Journal: BMC Cancer

Article Title: Quercetin abrogates chemoresistance in melanoma cells by modulating ΔNp73

doi: 10.1186/1471-2407-10-282

Figure Lengend Snippet: Qct causes changes in p73 distribution . A) Non-quantitative PCR in DB-1 cells for TAp73 (Lane 1), ΔNp73 (Lane 2) and TAp73 positive control using SK Mel 28 cells (Lane 3) following treatment with TMZ. TAp73 was undetectable in the DB-1 cell line. B) Real time RT-PCR for ΔNp73 in DB-1 cell lines treated with TMZ 400 μM for 48 hrs followed Qct 75 μM for 24 hrs. Results are Mean ± SEM of triplicate experiments in DB-1 cell lines. C) p73 expression by western blot analysis in DB-1 and SK Mel 28 cell lines treated with vehicle (DMSO), TMZ 400 μM for 48 hrs followed by Qct 75 μM for 24 hrs. D) Immunocytochemical analysis of p73 localization in DB-1 cell lines treated with vehicle (DMSO), TMZ 400 μM for 48 hrs followed by Qct 75 μM for 24 hrs. Solid arrowheads indicate nuclear staining in cells without Qct treatment, while line-type arrowheads indicate cytoplasmic staining following Qct treatment.

Article Snippet: Antibody for GAPDH was purchased from Millipore-Chemicon (San Francisco, CA, USA). p73 antibody for western blotting and immunocytochemistry (ICC) was obtained from IMGENEX (San Diego, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Expressing, Western Blot, Staining

Journal: BMC Cancer

Article Title: Quercetin abrogates chemoresistance in melanoma cells by modulating ΔNp73

doi: 10.1186/1471-2407-10-282

Figure Lengend Snippet: Knockdown of p73 with siRNA restores PARP cleavage . Western blot analysis after transfection with p73 siRNA in DB-1 cell lines A) at basal level and B) after TMZ treatment and C) Quantification of p73 to GAPDH and Cleaved PARP relative to uncleaved PARP. D) Transient transfection of tyrosinase DNA in DB-1 cell lines and western blot analysis of p53 and 73 expression.

Article Snippet: Antibody for GAPDH was purchased from Millipore-Chemicon (San Francisco, CA, USA). p73 antibody for western blotting and immunocytochemistry (ICC) was obtained from IMGENEX (San Diego, CA, USA).

Techniques: Knockdown, Western Blot, Transfection, Expressing

Journal: PLoS ONE

Article Title: The p53 R172H Mutant Does Not Enhance Hepatocellular Carcinoma Development and Progression

doi: 10.1371/journal.pone.0123816

Figure Lengend Snippet: A) Immunoblot demonstrating knockdown of p53 in cell lines derived from p53 R172H HCCs as compared to pGIPZ empty vector control infections. B) Quantitative RT-PCR analysis of p53 family transcriptional targets in four mouse HCC cell lines with knockdown of p53 R172H . Ct values in each sample were normalized to β-Actin as an endogenous reference. Fold changes were calculated by normalizing all samples to the average Ct value of the cell line’s pGIPZ control. The Comparative Ct method was used for fold change calculation. C) Immunoblots demonstrating the expression of p63 and p73 isoforms in p53 fl/fl and p53 R172H HCC cell lines. Note the presence of multiple isoforms within the cell lines. GAPDH is used as a loading control. MW = molecular weight marker.

Article Snippet: Antibodies used were: p53 antibody (1:2000, Cell Signaling 1C12), p21 antibody (1:1000, Santa Cruz sc-6246), TAp63 antibody (1:500, Biolegend 618901), ΔNp63 antibody (1:500, Biolegend 619001), Total p63 antibody (1:500 Santa Cruz, ac-8431), TAp73 antibody (1:500 Novus 24737), Total p73 antibody (1:500 Imgenex).

Techniques: Western Blot, Derivative Assay, Plasmid Preparation, Quantitative RT-PCR, Expressing, Molecular Weight, Marker

Journal: Archives of ophthalmology (Chicago, Ill. : 1960)

Article Title: Transducible peptide therapy for uveal melanoma and retinoblastoma.

doi: 10.1001/archopht.120.10.1341

Figure Lengend Snippet: Figure 3. A, Cell death (measured by MTS assay [Promega Inc, Madison, Wis]) in U2OS cells treated with the indicated peptides and the caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone (Z-VAD). Error bars represent SEMs. B, TUNEL (terminal deoxynucleotidyl transferase–mediated fluorescein-dUTP nick-end labeling) staining of U2OS cells that were untreated (left) or treated for 8 hours with 200µM Tat-HDM2 (right). Dark brown nuclear staining indicates a positive TUNEL reaction (original magnification 40). Note the nuclear blebbing and DNA margination consistent with apoptosis. C, Western blot analysis using anti-p53 or anti-p73 antibodies and semiquantitative reverse transcription–polymerase chain reaction using primers for p21, Pig3, Bax, and GAPDH (control).

