection mixture for 30 min at room temperature and add to cells and shake for 10-30 seconds.  Replace medium after 6h and re-incubate cells for 72 h

Publication protocol

Unless otherwise mentioned, all cultures were carried out in 100-mm culture plates pre-coated with laminin-5 (4 µg/mL). All transfections were carried out using FuGene HD transfection reagent as per the manufacturer's protocol (Roche Applied Science, Madison, WI). Briefly, the cells were cultured in a 100 mm dish to 75% confluence. Then, 21 µL of Fugene (diluted in 100 µL of serum-free medium) was added dropwise to 7 µg of plasmid DNA (in 100 µL of serum-free medium). This mixture was incubated for 30 min and was then used to transfect each plate in the absence of serum. After six hours, the medium was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were transfected for 72 hrs with scrambled vector (pSV), shRNA against uPAR (pU), shRNA against cathepsin B (pC) and a bicistronic shRNA construct that targets both uPAR and cathepsin B (pCU) and CD151 siRNA [15]. For overexpression of uPAR and cathepsin B, cells were transfected with a plasmid expressing full-length human cDNA clone of uPAR (FluPAR) (SC319092) and cathepsin B (FlCath B) (SC109129).

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Manufacturer protocol

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