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. 2001 Dec 15;29(24):5207–5215. doi: 10.1093/nar/29.24.5207

Figure 4.

Figure 4

TR sequences enhance the transcriptional activity of wild type p53. (A) H1299 cells were seeded (45 000 cells/well) in a 12 well plate and transiently transfected 16–24 h later with Waf-1-Luc or Mdm-2-Luc (0.2 µg/well), pBlueScript (white bars) or pBlueScript-TEL (black bars) (2.8 µg/well), and pCMV-p53 (1 ng/well when indicated). pCMVβ-GAL (0.2 µg/well) was included in each transfection as an internal control. Cells were assayed for luciferase and β-galactosidase activity 24–36 h after transfection. Relative luciferase activity was calculated by dividing the luciferase activity by the β-galactosidase activity of the same sample. Histograms show the mean of typical experiment performed in triplicate. Bars indicate the standard deviation of the mean. (B) TR sequences enhance the transcriptional activity of wild-type p53 in MCF-7 cells. MCF-7 cells were seeded (140 000 cells/well) in a 12 well plate and transiently transfected 16–24 h later with Waf-1-Luc in the presence of 2.8 µg/well pBlueScript (white bars) or pBlueScript-TEL (black bars). Luciferase and β-galactosidase assays were carried out as described above. (C) E6 inhibition of wild type p53 induced Waf-1-Luc in MCF-7 cells. Indicated amounts of pSG5-E6 were co-transfected with Waf-1-Luc (0.2 µg/well). pCMVβ-GAL (0.2 µg) was included in each transfection as an internal control. Luciferase and β-galactosidase assays were carried out as described before. (D) TR sequences enhance the transcriptional activity of wild type p53 in normal human fibroblasts. WI-38 cells, (passage 21–23) were seeded (100 000 cells/well) in a 6 well plate and transiently transfected 16–24 h later with Mdm-2-Luc or Waf-1-Luc (0.5 µg/well), 4.5 µg/well of pBlueScript (white bars) or pBlueScript-TEL (black bars). pCMVβ-GAL (0.2 µg/well) was included in each transfection as an internal control. Luciferase and β-galactosidase assays were carried out as described above.