Chondrocyte transfection with siRNAs
Chondrocytes (25000/cm2) were cultured in 24-well plates and transfected with Tmem251 specific or control siRNA when 60% confluency was reached, as described.(Lui et al., 2016) Briefly, cells were washed with PBS 1h prior to transfection with PBS and medium was changed to DMEM/F12 without any antibiotics. Chondrocytes were separately transfected with 40nM of negative control siRNA, two siRNAs against TMEM251 (s179664 or s179666) and GapdhsiRNA (Thermo Fisher Scientific) using Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s instructions. 16h post transfection, chondrocyte culture medium was replaced back to DMEM/F12 with GlutaMAX supplemented with 10% FBS, 50µg/ml ascorbic acid, 1% penicillin/streptomycin, and 50ng/ml fungizone.