Figure 7.
Figure 7. Regulation of p53 and Mdm2 mRNA expression by STAT1α and p53/STAT1α proapoptotic complex. (A) Determination by RQ-PCR of the relative expression levels of p53 and Mdm2 in EGFP-positive and -negative cells from CKR516-STAT1β–transfected cells. Induced (+) and noninduced (–) cells were treated with doxycycline (Doxy), IFN-γ (250 U/mL), or 15 μM fludarabine for 16 hours. Relative mRNA expression levels were assessed by RQ-PCR using Abl1 mRNA and a pool of RNA for normalization. Results are representative of at least 2 independent experiments. (B) p53/Mdm2 ratios of mRNA measured by RQ-PCR in EGFP-positive and -negative cells from CKR516-STAT1β–transfected cells. p53/Mdm2 mRNA ratios were calculated from the experiment described for panel A. Results are representative of at least 2 independent experiments. (C) Detection of p53 by immunofluorescence staining in EGFP-positive cells from CKR516-STAT1β– and STAT1α–transfected cells. Cells were transfected with CKR516-STAT1α (top panels) or CKR516-STAT1β (bottom panels), pretreated with doxycycline for 24 hours (inducibility reached 70%), and treated with fludarabine for 24 hours. Cytospins were stained with AlexaFluor 594 streptavidin for the detection of the secondary biotinylated immune serum against the p53 antibody (red) and with DAPI (blue) for nucleus staining. Induced cells are recognized by the presence of EGFP (green) in the cytoplasm. In CKR516-STAT1α EGFP-positive cells, high levels of p53 (red) were localized in the nucleus when compared with EGFP-negative cells. Conversely, in CKR516-STAT1β EGFP-positive cells, no p53 was visible in the nucleus, similar to noninduced cells. (D) Physical association of STAT1α to p53 bound to the p53 DNA-specific probe. Nuclear extracts were obtained from nontransfected (PRI) cells treated (+) or not (–) with fludarabine. Extracts were incubated with biotinylated p53 DNA–specific probe. Complexes were captured on agarose–streptavidin, then washed and separated by electrophoresis. Western blot analysis was performed to detect p53 and STAT1. In the absence of fludarabine (lane 1), p53 binds to the probe. In the presence of fludarabine (lane 2), p53 binding is increased. In both cases, STAT1 is detected. In lane 3, an excess of nonbiotinylated p53 probe was added before the biotinylated p53 probe to detect nonspecific binding.

Regulation of p53 and Mdm2 mRNA expression by STAT1α and p53/STAT1α proapoptotic complex. (A) Determination by RQ-PCR of the relative expression levels of p53 and Mdm2 in EGFP-positive and -negative cells from CKR516-STAT1β–transfected cells. Induced (+) and noninduced (–) cells were treated with doxycycline (Doxy), IFN-γ (250 U/mL), or 15 μM fludarabine for 16 hours. Relative mRNA expression levels were assessed by RQ-PCR using Abl1 mRNA and a pool of RNA for normalization. Results are representative of at least 2 independent experiments. (B) p53/Mdm2 ratios of mRNA measured by RQ-PCR in EGFP-positive and -negative cells from CKR516-STAT1β–transfected cells. p53/Mdm2 mRNA ratios were calculated from the experiment described for panel A. Results are representative of at least 2 independent experiments. (C) Detection of p53 by immunofluorescence staining in EGFP-positive cells from CKR516-STAT1β– and STAT1α–transfected cells. Cells were transfected with CKR516-STAT1α (top panels) or CKR516-STAT1β (bottom panels), pretreated with doxycycline for 24 hours (inducibility reached 70%), and treated with fludarabine for 24 hours. Cytospins were stained with AlexaFluor 594 streptavidin for the detection of the secondary biotinylated immune serum against the p53 antibody (red) and with DAPI (blue) for nucleus staining. Induced cells are recognized by the presence of EGFP (green) in the cytoplasm. In CKR516-STAT1α EGFP-positive cells, high levels of p53 (red) were localized in the nucleus when compared with EGFP-negative cells. Conversely, in CKR516-STAT1β EGFP-positive cells, no p53 was visible in the nucleus, similar to noninduced cells. (D) Physical association of STAT1α to p53 bound to the p53 DNA-specific probe. Nuclear extracts were obtained from nontransfected (PRI) cells treated (+) or not (–) with fludarabine. Extracts were incubated with biotinylated p53 DNA–specific probe. Complexes were captured on agarose–streptavidin, then washed and separated by electrophoresis. Western blot analysis was performed to detect p53 and STAT1. In the absence of fludarabine (lane 1), p53 binds to the probe. In the presence of fludarabine (lane 2), p53 binding is increased. In both cases, STAT1 is detected. In lane 3, an excess of nonbiotinylated p53 probe was added before the biotinylated p53 probe to detect nonspecific binding.

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