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. 2010 Oct 6;20(1):80–89. doi: 10.1093/hmg/ddq434

Figure 2.

Figure 2.

PTEN ATP-binding mutations downregulate p53 in the nucleus. (A) Nuclear (N) and cytoplasmic (C) fractions in the absence of Tet were prepared and examined for protein levels of p53 and P-RB. The data represent the fold change of the normalized nuclear p53 (p53/PARP-1) and nuclear P-RB (P-PB/PARP-1) expressed in MCF-7 cells overexpressing the PTEN mutants, compared with those in MCF-7 cells overexpressing PTENWT, respectively. (B) p53 protein localization and intensity in MCF-7 cells determined by confocal microscope imaging and immunofluorescence. MCF-7 cells stably transfected with PTENWT or PTEN ATP-binding mutations were fixed and analyzed by immunofluorescence for endogenous p53 protein staining. p53 was detected with a polyclonal antibody followed by a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 568 (red). Nuclei were counterstained with DAPI (blue). (C) Quantification of immunofluorescence intensity for p53 in MCF-7 cells overexpressing PTENWT and mutants. The data represent the average of three independent measurements. (D) Luciferase reporter assay. PTENWT but not ATP-binding mutants enhances p53 transcriptional activity. MCF-7 cells stably expressing PTENWT or PTEN ATP-binding mutants, as well as the empty vector, were transfected with PG13-luc plasmid. Forty-eight hours later, luciferase activity was measured in cell lysates using equal amounts of protein. *P < 0.005 versus PTENWT. (E) Transient transfection of PTEN ATP-binding mutants into HEK293 cells decreases Bax mRNA transcription. Following transfection with the PTENs or control vector DNA, total RNA was extracted to measure Bax mRNA levels by qRT–PCR. *P < 0.05 versus PTENWT. (F) Transient transfection of PTEN ATP-binding mutants into HEK293 cells does not change p53 mRNA transcription. Total RNA from the above experiment was also used to measure p53 mRNA levels by qRT–PCR (P > 0.05). (G) Total protein lysates were extracted from MCF-7 cells stably expressing PTENWT, K62R, Y65C, K125E or the empty vector. Immunoblots were performed for p53, MDM2, P-MDM2, FLAG and tubulin.