Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 1 Docking model of p28 and p63 DBD. Notes: (A) The structure for the p28-p63 DBD complex was obtained from the best docking model. Blue: overall ribbon diagram of the p63 DBD, green: S4, S6, S7, S9, and S10 loops. (B) Ribbon diagram of the p63 DBD (yellow) superimposed on the electrostatic potential plot. p28 (green) binds to the hydrophobic region. (C) Relationship of p28 (green) and S4, S6, S7, S9, and S10 (orange) of the p63 DBD. (D) Amino acid sequence alignment of the DBD of p53 and p63, data from Chen et al.41 p28 binding residues are indicated in red on the aa sequence of p53 DBD (top) and p63 DBD (bottom). The Pirh2 binding motif on p53 is indicated by the blue bar. S, sheet structure; orange box, DNA-binding sites; green box, zinc finger sites. Abbreviation: DBD, DNA-binding domain.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Sequencing, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 2 Tapping mode AFM images of protein monolayer. Notes: Tapping mode AFM images of: (A) p63 DBD monolayer immobilized on an amino-silane and glutaraldehyde functionalized glass slide; (B) p63 DBD monolayer after p28 deposition. All images were acquired in ambient air. Abbreviations: AFM, atomic force microscopy; DBD, DNA-binding domain.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Microscopy, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 3 Schematic representation of AFS analysis. Notes: Left panel shows illustration of AFS analysis showing the p28 bound to the tip via a flexible linker and p63, p73, or p63 DBD immobilized on glass substrates. Right panel shows schematic representation of a force–piezo displacement cycle, showing a typical unbinding event. Abbreviations: AFS, atomic force spectroscopy; DBD, DNA-binding domain.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Force Spectroscopy, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 6 Competitive immunoprecipitation assay for Cop1 and Pirh2. Notes: GST-p63 DBD and GST alone were immobilized on glutathione-Sepharose 4B beads and incubated in absence (−) or presence of p28 (+: 10, ++: 100 mole excess), followed by addition of MCF-7 lysates containing Cop1 and Pirh2. Samples were separated by SDS-PAGE and immunoblotted with either anti-Cop1 or anti- Pirh2 antibodies. Lysate: whole-cell lysates of MCF-7 used in assay stably expressed Cop1 and Pirh2. Numbers below Pirh2 bands are the relative percentage to the level of Pirh2 bound to p63 DBD in the absence of p28. Abbreviations: DBD, DNA-binding domain; GST, glutathione S-transferase; SDS- PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; IB, immunoblotting.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Immunoprecipitation, Incubation, SDS Page, Stable Transfection, Binding Assay, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 5 Interaction of p28 with p53, p63, and p73. Notes: GST pull-down assay showing complex formation between GST-p28 and p53, p63, and p73. p53, p63, or p73 were detected with antibodies to anti-p53, p63, or p73. Whole-cell lysates (Lysate) from each cell line served as a positive control. The numbers indicated below each band are expressed relative to each control (Lysate) expressed as 100%. Abbreviations: GST, glutathione S-transferase; wt, wild-type; mut, mutant.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Pull Down Assay, Positive Control, Control, Mutagenesis

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 7 Effect of p28 on p63, p73, and E3 ligases on p53wt, mut breast cancer cells. Notes: MCF-7, MDD2, MDA-MB-231, and MCF-10A cells were treated with 50 µM p28 for 24–72 hours, and protein levels determined by immunoblotting (A). The expression of each gene was determined by RT-PCR (B). The numbers indicated below each band represent the level of expression relative to the control (control expressed as 100%). Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; wt, wild-type; mut, mutant; dom, dominant; neg, negative.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription, Polymerase Chain Reaction, Mutagenesis

Journal: International Journal of Nanomedicine

Article Title: A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

doi: 10.2147/ijn.s58465

Figure Lengend Snippet: Figure 8 Effect of p28 on p63, p73, and E3 ligases on p53wt, mut melanoma cells. Notes: (A) Mel-29, Mel-23, and Mel-6 cells were treated with 50 µM p28 for 24–72 hours, and protein levels determined by immunoblotting. (B) The expression of each gene was determined by RT-PCR. The numbers below each band represent the level of expression relative to the control (control expressed as 100%). Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; wt, wild-type; mut, mutant.

Article Snippet: of p63 and its DBD Plasmid DNA carrying the human p63 gene was purchased from OriGene Technologies, Inc., (Rockville, MD, USA).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription, Polymerase Chain Reaction, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Phosphorylated TP63 Induces Transcription of RPN13, Leading to NOS2 Protein Degradation

doi: 10.1074/jbc.m110.158642

Figure Lengend Snippet: FIGURE 1. Cisplatin induces RPN13 expression at the RNA level. Wild- type Np63 and Np63-S385G cells were treated with control medium () or 10 g/ml cisplatin (CIS; ) for 12 h. GAPDH was used as a loading control. A, RT-PCR analysis for RPN13 transcription. B, qPCR. Values for RPN13 (in relative units (RU)) were normalized to values for GAPDH, and values obtained from the control untreated samples were designated as 1. Experiments were performed in triplicate. FIGURE 2. Schematic representation of the human RPN13 gene pro- moter. The sequence of the human 1500-bp RPN13 promoter was found on the UCSC Genome Bioinformatics human genome web site, and certain potential TF-responsive elements (RE) were defined using TFSEARCH soft- ware. TF sequences are shown in boldface. The TSS is shown as an uppercase letter. The following responsive elements were located in the RPN13 pro- moter: TP63 (1376/1354, 1231/1216, 1189/1167, and 500/ 481), NF-Y/DDIT3 (1267/1246, 93/71, and 65/37), NF-B (995/ 985 and 724/716), STAT (857/843), and GAS (gamma-activated site; 971/862).

