2.9 ALP activity assay
Human or rat DP cells were seeded in 24-well plates and incubated with KY19382 for 48 hours. Next, the cells were washed twice with cold PBS and lysed with 55 µl 1x reporter lysis buffer (Promega) per well. Cell lysates were centrifuged at 10,000x g at 4°C for 30 minutes. Thirty microliters of each supernatant was incubated with 30 µl of p-nitrophenyl phosphate (pNPP) liquid substrate (Sigma Aldrich) for 1 hour. ALP activity was measured at 405 nm using the FLUOstar OPTIMA luminometer and normalized by the protein concentration from the Bradford assay (Bio-Rad Laboratories, Hercules, CA).
siRNA preparation and transfection
The cells were transfected with siRNA or the negative control (Bioneer, Daejeon, Korea) using Lipofectamine Plus (Invitrogen, Carlsbad, CA) in serum-free Opti-MEM (Gibco) according to the manufacturer’s instructions at a final concentration of 100 nM. The siRNA sequences targeting β-catenin were 5’-GAAACGGCTTTCAGTTGAG-3’ and 5’-AAACTACTGTGGACCACAAGC-3’ (Bioneer). At 12 hours after transfection with β-catenin siRNA, cells were treated with KY19382 for 48 hours. ALP assay and immunoblotting were performed to examine changes in ALP activity and to confirm the transfection efficiency of β-catenin siRNA.