Journal: Cancer research

Article Title: p53 is a Direct Transcriptional Target of MYCN in Neuroblastoma

doi: 10.1158/0008-5472.CAN-09-2598

Figure Lengend Snippet: MYCN amplification and expression is associated with p53 protein expression in 82 neuroblastomas

Article Snippet: Plasmids used were the p53-dependent pGL3-P2 reporter construct 23 , pCMV-β-galactosidase (Stratagene, La Jolla, CA), pRenilla-TK vector (Promega, Madison, WI) and p53 promoter reporter constructs pGL2-200bp and pGL2-356bp originally described by Wang & El-Deiry 24 and purchased from Addgene (Addgene, Cambridge, MA). pGL2-Δ200bp and pGL2-Δ356bp were generated by replacing the E-Box CATGTG with CTGCAG, and confirmed by sequencing.

Techniques: Amplification, Expressing

Journal: Cancer research

Article Title: p53 is a Direct Transcriptional Target of MYCN in Neuroblastoma

doi: 10.1158/0008-5472.CAN-09-2598

Figure Lengend Snippet: A) Western blot showing p53 and MYCN protein expression in neuroblastoma cell lines Con = control. B) Correlation between p53 protein and MYCN protein expression in neuroblastoma cell lines (Spearman Correlation, r=0.7924, p<0.005). p53 and MYCN expression was determined using densitometry, and normalized to actin. Knockdown of MYCN expression using siRNA (50nM) (M) in two MYCN amplified neuroblastoma cell lines, SMSKCNR and LAN5 compared with scrambled siRNA (SCR) led to decreased C) p53 protein expression and D) mRNA expression in both cell lines.

Article Snippet: Plasmids used were the p53-dependent pGL3-P2 reporter construct 23 , pCMV-β-galactosidase (Stratagene, La Jolla, CA), pRenilla-TK vector (Promega, Madison, WI) and p53 promoter reporter constructs pGL2-200bp and pGL2-356bp originally described by Wang & El-Deiry 24 and purchased from Addgene (Addgene, Cambridge, MA). pGL2-Δ200bp and pGL2-Δ356bp were generated by replacing the E-Box CATGTG with CTGCAG, and confirmed by sequencing.

Techniques: Western Blot, Expressing, Control, Knockdown, Amplification

Journal: Cancer research

Article Title: p53 is a Direct Transcriptional Target of MYCN in Neuroblastoma

doi: 10.1158/0008-5472.CAN-09-2598

Figure Lengend Snippet: A) p53 (Left) mRNA and (Right) protein expression in the presence of MYCN, (paired t-test, p<0.05). B) (Left) p53 DNA binding capacity (paired t-test, p<0.005) and (Right) p53-dependent reporter gene activity (paired t-test, p<0.05) in the presence of MYCN. The luciferase activity of the p53-dependent pGL3-P2 reporter construct was normalized to β-galactosidase activity of pCMV-β-galactosidase plasmid construct. C) Bar chart showing higher mRNA expression of p53 target genes MDM2 and PUMA in the presence of MYCN (paired t-test, p<0.05). D) (Left) Western blot and (Right) densitometry analysis normalized to actin showing higher protein expression of p53 target genes MDM2 and PUMA in the presence of MYCN (paired t-test, p<0.05).

Article Snippet: Plasmids used were the p53-dependent pGL3-P2 reporter construct 23 , pCMV-β-galactosidase (Stratagene, La Jolla, CA), pRenilla-TK vector (Promega, Madison, WI) and p53 promoter reporter constructs pGL2-200bp and pGL2-356bp originally described by Wang & El-Deiry 24 and purchased from Addgene (Addgene, Cambridge, MA). pGL2-Δ200bp and pGL2-Δ356bp were generated by replacing the E-Box CATGTG with CTGCAG, and confirmed by sequencing.

