Sicontrol non targeting pool sirna

To silence expression of Smad1/5, specific siRNAs targeting Smad1 and Smad5 (ON-TARGETplus SMARTpool) and a negative control siRNA (siCONTROL Non-Targeting siRNA Pool) were purchased from Dharmacon (Chicago, IL, USA). The siRNAs were introduced into the proHp-overexpressing SK-Hep1 cells according to the manufacturer’s protocol. Briefly, 25 nM (final concentration) of a mixture of Smad1- and Smad5-targeting siRNAs or a negative control siRNA was mixed with DharmaFECT4 transfection reagent and incubated for 20 min at room temperature. The transfection mixture was applied to the SK-Hep1 cells which were previously transfected with Hp2 gene for 24 h. After the cells were further incubated for 24 h, the cells were treated with TGF-β (5ng/mL) for 48 h (E-cadherin and vimentin) or 1 h (Smad phosphorylation). In experiments to analyze Smad phosphorylation, the cells were preincubated for 3 h in serum-free medium before TGF-β treatment.

Cells were transfected with 100 nM each of siGENOME Smart-pool for Pak1, Pak4 and siControl nontargeting siRNA pool (Dharmacon, Lafayette, CO), using SilentFect (Bio-Rad Laboratories, Hercules, CA) per manufacturer’s instructions for 48 hours before RNA and protein extraction, cell counting and cell plating [10 (link)].

To knock down endogenous SMAD3, SMAD4 or CTGF, cells were transfected with 50 nM ON-TARGETplus SMARTpool SMAD3, SMAD4 or CTGF siRNA (Dharmacon, Lafayette, CO) using Lipofectamine RNAiMAX (Invitrogen, Burlington, ON). The siCONTROL non-targeting siRNA pool (Dharmacon) was used as a transfection control. The knockdown efficiency was examined by RT-qPCR or western blot analysis.

We obtained siGENOME ON-TARGETplus SMARTpool human GnRH-I receptor siRNA, human EGF siRNA, human Twist siRNA, human N-cadherin siRNA, and siCONTROL NON-TARGETINGpool siRNA from Dharmacon (Lafayette, CO, USA). We transfected the cells with siRNA (100 nM) using Lipofectamine RNAiMAX. After a 24 h transfection, the medium was removed and changed to a fresh serum-free medium. Cells were transfected with 100 nM si-GLO (Dharmacon) for 24 h, and the siRNA transfection efficiency was verified via fluorescent microscopy.

To knock down endogenous TGF-β receptor type I, Smad4, Smad2, Smad3 or ERK1/2, cells were transfected with 50 nM ON-TARGETplus SMARTpool TGF-β receptor I, Smad4, Smad2, Smad3 or ERK1/2 siRNA (Dharmacon, Lafayette, CO) using Lipofectamine RNAiMAX (Invitrogen, Life Technologies). siCONTROL NON-TARGETING pool siRNA (Dharmacon) was used as the transfection control.

To knockdown endogenous ALK4, ALK5 or SMAD4, cells were transfected with 50 nM ON-TARGETplus SMARTpool siRNA targeting a specific gene (Dharmacon) using Lipofectamine RNAiMAX (Invitrogen). The siCONTROL NON-TARGETING pool siRNA (Dharmacon), was used as the transfection control.