Journal: Cell Death & Disease

Article Title: The sodium/iodide symporter NIS is a transcriptional target of the p53-family members in liver cancer cells

doi: 10.1038/cddis.2013.302

Figure Lengend Snippet: NIS is a target of the p53-family members. ( a ) Real-time RT-PCR for NIS mRNA in primary hepatocytes (PHH), hepatocellular carcinoma (Hep3B, HuH7 and HepG2) and CCA (CCSW1 and CCLP1) cell lines. Data are expressed as mean values±S.E.M. of three independent experiments performed in duplicate. ( b ) Anti-NIS immunoblot of total protein extracts from the indicated cells. ( c ) Anti-NIS and anti-Na + -K + ATPase immunoblots after biotinylation and isolation of cell surface proteins in HepG2 cells. ( d ) Schematic representation of the human NIS proximal regulatory region (−1000/+5000 relative to NIS gene TSS sequence). In silico analysis was performed using the Genomatix package (cutoff score of 80%). Solid circles: p53-binding sites. Open circles: Sp1 consensus sites. Hatched rectangles: human NIS upstream enhancer (hNUE) and NIS proximal promoter regions. Solid rectangles: location of the PCR products obtained with the two ChIP primer pairs called A and B. ( e ) ChIP qPCR analysis of the interaction between NIS promoter and the p53-family proteins. Cross-linked chromatin from HepG2 and CCSW1 cells and PHHs (PHH) was immunoprecipitated with specific anti-p53 ( α TP53), anti-p63 ( α TP63), and anti-p73 α ( α TP73 α ) antibodies, or the relevant IgG controls, and then analyzed by real-time qPCR using the NIS promoter (Pr) A or B primer pairs. Control reactions using distant primers did not amplify anti-p53, anti-p63 or anti-p73 α ChIPped products (data not shown). Data are means±S.D. from three independent experiments

Article Snippet: The following antibodies were used: anti-p53 (DO1) mouse monoclonal, anti-p63 (4A4) rabbit polyclonal from Santa Cruz Biotechnology Inc. (San Diego, CA, USA); anti-p73 mouse monoclonal (IMG-259A) from Imgenex (San Diego, CA, USA), anti-cleaved PARP rabbit polyclonal from Cell Signaling Technology (Danvers, MA, USA); anti-NIS LP10 rabbit polyclonal., pcDNA-HA-P53, pcDNA-HA-P63 α , pcDNA-HA-P73 α expression vectors are transfected into cells using TransIT-TKO and TransIT-LT1 reagents from Mirus (Mirus Bio LLC). pcDNA-NIS expression vectors are described elsewhere.

Techniques: Quantitative RT-PCR, Western Blot, Isolation, Sequencing, In Silico, Binding Assay, ChIP-qPCR, Immunoprecipitation, Control

Journal: Cell Death & Disease

Article Title: The sodium/iodide symporter NIS is a transcriptional target of the p53-family members in liver cancer cells

doi: 10.1038/cddis.2013.302

Figure Lengend Snippet: p53-family members activate transcription of NIS promoter in liver cancer cell lines. ( a ) The indicated HCC (HepG2, HUH7 and Hep3B) and CCA (CCSW1 and CCLP1) cell lines were co-transfected with 300 ng of the −2000/+375 NIS promoter luciferase construct and either p53, p63 α or p73 α expression vectors. Luciferase activity was assayed 24 h after transfection and expressed as fold induction relative to the control after normalization for transfection efficiency using the dual-luciferase assay system. Histograms show the means of at least three experiments performed in quadruplicate. Bars indicate S.D. ( b ) NIS mRNAs levels were determined by real-time qPCR using NIS-specific primers in HepG2 and CCSW1 cells transfected with the −2000/+375 NIS promoter luciferase construct and the indicated expression vectors. Results are expressed as fold induction relative to the mock-transfected controls after normalization towards endogenous human β -actin mRNAs (beta actin FAM Probe Roche) mRNAs. Data are means±S.D. from at least three independent experiments performed in duplicate. ( c ) Anti-NIS immunoblot of HepG2 cells transfected with p53, p63 α or p73 α expression vectors (upper panel). α -Actin protein levels were used to normalize equal loadings from lysate samples. Histograms (lower panel) represent the means of three independent experiments. Bar, S.D. ( d ) HepG2 cells were co-transfected with the −2000/+375 NIS luciferase construct and 100 nM of p53, p63 and p73 small interfering RNAs or a control siRNA smart pool. Luciferase activity was determined as in ( a ). Data are means±S.D. of at three independent experiments performed in duplicate. P -values were determined using the two-tails Student's t- test. * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The following antibodies were used: anti-p53 (DO1) mouse monoclonal, anti-p63 (4A4) rabbit polyclonal from Santa Cruz Biotechnology Inc. (San Diego, CA, USA); anti-p73 mouse monoclonal (IMG-259A) from Imgenex (San Diego, CA, USA), anti-cleaved PARP rabbit polyclonal from Cell Signaling Technology (Danvers, MA, USA); anti-NIS LP10 rabbit polyclonal., pcDNA-HA-P53, pcDNA-HA-P63 α , pcDNA-HA-P73 α expression vectors are transfected into cells using TransIT-TKO and TransIT-LT1 reagents from Mirus (Mirus Bio LLC). pcDNA-NIS expression vectors are described elsewhere.

Techniques: Transfection, Luciferase, Construct, Expressing, Activity Assay, Control, Western Blot

Journal: Cell Death & Disease

Article Title: The sodium/iodide symporter NIS is a transcriptional target of the p53-family members in liver cancer cells

doi: 10.1038/cddis.2013.302

Figure Lengend Snippet: p53 and p73 are actively recruited to NIS proximal promoter in response to doxorubicin in liver cancer cells. Cross-linked chromatin from untreated and doxorubicin-treated (2 μ M; 24 h) HepG2 cells, CCSW1 cells and PHHs was immune-precipitated with the relevant control IgG or specific anti-p53 ( α TP53), anti-p63 ( α TP63) and anti-p73 α ( α TP73 α ) antibodies and analyzed by real-time PCR with the A and B pairs of NIS promoter selective primers. Control reactions using distant primers did not amplify anti-p53, anti-p63 or anti-p73 α ChIPed products (data not shown). Histograms represent the means of three independent experiments. Bar, S.D.

Article Snippet: The following antibodies were used: anti-p53 (DO1) mouse monoclonal, anti-p63 (4A4) rabbit polyclonal from Santa Cruz Biotechnology Inc. (San Diego, CA, USA); anti-p73 mouse monoclonal (IMG-259A) from Imgenex (San Diego, CA, USA), anti-cleaved PARP rabbit polyclonal from Cell Signaling Technology (Danvers, MA, USA); anti-NIS LP10 rabbit polyclonal., pcDNA-HA-P53, pcDNA-HA-P63 α , pcDNA-HA-P73 α expression vectors are transfected into cells using TransIT-TKO and TransIT-LT1 reagents from Mirus (Mirus Bio LLC). pcDNA-NIS expression vectors are described elsewhere.

Techniques: Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: TAp73 Induction by Nitric Oxide

doi: 10.1074/jbc.M110.184879

Figure Lengend Snippet: Mitochondrial membrane potential is compromised by knocking down TAp73. A, cells were transfected with 500 nm concentrations of a TAp73 siRNA or a siRNA control (Ctrl). Mitochondrial membrane potential ΔΨm was measured by flow cytometry 48 h later using the JC-1 probe. Some samples were treated by 0.5 mm DETA-NO or 0.1 mm H2O2 during the last 24 h or the last 6 h, respectively. The uncoupling agent carbonyl cyanide p-chlorophenylhydrazone (CCCP) at 50 μm was used as a positive control. Flow cytometry diagrams show the green fluorescent signal of JC-1 monomers in apoptotic cells on the x axis versus the red fluorescent signal of JC-1 oligomers in intact cells on the y axis. B, JC-1 green (gray bars) and red (black bars) fluorescence is shown. siCtrl and siTAp73 refer to a siRNA control and a siRNA against TAp73 mRNA, respectively. Results are the mean ± S.D. of three determinations. C, red-to-green fluorescence ratio was determined from values shown in B. This ratio is proportional to ΔΨm. D, cells were either not transfected (white bars) or transfected with a p53 (gray bars) or a p73 (black bars) siRNA. One day later they were treated with 6 μg/ml paraquat or 0.1 mm H2O2 as indicated. ROS production was analyzed after 24 h by flow cytometry using the H2DCFDA fluorogen. The results shown are the mean ± S.E. of three determinations. p < 0.05 (*) and p < 0.01 (**), compared with untreated and non-transfected cells.

