Fig. 1

Screening of potential activators of Oct4. A The activities of the Oct4 promoter-driven firefly luciferase reporter. HEK-293 T cells were transfected with the pGL3-Oct4 and pGL3-basic reporter plasmid, plus with pCMV-β-galactosidase plasmids. 48 h later, the cells were lysated and the luciferase activities was detected. B The activities of the pEZX-MT01-Oct4 mRNA 3'UTR reporter plasmid. HEK-293 T cells were transfected with pEZX-MT01 and pEZX-MT01-Oct4 mRNA 3'UTR plasmids, plus with pCMV-β-galactosidase plasmids. 48 h later, the cells were lysated and the luciferase activities was detected. C Primary screening using Oct4 promoter-driven firefly luciferase reporter. HEK-293 T cells were transfected with the pGL3-Oct4p plasmid for 24 h and then treated with small-molecule compounds (5 μg/mL) for 24 h. DMSO was used as a negative control. Subsequently, the luciferase activity was measured. The results were expressed as the fold induction (over the activity of the negative control). D Secondary screening for compounds obtained from (C). P19 cells were co-transfected with pGL3-Oct4p or pGL3-basic vector and pCMV-β-galactosidase plasmid for 24 h and then treated with the identified compounds (5 μg/mL) for 24 h. DMSO was used as a negative control. Subsequently, the luciferase activity was measured and normalized to β-galactosidase activity. The results were expressed as the fold induction (over the activity of the negative control). E Primary screening using Oct4 mRNA 3'UTR reporter plasmid. HEK-293T cells were transfected with a pEZX-Oct4 mRNA 3'UTR plasmid for 24 h and then treated with 480 small-molecule compounds (5 μg/mL) for 24 h. DMSO was used as a negative control. Subsequently, the luciferase activity was measured. The results were expressed as the fold induction (over the activity of the negative control). F Secondary screening for compounds obtained from (E). P19 cells were co-transfected with pEZX-Oct4 mRNA 3'UTR or pEZX-MT01 vector and pCMV-β-galactosidase plasmid for 24 h and then treated with the identified compounds (5 μg/mL) for 24 h. DMSO was used as a negative control. Subsequently, the luciferase activity was measured and normalized to β-galactosidase activity. The results were expressed as the fold induction (over the activity of the negative control). *P < 0.05, **P < 0.01, ***P < 0.001, compared with the negative control