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1.
<b>Figure 5.</b>

Figure 5.. From: Urokinase Expression by Tumor Suppressor Protein p53.

Identification of the p53 protein binding sequence on urokinase-type plasminogen activator (uPA) 3′ untranslated region (UTR) mRNA. (A) Deletion map indicating the p53 protein binding site on uPA mRNA. Pst1, restriction site of enzyme Pst I. (B) Recombinant p53 (rp53) protein expressed in E. coli was affinity purified. The rp53 was incubated with 32P-labeled uPA mRNA full-length coding region (CDR-FL; 1) or full-length 3′UTR (3′UTR-FL; 2) or 3′UTR deletion transcripts (3′UTR-DL; 3–9), and the RNA–protein complex was analyzed by gel mobility shift assay. Arrow indicates the uPA mRNA–p53 protein complex. The 32P-labeled uPA mRNA probe (Fp) is incubated with buffer alone. Data are representative of four independent analyses.

Praveenkumar Shetty, et al. Am J Respir Cell Mol Biol. 2008 September;39(3):364-372.
2.
<b>Figure 2.</b>

Figure 2.. From: Urokinase Expression by Tumor Suppressor Protein p53.

The protein p53 inhibits urokinase-type plasminogen activator (uPA) expression in Beas2B cells. (A) Cells lacking p53 (p53−/−) were transfected with vector cDNA (pcDNA3.1) or p53 cDNA (p53) in pcDNA3.1. Stably transfected cell lines expressing vector cDNA or p53 cDNA were analyzed for p53 expression by Western blotting as described in Figure 1A. (B) p53−/− Cells transfected with vector pcDNA3.1 or Wt. p53 cDNA in pcDNA3.1 were treated with phosphate-buffered saline (PBS) or amino-terminal fragment (ATF) (1 μg/ml). The conditioned media (CM) and cell lysates (CL) were subjected to Western blotting using an anti-uPA antibody. (C) RNA isolated from p53−/− cells transfected with vector cDNA or p53 cDNA treated with PBS or uPA (1 μg/ml) for 12 hours was subjected to Northern blotting using 32P-labeled uPA cDNA and 32P-labeled β-actin cDNA. (D) Beas2B cells transfected with control nonspecific silencing RNA (NspSiRNA) or p53 SiRNA were treated with PBS or 1 μg/ml of uPA for 24 hours in methionine-deficient medium containing 35S-methionine. The CM and CL were subjected to immunoprecipitation using anti-uPA and anti–β-actin antibodies as described in Figure 1B. (E) Beas2B cells transfected with control SiRNA or p53 SiRNA were treated with PBS or uPA (1 μg/ml) for 12 hours. The total RNA isolated from these cells was subjected to Northern blotting using 32P-labeled uPA cDNA and β-actin cDNA. The figure shown is a representation of four independent experiments.

Praveenkumar Shetty, et al. Am J Respir Cell Mol Biol. 2008 September;39(3):364-372.
3.
<b>Figure 1.</b>

Figure 1.. From: Urokinase Expression by Tumor Suppressor Protein p53.

Regulation of urokinase-type plasminogen activator (uPA) protein and mRNA expression by p53 in lung epithelial cells. (A) Expression of p53 protein by lung-derived cells. Lysates of Beas2B (p53+/+) and H1299, p53-deficient non–small cell carcinoma (p53−/−) cells treated with phosphate-buffered saline (PBS) or amino-terminal fragment (ATF) of uPA (50 ng/ml) for 24 hours were subjected to Western blotting using anti-p53 antibody. The same membrane was stripped and developed with an anti–β-actin antibody for equal loading. (B) Increased uPA expression in p53-deficient cells. Total proteins from conditioned media (CM) and cell lysates (CL) of p53+/+ and p53−/− cells treated with PBS or 1 μg/ml of uPA for 24 hours in the presence of 35S-labeled methionine were subjected to immunoprecipitation using anti-uPA antibody followed by β-actin antibody for equal loading. The immunoprecipitated proteins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and autoradiography. (C) RNA isolated from p53+/+ and p53−/− cells treated with PBS or uPA (1 μg/ml) for 12 hours was subjected to Northern blotting using 32P-labeled uPA cDNA and 32P-labeled β-actin cDNA. Experiments were repeated three times and representative data are shown.

Praveenkumar Shetty, et al. Am J Respir Cell Mol Biol. 2008 September;39(3):364-372.
4.
<b>Figure 6.</b>

Figure 6.. From: Urokinase Expression by Tumor Suppressor Protein p53.

Determining the destabilizing function of p53 binding uPA 3′untranslated region (UTR) mRNA sequence. (A) Structure of β-globin/uPA 3′UTR chimeric mRNA. The p53 protein binding 35-nucleotide (nt) sequence corresponding to nt 1585 to 1620 of uPA 3′UTR cDNA (C3) and a control sequence of similar size corresponding to the nonbinding region (C4) were inserted into the 3′UTR of β-globin cDNA. The chimeric β-globin/uPA 3′UTR cDNA was subcloned into pcDNA3.1. (B) Nucleotide sequence of p53 binding region nt 1585–1620 (C3) or nonbinding control sequence (C4). (C) Decay of β-globin/uPA 3′UTR chimeric mRNA. Beas2B cells were transfected with the chimeric β-globin/uPA 3′UTR gene containing the 35-nt (nt 1585 to 1620) p53 binding sequence (C3) or nonbinding control sequence (C4) in pcDNA3.1. Total RNA was isolated at different time intervals after treatment with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) as described above (Figure 3B) and analyzed the level of chimeric mRNA by Northern blotting using a 32P-labeled probe. (D) Effect of p53 binding uPA mRNA 3′UTR sequence on uPA expression. Stably transfected H1299 cells overexpressing p53 cDNA in pcDNA 3.1 were transfected with non-specific control (C4, NSP) or p53 binding (C3, p53) uPA mRNA 3′UTR sequences. The conditioned medium samples were analyzed for uPA expression by Western blotting using an anti-uPA antibody. Data are representative of at least four independent analyses.

