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.