Article Snippet: Primary antibodies to p73 (IMG-246, Imgenex, and A300-126A, Bethyl Laboratories), Chk1, NQO1, and Crk-L (sc-8408, sc-32793, and sc-319 respectively, Santa Cruz Biotechnology), phospho-Chk1 and phospho-Chk2 (2348 and 2661 respectively, Cell Signaling), β-actin and α-tubulin (A 5316 and T 9026, Sigma) were used for immunoblotting.

Techniques: Membrane, Transfection, Control, Flow Cytometry, Positive Control, Fluorescence

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Eighty-six genes (brown) were down regulated in the absence of all three p53 family members. A number of genes were down regulated in the absence of each p53 family member individually; 109 in p53 deficient cells (red), 148 in p63 deficient cells (yellow), and 131 in p73 deficient cells (blue). Several genes were down regulated in the absence of two family members; 47 in the absence of p53 and p63 (orange), 58 in the absence of p63 and p73 (green), and 41 in the absence of p53 and p73 (purple).

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques:

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Each row represents the specified gene. Each column represents the expression level of a specified knock out MEF line relative to the expression level of wild-type MEFs after DNA damage. The red color indicates upregulation, the green color indicates down regulation, while black indicates no significant change of the indicated gene expression. Clustering based on Euclidean distance indicates that p63- and p73- deficient E1A MEFs are more similar to each other than to p53−/− E1A MEFs. Genes of interest are listed in boxes and are associated with their corresponding location on the heatmap.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Expressing, Knock-Out, Gene Expression

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: p53 family response elements assayed by ChIP.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques:

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Real time PCR analysis of E1A MEFs of the following genotypes (wild-type, p53−/− , p63−/−, p73−/− and p63−/−;p73−/− ) after treatment with (A) doxorubicin (0.34 µM) for 12 hours or (B) γ radiation (12 hours). The Y-axis shows the fold induction. Bars represent 3 MEF lines for each genotype, each performed in triplicate. Data represent the mean ± SEM. The asterisk denotes statistical significance compared to wild-type, p<0.001.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: (A) Western blot analysis for Rad51 using whole cell lysates from wild-type and p63−/−;p73−/− MEFs treated with 0 Gy or 10 min (m), 30 m, 1 hour (h), 2 h and 4 h after 5 Gy of gamma irradiation. Actin was used as a control for equal loading. (B–I) Immunohistochemistry (IHC) of normal mammary tissue or mammary adenocarcinomas from p63+/−;p73+/− mice using antibodies as follows: (B) normal mammary tissue from p63+/−;p73+/− mouse using Rad51 antibody, (C) mammary adenocarcinoma from p63+/−;p73+/− mouse using Rad51 antibody, (D) normal mammary tissue from p63+/−;p73+/− mouse using BRCA2 antibody, (E) mammary adenocarcinoma from p63+/−;p73+/− mouse using BRCA2 antibody, (F) normal mammary tissue from p63+/−;p73+/− mouse using p63 antibody, (G) mammary adenocarcinoma from p63+/−;p73+/− mouse using p63 antibody, (H) normal mammary tissue from p63+/−;p73+/− mouse using p73 antibody, (I) mammary adenocarcinoma from p63+/−;p73+/− mouse using p73 antibody.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Western Blot, Irradiation, Control, Immunohistochemistry

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) analysis using wild-type E1A MEFs (WT) and E1A MEFs deficient for the p53 family members ( p53−/− , p63−/− and p73−/− ) before (U) and after treatment with doxorubicin (D) for 12 hours. Antibodies used to immunoprecipitate protein-DNA complexes in each cell line are shown in various colors: p53 (red), p63 (blue), and p73 (green). Total input chromatin is shown for each sample (input). Each ChIP was performed using 3 independent MEF lines in triplicate.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Chromatin Immunoprecipitation

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Bar graphs showing fold induction for each luciferase reporter gene in (A–D) p63−/−; p73−/− or (E,F) p53−/−;p73−/− primary MEFs. Reporter genes used are as follows: (A,E) pGL3-Rad51-1 containing the binding elements in intron 1, (B) pGL3-Rad51-2 containing the binding elements in intron 2, (C,F) pGL3-BRCA2 containing the binding element in intron 2, and (D) pGL3-mre-11 containing the binding element in intron 1. Pluses above each bar graph indicate which isoforms of p63 or p73 were transfected in cells with the firefly-luciferase reporter genes. Renilla-luciferase was used as a control for transfection efficiency, and pPERP-luc was used as a positive control. Each experiment was performed 6 times using 3 independent MEF lines. Data are represented as the mean ± SEM.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Luciferase, Binding Assay, Transfection, Control, Positive Control