Tgf β1 shrna

The TGF-β1 shRNA and a scrambled sequence as a control were synthesized by GenePharma (Shanghai, China) and inserted into the lentiviral vector pGIPZ (Open Biosystems, Huntsville, Alabama, USA). The sequences were as follows: TGF-β1 shRNA1 GCGGCAGCTGTACATTGACTT. TGF-β1 shRNA2 GCAACAATTCCTGGCGATACC. Control sequence TTCTCCGAACGTGTCACGT. The resulting recombinant plasmid pGIPZ-TGF-β1 shRNAs were confirmed by restriction endonuclease analysis and DNA sequencing. HEK-293 cells were co-transfected with pGIPZ-TGF-β1 shRNAs and packaging plasmids pLP1, pLP2 and pLP/VSVG (Invitrogen, Carlsbad, CA, USA). The supernatant containing lentiviral particles was harvested to determine the virus titer and used to infect MSCs. The expression of TGF-β1 was determined using real-time PCR and ELISA.

To silence TGF‐β1, the cells were transfected with a TGF‐β1 shRNA or with a scramble control (GenePharma, Hi‐Tech Park, Shanghai, China) using a Lipofectamine reagent (Invitrogen, Waltham, MA, USA) following the manufacturer's instructions. A stable TGF‐β1‐silenced cell line was obtained using puromycin selection. MCF‐7 cells were treated with 10 ng·mL⁻¹ of recombinant human TGF‐β1 (rhTGF‐β1) (PeproTech, Rocky Hill, NJ, USA) for 24 h and then removed by replacing cultural medium.