Compound screening

In the primary screening, HEK-293T cells were plated onto a six-well plate at 1 × 10⁵ cells/well. After 24 h, the cells were transfected with pGL3-Oct4p or pEZX-MT01-Oct4 mRNA 3'UTR reporter using Entranster-D and maintained in DMEM. After 4 h, the transfected cells were replated onto 96-well plates at a density of 8 × 10³ cells/well. After 24 h, the cells were treated with compounds at final concentrations of 5 μg/mL in DMEM with 3% FBS for 24 h. The luciferase activity was assayed described in a previous study [24].

In the secondary screening, P19 cells were plated onto a 24-well plate at a density of 4 × 10⁴ cells/well. After 24 h, the cells were transfected with pGL3-Oct4p (pEZX-Oct4 mRNA 3'UTR) plasmids and pGL3-basic (pEZX-MT01) vector plasmids per well plus pCMV-β-galactosidase plasmids using Lipofectamine 2000. After 24 h, the cells were treated with the identified compounds at a final concentration of 5 μg/mL or DMSO (the negative control). After 44 h of incubation, the luciferase activity of the cells was assayed and normalized to β-galactosidase activity using the FLUOstar OPTIMA system (BMG Labtech, Offenburg, Baden-Wuerttemberg, Germany).