Techniques: Expressing, Binding Assay, Activity Assay, Luciferase, Construct, Plasmid Preparation, Western Blot

Journal: Cancer research

Article Title: p53 is a Direct Transcriptional Target of MYCN in Neuroblastoma

doi: 10.1158/0008-5472.CAN-09-2598

Figure Lengend Snippet: A) (Left) Western blot and (Right) representative graph showing the positive correlation between MYCN protein and p53 protein expression in Tet21N cells harvested after tetracycline removal from growth media (Spearman Correlation, r=0.7924, p<0.005). Con, cells cultured continuously in the presence of tetracycline. B) Western analysis showing p53 is functional in both Tet21N MYCN+ and MYCN− cells after DNA damage, leading to induction of target genes p21WAF1 and MDM2. Con, non-irradiated samples. C) qRT-PCR analysis of 7 p53 regulated genes identified using microarray analysis, upregulated in the presence of MYCN and showed a decrease in expression after p53 siRNA treatment (50nM) for 24 hours. D) MYCN amplified NGP cells treated with MYCN siRNA (40nM), p53 siRNA (50nM), p53 and MYCN siRNA (35nM of each siRNA) or SCR siRNA (70nM) for 48 hours prior to irradiation induced DNA damage. 24 hours post irradiation cells were harvested and analyzed for the expression of apoptosis mediators cleaved caspase 3 and PUMA (Left) and Capsase-3/7 activity (Right).

Article Snippet: Plasmids used were the p53-dependent pGL3-P2 reporter construct 23 , pCMV-β-galactosidase (Stratagene, La Jolla, CA), pRenilla-TK vector (Promega, Madison, WI) and p53 promoter reporter constructs pGL2-200bp and pGL2-356bp originally described by Wang & El-Deiry 24 and purchased from Addgene (Addgene, Cambridge, MA). pGL2-Δ200bp and pGL2-Δ356bp were generated by replacing the E-Box CATGTG with CTGCAG, and confirmed by sequencing.

Techniques: Western Blot, Expressing, Cell Culture, Functional Assay, Irradiation, Quantitative RT-PCR, Microarray, Amplification, Activity Assay

Journal: Cancer research

Article Title: p53 is a Direct Transcriptional Target of MYCN in Neuroblastoma

doi: 10.1158/0008-5472.CAN-09-2598

Figure Lengend Snippet: A) qRT-PCR analysis of p53 mRNA expression in Tet21N MYCN+ and MYCN− cells after treatment with 1μg/ml Actinomycin D, shows a decrease in p53 mRNA expression, suggesting that MYCN regulates p53 transcription. B) Western blot showing p53 and MYCN protein expression in (Top) Tet21N MYCN+ and (Bottom) MYCN− cells harvested after treatment with 25μM of Cycloheximide (CHX), demonstrates a decrease in p53 expression suggesting that p53 is not predominantly post-translationally stabilized. C) MYCN ChIP analysis of the p53 promoter in (Left) Tet21N MYCN+ cells, (second from left) Tet21N MYCN− cells and (second from right) MYCN amplified LAN5 cells. (Right) As a positive control direct binding of MYCN to the nucleolin promoter in Tet21N MYCN+ cells is shown. D) Relative luciferase activity of p53 promoter constructs (pGL2-200bp, pGL2-Δ200bp, pGL2-356bp, pGL2-Δ356bp) transfected into (Left) Tet21N MYCN+ and MYCN− cells, and (Right) co-transfected with pCMV14-MYCN expression plasmid into SHEP cells.

Article Snippet: Plasmids used were the p53-dependent pGL3-P2 reporter construct 23 , pCMV-β-galactosidase (Stratagene, La Jolla, CA), pRenilla-TK vector (Promega, Madison, WI) and p53 promoter reporter constructs pGL2-200bp and pGL2-356bp originally described by Wang & El-Deiry 24 and purchased from Addgene (Addgene, Cambridge, MA). pGL2-Δ200bp and pGL2-Δ356bp were generated by replacing the E-Box CATGTG with CTGCAG, and confirmed by sequencing.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Amplification, Positive Control, Binding Assay, Luciferase, Activity Assay, Construct, Transfection, Plasmid Preparation