Figure 5
Oroxylin A inhibits the transcription of MDM2 via PTEN. (A) MCF-7 and HCT116 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was normalized to Renilla activity and expressed as luciferase/Renilla relative units. (B) H1299 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was measured. (C) Wt-PTEN plasmids were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into PC3M cells. Luciferase activity was measured. (D) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Luciferase activity was measured. (E) Cells were treated with oroxylin A for 48 h. Western blot assays were performed for PTEN. (F) Nucleus and cytosolic fractions were isolated after treatment and subjected to Western blot analysis for PTEN. (G) Immunofluorescence experiment performed in MCF-7 and HCT116 cells upon oroxylin A treatment using antibodies specific to PTEN and DAPI. (H) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Cells were then treated with 200 μΜ oroxylin A for 48 h. Luciferase activity was measured. (I) MCF-7 and HCT116 cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA, then incubated with 200 μM oroxylin A for 48 h. The mRNA expression of MDM2 was detected by Quantitative RT-PCR. (J) Cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA and incubated with 200 μM oroxylin A for 48 h. Western blot assays were performed for p53, MDM2, and PTEN. All the Western Blot bands were quantified. Bars, SD; *p < 0.05 or **p < 0.01 versus non-treated control.