Tgfbr2 sirna

The coding sequence of PART‐1 and TGFBR2 was subcloned into the pcDNA3.1 (+) vector (Invitrogen). PART‐1 siRNA, TGFBR2 siRNA, nonsense siRNA, miR‐590‐3p mimic, miR‐590‐3p inhibitor and respective controls were purchased from Guangzhou Ribobio company. Chondrocytes were transfected with the above plasmids, siRNAs or miRNAs using Lipofectamine 3000 (Invitrogen), and transfected chondrocytes were collected for in vitro assays at 24‐72 hours after transfection. In some experiments, the cells were stimulated with 5 ng/mL interleukin 1β (IL‐1β; Sigma‐Aldrich) for 24 hours.

Tgfbr2-siRNA and non-silencing siRNA were purchased from Ribo (RiboBio, Guangzhou, China). Active siRNA against Tgfbr2 used in the in vitro studies had sequences 5′-CCUGUUGCCUGUGUGACUU-3′ (sense) and 3′-GGACAACGGACACACUGAA-5′ (antisense). Tgfbr2-siRNA transfection of siRNA was performed using lipofectamine transfection regent 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s protocol. Control cells were mock transfected with lipofectamine transfection regent 2000 only. Chondrogenic ATDC5 cells were seeded in a 6-well plate until cells reached 50% confluence. The full culture medium was changed with serum-free and antibiotic-free medium at 20 min before transfection. The cells were incubated with transfection mixtures containing Tgfbr2-siRNA or non-silencing siRNA for 6 h, and then the mixtures were replaced with complete culture medium.