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Figure 6 | Journal of Hematology & Oncology

Figure 6

From: Oroxylin A promotes PTEN-mediated negative regulation of MDM2 transcription via SIRT3-mediated deacetylation to stabilize p53 and inhibit glycolysis in wt-p53 cancer cells

Figure 6

Oroxylin A inhibited transcription of MDM2 by promoting SIRT3-regulated lipid phosphatase activity of PTEN. (A) Cells were treated with oroxylin A (OA) for 48 h. Nuclei were isolated and PTEN was immunoprecipitated using anti-FL SIRT3 antibody. Western blot assays were performed for PTEN and FL SIRT3. (B) Nuclei were isolated and acetylated PTEN was immunoprecipitated using anti-PTEN antibody. Western blot assays were performed for acetylated-lysine and PTEN. (C) Cells were co-transfected with MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and siRNA targeting SIRT3, then incubated with OA for 48 h. Luciferase activity was measured. (D, E) Cells were transfected with SIRT3 cDNA or treated with OA firstly. Then both were treated with NAM for 48 h. (D) Nuclei were isolated and Western blot assays were performed for MDM2, acetylated PTEN, and FL SIRT3. (E) Before treatments, MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) was co-transfected into cells. Luciferase activity was measured. (F) Cells were treated with sodium orthovanadate (SO) for 48 h. The mRNA expression of MDM2 was detected. (G) Cells were transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc or pGL3Basic-Mdm-T1-luc), and then treated with SO for 48 h. Luciferase activity was measured. (H) Cells were treated as that in (D). Lipid phosphatase activity of PTEN was assayed. (I, J, K) H1299 cells were treated with OA in/without the presence of tenovin-1for 48 h. (I) Nuclei were isolated and Western blot assays were performed for MDM2 and acetylated PTEN. (J) Before treatment, cells were transfected with MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc). Luciferase activity was measured. (K) Lipid phosphatase activity of PTEN was assayed. (L) Cells were transfected with siRNA targeting SIRT3 and incubated with OA for 48 h. Lipid phosphatase activity of PTEN was assayed. All the Western blot bands were quantified. Bars, SD; *p < 0.05 or **p < 0.01 versus non-treated control.

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