Lipofectamine 3000 transfection reagent

Sangon Biotech (Shanghai, China) synthesizes the random sequences of TLR9 siRNA and the negative controls (Scra siRNA). When the cell confluency reached about 80%, TLR9 siRNA or Scra siRNA was transfected into 3D4/21 cells by using the Lipofectamine™ 3000 Transfection Reagent (Takara, Liaoning, China). Taken briefly, 3D4/21 cells were randomly divided into 4 groups: Scra group—cells were transfected with Scra siRNA (2 μg) for 48 h, which serves as a negative control; TLR9 siRNA group—cells were transfected with TLR9 siRNA (2 μg) for TLR9 silencing for 48 h; HD-13+Scra group—cells were infected with HD-13 and transfected with Scra siRNA (2 μg) for 48 h; and HD-13+TLR9 siRNA group—cells were infected with HD-13 and transfected with TLR9 siRNA for TLR9 silencing for 48 h. At 48 h posttreatment, 1 × 10⁶ 3D4/21 cells were applied to subsequent experiments in each group. Furthermore, in vivo, plasmids (1 × 10¹² PFU/mL) were injected into piglets via tail vein injection.

PcDNA3.1-ASPN plasmids and its negative control pcDNA3.1-NC (synthesized by Sangon Biotech, Shanghai, China) were transfected in 6-well plates at 500 ng each well. The miR-4303 mimics (synthesized by Sangon Biotech, Shanghai, China) and their corresponding controls were transfected at 100 nM each well. All the transfection was performed using Lipofectamine™ 3000 Transfection Reagent (Takara, Kusatsu, Japan). 48-h post-transfection, chondrocytes were treated with LPS for subsequent experiments.