Article Snippet: Western blot analyses were performed using a polyclonal p53 antibody (sc-6243; Santa Cruz Biotechnology Inc, Santa Cruz, Calif), dilution 1:500, and a polyclonal p73 antibody (sc-7957; Santa Cruz Biotechnology), 1:500 dilution.

Techniques: MTS Assay, TUNEL Assay, End Labeling, Staining, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Control

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: ( A ) Analysis of TP73 , c-MYC and MYCN genes transcript levels in a panel of human neuroblastoma (NB) cell lines. Semi-quantitative reverse transcription-polymerase chain reaction (RT–PCR) was used to monitor the levels of p73α and p73β transcripts. TAp73 , Δex2-3p73 , ΔNp73 , c-MYC and MYCN transcript levels were evaluated using real-time quantitative RT–PCR (qRT–PCR). ( B ) Western blot analysis of p73, MYCN and p53 protein levels and status (isoform, mutation) in human NB cell lines. For p53, WT refers to the wild-type protein; dupl. ex 7–9 and Δ 373–393 correspond to mutations with duplication of exons 7–9 and deletion of aminoacids 327–393, respectively; the β isoform is a recently described p53 variant generated by alternative splicing in a cryptic site causing a premature stop codon in the C-terminal region. ( C ) Table summarizing the expression and isoform/mutational status of p73, MYCN and p53 protein in this series of human NB cell lines.

Article Snippet: The primary antibodies used, all mouse monoclonal, except for p73 (rabbit polyclonal), were: anti-p73 (a kind gift from Dr Mourad Kaghad, Sanofi-Aventis Recherche, Labège, France), anti-p73β (Ab-3, Oncogene Research), anti-MYCN (Ab-1, Oncogene Research), anti-p53 (DO-7, DAKO), all 4 at a dilution of 1/1000 and anti-β-actin (Chemicon) at a dilution of 1/5000 as a loading control.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Mutagenesis, Variant Assay, Generated, Alternative Splicing, Expressing

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: P73 inhibits MYCN expression at the protein and RNA levels in human neuroblastoma cells. ( A ) Effects of ectopic expression of p73 isoforms (top panel) on MYCN protein (top panel) and MYCN RNA (bottom panel) levels in IMR32 neuroblastoma (NB) cells. Protein levels were monitored by western blotting (top panel). As the first p73 antibody used preferentially recognizes the α isoform, western blot analysis was also performed with an anti-p73β antibody. Ectopic expression of TAp73β also resulted in a moderate up-regulation of p73α, likely as a consequence of transactivation of genes such as that encoding E2F1 that is known to activate TAp73α expression. MYCN transcript levels were evaluated in matching RNA samples using real-time quantitative RT–PCR (qRT–PCR, bottom panel). ( B ) Similar analyses were performed to evaluate the impact of small interfering RNA (siRNA)-mediated depletion of endogenous TAp73 on MYCN protein (top panel) and MYCN RNA (bottom panel) levels in Kelly and LAN-1 NB cells. Plotted qRT–PCR values are the means ± SEM of three replicates.

Article Snippet: The primary antibodies used, all mouse monoclonal, except for p73 (rabbit polyclonal), were: anti-p73 (a kind gift from Dr Mourad Kaghad, Sanofi-Aventis Recherche, Labège, France), anti-p73β (Ab-3, Oncogene Research), anti-MYCN (Ab-1, Oncogene Research), anti-p53 (DO-7, DAKO), all 4 at a dilution of 1/1000 and anti-β-actin (Chemicon) at a dilution of 1/5000 as a loading control.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Small Interfering RNA

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: P73α but not β isoforms lead to activation of MYCN gene transcription in human neuroblastoma cells. Effects of ectopic expression of p73 isoforms ( A and C ) or small interfering RNA (siRNA)-mediated depletion of endogenous TAp73 ( B and D ) on MYCN gene promoter activity in neuroblastoma cell lines. Values represent the mean Luciferase reporter gene activity ± SEM of three replicates.

Article Snippet: The primary antibodies used, all mouse monoclonal, except for p73 (rabbit polyclonal), were: anti-p73 (a kind gift from Dr Mourad Kaghad, Sanofi-Aventis Recherche, Labège, France), anti-p73β (Ab-3, Oncogene Research), anti-MYCN (Ab-1, Oncogene Research), anti-p53 (DO-7, DAKO), all 4 at a dilution of 1/1000 and anti-β-actin (Chemicon) at a dilution of 1/5000 as a loading control.

Techniques: Activation Assay, Expressing, Small Interfering RNA, Activity Assay, Luciferase

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: The p73 protein interacts with MYCN mRNA. Binding of the p73 protein to MYCN transcript was tested by RNA immunoprecipitation (RNA IP) in LAN-1 cells. RNA samples were purified from non-precipitated cellular lysates (input) or extracts precipitated with an antibody raised against p73 (anti-p73 Ab), or a pre-immune serum (Control Ab). Immunoprecipitated MYCN transcripts were detected using real-time quantitative reverse transcription-polymerase chain reaction (qRT–PCR). cDNA: RNA subjected to reverse transcription; RT(−): samples in which reverse transcriptase was omitted from the reaction, used as negative controls. Plotted qRT–PCR values (ratios of RNA IP/input) are the means ± SEM of three replicates.

Article Snippet: The primary antibodies used, all mouse monoclonal, except for p73 (rabbit polyclonal), were: anti-p73 (a kind gift from Dr Mourad Kaghad, Sanofi-Aventis Recherche, Labège, France), anti-p73β (Ab-3, Oncogene Research), anti-MYCN (Ab-1, Oncogene Research), anti-p53 (DO-7, DAKO), all 4 at a dilution of 1/1000 and anti-β-actin (Chemicon) at a dilution of 1/5000 as a loading control.

Techniques: Binding Assay, RNA Immunoprecipitation, Purification, Control, Immunoprecipitation, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: TAp73α inhibits MYCN mRNA stability in human neuroblastoma cells. Effects of ectopic expression of TAp73α ( A ) or small interfering RNA (siRNA)-mediated depletion of endogenous TAp73 ( B and C ) on MYCN mRNA stability in neuroblastoma (NB) cell lines. NB cells were transfected with the indicated control or p73 isoforms expression vectors (A) or siRNA (B, C), then treated with Actinomycin D (5 µM) to block transcription, and collected at different time points for RNA analysis. Logarithms of MYCN mRNA levels (determined by real-time quantitative reverse transcription-polymerase chain reaction—qRT–PCR—. Plotted values are the means ± standard error of the mean—SEM—of three replicates) are displayed as a function of time. The slope of the straight lines obtained in the different experimental conditions (top panels) allowed us to calculate the half-life of MYCN mRNA in response to p73 levels engineering in the three studied cell lines (bottom panels).

Article Snippet: The primary antibodies used, all mouse monoclonal, except for p73 (rabbit polyclonal), were: anti-p73 (a kind gift from Dr Mourad Kaghad, Sanofi-Aventis Recherche, Labège, France), anti-p73β (Ab-3, Oncogene Research), anti-MYCN (Ab-1, Oncogene Research), anti-p53 (DO-7, DAKO), all 4 at a dilution of 1/1000 and anti-β-actin (Chemicon) at a dilution of 1/5000 as a loading control.

Techniques: Expressing, Small Interfering RNA, Transfection, Control, Blocking Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: TAp73 exerts different effects on the regulation of MYCN and c-MYC expression. Effect of small interfering RNA (siRNA)-mediated depletion of endogenous TAp73 on MYCN and c-MYC mRNA ( A ) and c-MYC protein levels ( B ) in SH-SY5Y neuroblastoma (NB) cells. RNA levels were evaluated using real-time quantitative reverse transcription-polymerase chain reaction (qRT–PCR, A). Plotted qRT–PCR values are the means ± SEM of three replicates. Protein levels were monitored by western blotting (B). Transfection with TAp73 siRNA led to inhibition of both TA- and ΔN-isoforms of p73, as expected from the fact that TAp73 is known to positively regulate the expression of the ΔNp73 isoform. Control and TAp73 siRNA-transfected SH-SY5Y cells were treated with Actinomycin D (5 µM) to block transcription and collected at different time points for RNA analysis. Logarithms of MYCN and c-MYC mRNA levels (plotted values being the means ± SEM of three replicates) are displayed as a function of time. The slope of the straight lines obtained ( C ) allowed us to calculate the half-life of MYCN and c-MYC transcripts in response to p73 depletion in the SH SY5Y cells ( D ).

Article Snippet: The primary antibodies used, all mouse monoclonal, except for p73 (rabbit polyclonal), were: anti-p73 (a kind gift from Dr Mourad Kaghad, Sanofi-Aventis Recherche, Labège, France), anti-p73β (Ab-3, Oncogene Research), anti-MYCN (Ab-1, Oncogene Research), anti-p53 (DO-7, DAKO), all 4 at a dilution of 1/1000 and anti-β-actin (Chemicon) at a dilution of 1/5000 as a loading control.

Techniques: Expressing, Small Interfering RNA, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Inhibition, Control, Blocking Assay