Tgf β1 sirna

TGF-β1 siRNA, mock siRNA and the transfection agent were purchased from SCBT (Santa Cruz, CA). The transfection protocol was followed according to the instructions of the manufacturer. Inhibition efficacy was determined by basal expression control of the TGF-β1-regulated genes SNAI1, SNAI2, and CTGF. Transfected cells were exposed to Emdogain at 100 µg/ml in serum-free medium for 24 hours. Gene expression analysis was performed targeting IL-11.

The siRNA experiments were performed with HIF-1α siRNA (sc-35561), TGF-β1 siRNA (sc-37192), and scramble control siRNA (sc-37007) following the protocol provided by Santa Cruz Biotechnology.

For inhibition of TGF-β1, MKP7 or CTGF expression, LX-2 cells in 6-well plates (5 × 10⁴ cells/well) were transfected with TGF-β1 siRNA (Santa Cruz) or Trilencer-27 siRNA duplexes against MKP7 or CTGF (Origene Technologies Inc., Rockville, MD, USA) using Lipofectamine RNAiMAX Reagent (Life Technologies Inc.) according to the instructions of the manufacturer. Transfection using universal scrambled negative control siRNA duplex (Origene) was used as a negative control. After transfection for 48 h, LX-2 cells were exposed to Ang II (10⁻⁷ M) for another 0.5, 1, 4, or 24 h, respectively. Transient siRNA-mediated attenuation of TGF-β1, MKP7 or CTGF expression was determined by immunoblotting analysis with the respective antibodies.