Praveenkumar Shetty, et al. Am J Respir Cell Mol Biol. 2008 September;39(3):364-372.
5.
<b>Figure 3.</b>

Figure 3.. From: Urokinase Expression by Tumor Suppressor Protein p53.

Effect of p53 on urokinase-type plasminogen activator (uPA) mRNA expression. (A) Effect of p53 on uPA mRNA synthesis by lung epithelial cells. Naive H1299 cells (p53−/−) or H1299 cells transfected with vector cDNA (pcDNA3.1) or p53 cDNA were switched to serum-free media for 12 hours. Nuclei isolated from H1299 cells were subjected to the transcription reaction in the presence of 32P-labeled uridine triphosphate (UTP) at 30°C for 30 minutes. 32P-labeled nuclear RNA was hybridized with uPA cDNA immobilized on nitrocellulose membrane. β-Actin and plasmid University of California (PUC)18 cDNAs were used as positive and negative loading controls, respectively. The data represent the findings of three independent analyses. (B) Expression of wild-type (Wt) p53 in non–small cell lung carcinoma cells lacking p53 (p53−/−) destabilizes uPA mRNA. Naive p53−/− cells (p53−/−) or stable p53−/− cells overexpressing vector cDNA (pcDNA3.1) or p53 cDNA (p53 cDNA) were subjected to transcription chase experiment by treating with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) (20 μg/ml). The level of uPA mRNA was measured by Northern blotting at 0–12 hours. Experiments were repeated four times. (C) Inhibition of p53 expression enhances both basal and uPA-mediated stabilization of uPA mRNA. Beas2B cells transfected with control nonspecific (Nsp) silencing RNA (SiRNA) or p53 SiRNA were treated with PBS or uPA (1 μg/ml) for 12 hours. Ongoing transcription was inhibited by treatment with DRB and the decay of uPA mRNA was analyzed for 0–12 hours by Northern blotting. β-Actin mRNA expression is shown for equal loading. Densitometric scanning of individual bands is shown as a line graph, where mRNA level at 0 hours for each treatment is calculated as 100%. The experiments were repeated at least four times.

Praveenkumar Shetty, et al. Am J Respir Cell Mol Biol. 2008 September;39(3):364-372.
6.
<b>Figure 4.</b>

Figure 4.. From: Urokinase Expression by Tumor Suppressor Protein p53.

The protein p53 binds to urokinase-type plasminogen activator (uPA) 3′ untranslated region (UTR) mRNA. (A) The recombinant p53 protein (rp53) expressed in the prokaryotic system was incubated with the 32P-labeled uPA mRNA coding (CDR) or 3′UTR, and the mRNA–rp53 complexes were analyzed by gel mobility shift assay on 5% nondenaturing polyacrylamide gel electrophoresis. Lanes: uPA mRNA was incubated with rp53 protein (p53) or buffer alone (free probe [Fp]). Arrow indicates RNA–protein complex. (B) Beas2B cells treated with varying amounts (0–1 μg/ml) of uPA for 24 hours were lysed and the lysates were immunoprecipitated using anti-p53 monoclonal antibody (p53 mAb) or nonspecific mouse IgG (NSp mIgG). The p53 protein–associated uPA mRNA was detected by reverse transcriptase–polymerase chain reaction (PCR) using 32P-labeled 2′-deoxycytidine 5′-triphosphate (dCTP) and verified by nucleotide sequencing of the corresponding nonradioactive PCR product. Experiments were repeated at least three times. Competitive inhibition of p53–uPA mRNA interaction using unlabeled uPA mRNA sense and antisense transcripts to determine the specificity of p53-uPA mRNA 3′UTR interaction. p53 Protein was incubated with 32P-labeled probe uPA 3′UTR mRNA in the presence of 0- to 125-fold molar excess of unlabeled sense (C) or antisense (D) transcript. (E) Effects of polyribonucleotides, proteinase K, and sodium dodecyl sulfate (SDS) on uPA mRNA interaction with the binding protein. p53 Protein incubated with a 125-fold excess of unlabeled sense, poly(A), (C), (G), and (U); proteinase K (2.5 mg/ml); and 0.1% SDS for 30 minutes at 30°C. 32P-labeled uPA mRNA probe was added, and the mixture was digested with RNase T1 and analyzed by gel mobility shift assay. (F) rp53 Protein was incubated with 200-fold molar excess of p53 consense sequence (5′ prom) or 32P-labeled uPA 3′UTR mRNA predigested with RNase T1 before exposure to p53 (RNase T1). Experiments are representations of four independent analyses.

Praveenkumar Shetty, et al. Am J Respir Cell Mol Biol. 2008 September;39(3):364-372.

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