Article Snippet: Antibodies—We used a rabbit anti- Np63 polyclonal antibody (Ab-1, EMD Chemicals); a rabbit anti-TP63 (TP73L) monoclonal antibody (clone Y289, NB110-57309) and a rabbit anti-DDIT3 (DNA damage-inducible transcript 3) polyclonal antibody (NB100-78344) (Novus Biologicals); mouse monoclonal antibodies against -actin (Sigma) and NF-YA (Rockland Immunochemicals); and rabbit polyclonal antibodies against DNA topoisomerase II (TOP2A, ab74715), RPN13 (ab91567), NF- B p65 subunit (ab32536), NF- B p50 subunit (ab7549), STAT3 (signal transduction activator of transcription 3; ab32500), and UCH37 (ab38528) (Abcam).

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Oral surgery, oral medicine, oral pathology and oral radiology

Article Title: Transforming Growth Factor-β1 activates ΔNp63/c-Myc to promote Oral Squamous cell carcinoma

doi: 10.1016/j.oooo.2016.05.018

Figure Lengend Snippet: (a,e,i,m) Proteins expression in normal oral gingival mucosa (red): PCNA, Ki67 and ΔNp63 revealed nuclear positivity staining in epithelia cells, while cyclin A expressed in both the epithelial cells and ECM. (b,c,f,g,j,k,n,o) Proteins expression in well- and moderately-differentiated OSCC (red): All of them showed a very similar pattern as they were in normal oral gingival mucosa. But their expression decreased in the cells toward the center of the tumor nest. (d,h,l,p) Proteins expression in poorly-differentiated OSCC (red): In the tumor nest, these proteins expressed only in a small portion of cancer cells (red), showing a significantly decreased expression fashion except for Ki67. (q–t) TGFβ1 expression in OSCC (red): TGFβ1 showed both nuclear and cytoplasmic staining in the epithelium as well as in the ECM (red). (at) E-cadherin expression in OSCC (green): E-cadherin expression was strictly within the epithelial membrane of the squamous layers, except in poorly-differentiated OSCC, it was reduced and discontinuous (green, d, h, l, p and t). In all slides, nuclei were stained with DAPI (blue). Scale Bar at 400μm (p) for low magnification; 100μm (p, inset) for higher magnification.

Article Snippet: To activate, we transfected cells with ΔNp63 and c-Myc, full-length ΔNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, MA) for 24 h by themselves followed by an additional 24 h with TGFβ1.

Techniques: Expressing, Staining, Membrane

Journal: Oral surgery, oral medicine, oral pathology and oral radiology

Article Title: Transforming Growth Factor-β1 activates ΔNp63/c-Myc to promote Oral Squamous cell carcinoma

doi: 10.1016/j.oooo.2016.05.018

Figure Lengend Snippet: (A) TGFβ1 regulates PCNA, Ki67, cyclin E2, ΔNp63 and E-cadherin expression in UMSCC38 cells

Article Snippet: To activate, we transfected cells with ΔNp63 and c-Myc, full-length ΔNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, MA) for 24 h by themselves followed by an additional 24 h with TGFβ1.

Techniques: Expressing

Journal: Oral surgery, oral medicine, oral pathology and oral radiology

Article Title: Transforming Growth Factor-β1 activates ΔNp63/c-Myc to promote Oral Squamous cell carcinoma

doi: 10.1016/j.oooo.2016.05.018

Figure Lengend Snippet: (A) TGFβ1 uses both Smad dependent and Smad independent pathways during UMSCC cell proliferation. pSmad2 was expressed in UMSCC38 cells but the expression was indifferent with TGFβ1 treatment of different dosages, however, its expression in the UMSCC11B was negligible in 2 and 5 ng/mL treatments and very low in 10 ng/mL treatment. Subsequently, western blot analysis of phosphorylated proteins was performed to determine the effects of TGFβ1 on Smad-independent pathways. pAKT showed no expression in UMSCC38 cells with any doses of TGFβ1 treatments. UMSCC11B showed similarly increased expression of pAKT with no difference in the TGFβ1 treatment conditions. ΔNp63 expression in both UMSCC38 and 11B were comparable and showed increased expression in a dose dependent manner in comparison to control (actin).

Article Snippet: To activate, we transfected cells with ΔNp63 and c-Myc, full-length ΔNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, MA) for 24 h by themselves followed by an additional 24 h with TGFβ1.

Techniques: Expressing, Western Blot, Comparison, Control

Journal: Oral surgery, oral medicine, oral pathology and oral radiology

Article Title: Transforming Growth Factor-β1 activates ΔNp63/c-Myc to promote Oral Squamous cell carcinoma

doi: 10.1016/j.oooo.2016.05.018

Figure Lengend Snippet: Based on our data, we hypothesized that in the initial stage, UMSCC cell proliferation (both in the primary, UMSCC38 and secondary tumor, UMSCC11B cell lines) is achieved by TGFβ1/Smads/ΔNp63/c-Myc pathway with higher proliferative attributes in UMSCC38. However, subsequently TGFβ1 switches it’s signaling via PI3K/AKT/ΔNp63 pathway at the inception stage for EMT/invasive, primarily in the secondary tumor, as seen in the UMSCC11B cell lines (that resulted from relapse and recurrence at the primary site) from. We further conclude that while TGFβ1 has distinct functions in cancer progression, its downstream signaling partners and transcription factors regulate ultimate cancer cell fate and achieve switch from tumor growth to invasion.

Article Snippet: To activate, we transfected cells with ΔNp63 and c-Myc, full-length ΔNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, MA) for 24 h by themselves followed by an additional 24 h with TGFβ1.

Techniques: