Knockdown of ALK2, ALK3, ID3 and SMAD4 in HESCs and HDSCs

RNA interference was enabled by the transfection of small interfering RNA (siRNA). ON-TARGET plus non-targeting control pool siRNA or an ON-TARGET plus SMART pool targeting ALK2, ALK3, ID3 and SMAD4 were purchased from Dharmacon Inc. A total of 2 × 10⁵ HESCs or primary HDSCs were simultaneously seeded with full culture media 1 day before transfection. Lipofectamine RNA iMAX (Life Technologies) was used to transfect control siRNA or siRNA against ALK2, ALK3, ID3 and SMAD4 into the cells at a dose of 25 nM in accordance with the manufacturer’s instructions. The cells were then cultured for 24 h at 37 °C in a CO2 incubator untill starvation (synchronization of all the cells to the same cell cycle phase and removal of various ligands in serum). RT‒qPCR or Western blot analysis was used to assess the knockdown effectiveness of each target.