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: Fig. 1. Different grades of oral squamous cell carcinoma (OSCC) express proliferating cell nuclear antigen (PCNA), Ki67, cyclin A, DNp63, and transforming growth factor-b1 (TGF-b1) in comparison to E-cadherin. (A, E, I, M): Proteins expression in normal oral gingival mucosa (red): PCNA, Ki67, and DNp63 revealed nuclear positivity staining in epithelia cells, and cyclin A expressed in both the epithelial cells and extracellular matrix (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

Article Snippet: To activate, we transfected cells with DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques: Comparison, Expressing, Staining

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: Fig. 2. Transforming growth factor-b1 (TGF-b1) regulates proliferating cell nuclear antigen (PCNA), Ki67, cyclin E2, DNp63, and E-cadherin expression in UMSCC38 cells (A). Column (a): PCNA, Ki67, cyclin E2, and DNp63 revealed negative staining in untreated UMSCC38 cells. Column (b): All the cells grown in 10% FBS/DMEM (positive control) were nuclear positive stained by PCNA, Ki67, cyclin E2, or DNp63. Columns (c-h): PCNA, Ki67, cyclin E2, and DNp63 expression in TGF-b1-treated cells. The number of nuclei positively stained cells increased in a time and dose-dependent manner, compared with untreated groups. Furthermore, as indicated by E-cadherin staining along the cell membrane (red), the cobblestone morphology was preserved in all treated UMSCC38 cells. TGF-b1 regulates PCNA, Ki67, cyclin E2, DNp63, and E-cadherin expression in UMSCC11B cells. Columns (a-b): PCNA, Ki67, cyclin E2, and DNp63 expressed very similarly as that in UMSCC38 untreated and positive control cells. Columns (c-e): 24 h TGFb1 treatment increased the number and intensity of PCNA, Ki67, cyclin E2, and DNp63 (green) expression in UMSCC11B cells (B) compared to untreated control groups. Also, the expression pattern of the epithelial marker, E- cadherin (red), was concentrated on the cell membrane, forming a continuous membranous, similar to that in the untreated cells. Columns (f-h): When treated with TGF-b1 for 48 hours, the number of positive cells decreased significantly, and the membranous expression of E-cadherin (red) was reduced and interrupted thereafter. (C): Percentage of immunofluorescence positively stained UMSCC38 and UMSCC11B cells. For both UMSCC38 and UMSCC11B cells, only very limited untreated cells express PCNA, Ki67, cyclin E2, or DNp63. (a-d): Percentage of positively stained UMSCC38 cells for PCNA (a), DNp63 (b), Ki67 (c), and cyclin E2 (d). Over 90% of UMSCC38 cells grown in 10% fetal bovine serum (FBS) were positively stained for these proteins. When cells were treated with TGF-b1 for 24 hours, the percentages increased as the dilutions of TGF-b1 were raised. At 48 hours, the percentages raised to very similar level with that of positive control (10% FBS) groups. (e-h): Percentage of positively stained UMSCC11B cells for PCNA (e), DNp63 (f), Ki67 (g), and cyclin E2 (h). Almost 95% of cells grown in 10% FBS were positive stained for PCNA, DNp63, Ki67, and cyclin E2. When cells were treated with TGF-b1 for 24 hours, the percentages increased obviously as the dilutions of TGF-b1 were raised. But at 48 hours, the percentages of positively stained cells reduced obviously, very similar to that of untreated cells.

Article Snippet: To activate, we transfected cells with DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques: Expressing, Negative Staining, Positive Control, Staining, Membrane, Control, Marker

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: Fig. 4. Pathways used by transforming growth factor-b1 (TGF-b1) during UMSCC cell proliferation. A, TGF-b1 uses both Smad- dependent and Smad-independent pathways during UMSCC cell proliferation. pSmad2 was expressed in UMSCC38 cells, but the expression was indifferent with TGF-b1 treatment of different dosages; however, its expression in the UMSCC11B was negligible in 2 and 5 ng/mL treatments and very low in the 10 ng/mL treatment. Subsequently, Western blot analysis of phosphorylated proteins was performed to determine the effects of TGF-b1 on Smad-independent pathways. pAKT showed no expression in UMSCC38 cells with any doses of TGF-b1 treatments. UMSCC11B showed similarly increased expression of pAKT with no difference in the TGF-b1 treatment conditions. DNp63 expression in both UMSCC38 and UMSCC11B were comparable and showed increased expression in a dose-dependent manner in comparison to control (actin). B, All three doses of TGF-b1 treatment groups differed significantly among themselves and between two cell lines (P .05, as indicated by *) from the untreated control cells (0.2% fetal bovine serum [FBS]) (P .005, as indicated by **). Results from the blots (see Figure 4A) and the intensity of the bands were measured using the Carestream Molecular Imaging Software version 5.3.1 (Rochester, NY). To perform a t test analysis of mean intensity measurements, a region of interest analysis was done from the data to Microsoft Excel software. Data points for all samples are paired by spatial arrangement on gel and compared pairwise to minimize the impact of subtle background artifacts on image analysis. C, Regulation of cyclin mRNA by TGF-b1. Cyclin D, E, A, and B mRNA expressions were determined by RT- PCR in TGF-b1 (5 ng/mL)etreated UMSCC38 and UMSCC11B cells every 12 hours for 48 hours. TGF-b1etreated cells had higher cyclins (D, E, A, and B) mRNA expression levels compared with the untreated control (0.2% FBS). Compared with UMSCC11B, TGF-b1 had chronologically higher cyclins (D, E, A, and B) mRNA levels in UMSCC38 cells in a time-dependent fashion till 48 hours. However, the levels sharply dropped after 24 hours in the UMSCC11B cell lines and continued to decline until 48 hours. D, Regulation of cyclin D mRNA by TGF-b1 in UMSCC cells: The levels of cyclin D mRNAs in response to TGF- b1, when DNp63 and c-Myc are either activated (by full-length cDNA) or repressed (by pRetrosuper-shRNA), was measured. To induce cyclins, TGF-b1 functions on both DNp63 and c-Myc, as activation and repression of DNp63 and c-Myc significantly affect cyclin D mRNA levels. The change in mRNA levels was determined by comparison to untreated control (UnTr, 0.2% FBS) and plotted as fold change/s (mean SD; n ¼ 3; *P < .05 compared with controls; **P < .005 compared with TGF-b1 treatments.

Article Snippet: To activate, we transfected cells with DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques: Expressing, Western Blot, Comparison, Control, Imaging, Software, Reverse Transcription Polymerase Chain Reaction, shRNA, Activation Assay

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: Fig. 4. (continued) E, TGF-b1 stimulates DNp63 gene activity in UMSCC cells: TGF-b1 increased DNp63 promoter activity chronologically, starting at 6 hours (five- and sixfold in UMSCC38 and UMSCC11B, respectively) and reaching the peak at 24 hours (103-fold and 98-fold in UMSCC38 and UMSCC11B, respectively). However, when both Smad-dependent and Smad- independent pathways were blocked, DNp63 promoter activity was repressed in both UMSCC38 and UMSCC11B cell lines. The results are shown as a mean SD obtained from three independent chromatin preparations (P .05 as indicated by * and P .005 as indicated by **).

Article Snippet: To activate, we transfected cells with DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques: Activity Assay

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: Fig. 6. Schematic diagram of the proposed mechanism of oral squamous cell carcinoma (OSCC) proliferation and invasion. On the basis of our data, we hypothesized that in the initial stage, UMSCC cell proliferation (both in the primary tumor, UMSCC38, and the secondary tumors, UMSCC11B cell lines) is achieved by TGF-b1/Smads/DNp63/c-Myc pathway with higher proliferative attributes in UMSCC38. However, subsequently, TGF-b1 switches its signaling via the PI3K/ AKT/DNp63 pathway at the inception stage for EMT/inva- sive, primarily in the secondary tumor, as seen in the UMSCC11B cell lines (that resulted from relapse and recur- rence at the primary site). We further conclude that although TGF-b1 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 DNp63 and c-Myc, full-length DNp63 (pCMV-Entry-p63; Origene, MD) and c-Myc cDNAs (pcDNA3.3 c-Myc; Addgene, Cambridge, MA) for 24 hours by themselves followed by an additional 24 hours with TGF-b1.

Techniques:

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: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: Col10a1-TAp63γ expression plasmid and establishment of stable TAp63γ expressing ATDC5 cell lines. ( A ) pCMV- TAp63γ and its derivative pCol10a1-TAp63γ expression plasmids are shown. Enzyme restriction sites for cloning are also shown. ( B ) Enzyme digestion confirmed the integration of TAp63γ in designated stable cell lines. ( C ) PCR using p63 and Taq sequence-specific primers confirmed the integration of TAp63γ into the stable cell lines: pCMV- TAp63γ and Col10a1-TAp63γ . ( D ) Western blot results further confirmed expression of TAp63γ in designated stable lines.

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Expressing, Plasmid Preparation, Cloning, Stable Transfection, Sequencing, Western Blot

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: TAp63γ upregulates Col10a1 expression in ATDC5 cells. ( A ) ATD5C cells were harvested for RNA isolation on day zero or after 4 days in culture. Col10a1 showed significant elevation in Col10a1-TAp63γ stable cell lines compared with the controls after 4 days in culture. ( B ) ATD5C cells were harvested for RNA isolation on day zero or after 4 days in culture. Col10a1 showed significant elevation in pCMV- TAp63γ stable cell lines compared with the controls after 4 days in culture. ( C ) The protein levels of Col10a1 in TAp63γ stable cell lines also showed upregulation of Col10a1 compared to blank and pCMV controls by western blot analysis.

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Expressing, Isolation, Stable Transfection, Western Blot

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: In vitro effect of TAp63γ on chondrocyte proliferation. ( A ) ATD5C cells were cultured for 0, 4, 7, 14, or 21 days and stained with Alcian blue. After 7 days in culture, the staining intensity of TAp63γ stable cell lines was much stronger than the blank and vector controls. Scale bar, 25 μm. ( B ) Sum object area of the staining by densitometry analysis (n=3, * p<0.05, ** p<0.01).

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: In Vitro, Cell Culture, Staining, Stable Transfection, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: TAp63γ promotes hypertrophic differentiation of ATDC5 cells. ( A ) ATD5C cells were cultured for 0, 4, 7, 14, or 21 days and stained for ALP (alkaline phosphatase). After 7 days in culture, the staining intensity of TAp63γ stable cell lines was much stronger than the blank and vector controls. Scale bar, 50 μm. ( B ) Sum object area of the staining by densitometry analysis (n=3, * p<0.05, ** p<0.01).

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Cell Culture, Staining, Stable Transfection, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: In vitro effect of TAp63γ on matrix mineralization. ( A ) ATD5C cells were cultured for 0, 4, 7, 14, or 21 days and stained with Alizarin red. After 4 and 7 days in culture, enhanced Alizarin red staining was observed in both Col10a1-TAp63γ and pCMV- TAp63γ stable cell lines compared with the blank and vector controls. Scale bar, 25 μm. ( B ) Sum object area of the staining by densitometry analysis (n=3, * p<0.05, ** p<0.01).

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: In Vitro, Cell Culture, Staining, Stable Transfection, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: Accelerated ossification in Col10a1-TAp63γ transgenic mice. ( A ) Col10a1 distal promoter and a shorter Col10a1 basal promoter (ShXBP) (from −220 to +45 bp) were used to generate a p63-expressing transgenic construct. ( B ) PCR genotyping was performed for the Col10a1-TAp63γ transgenic mice using DNA prepared from skin. ( C ) For mice at postnatal day 1 (P1), ossification signals of the fore- and hind-limb digits were evaluated. Tail ossification signals were observed up to the 11 th caudal vertebra in transgenic mice and up to the 8 th caudal vertebra in WT mice. ( D ) The statistical analyses of the ossified caudal vertebrae from four Col10a1-TAp63γ transgenic mouse lines at P1 are presented. Line 2: n = 9; Line 5: n = 9; Line 7: n = 10.

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Transgenic Assay, Expressing, Construct

Journal: Aging (Albany NY)

Article Title: TAp63γ influences mouse cartilage development

doi: 10.18632/aging.103190

Figure Lengend Snippet: Histological and immunofluorescent analysis of Col10a1-TAp63γ transgenic mice. ( A ) The proliferative and hypertrophic zones of the limb cartilage in Col10a1-TAp63γ transgenic and WT mice were evaluated by hematoxylin and eosin (H&E) staining. Scale bar, 100 μm. ( B ) Sagittal sections of the distal humerus from both WT and transgenic mouse limbs at postnatal day 1 were subjected to immunofluorescent analysis using an anti-Sox9 antibody. Scale bar, 50 μm. ( C ) Sagittal sections of the distal humerus from both WT and transgenic mouse limbs at postnatal day 1 were subjected to immunofluorescent analysis using an anti-Runx2 antibody (label as Sox9 and some description of the findings). Scale bar, 50 μm.

Article Snippet: The TAp63γ expression plasmid (pCMV- TAp63γ ) MR227536 was purchased from Origene.

Techniques: Transgenic Assay, Staining

Journal: Cell death & disease

Article Title: The Δ133p53β isoform promotes an immunosuppressive environment leading to aggressive prostate cancer.

doi: 10.1038/s41419-019-1861-1

Figure Lengend Snippet: Fig. 3 Δ133TP53β is expressed in cancer cells. a In situ hybridization using RNAscope detected Δ133TP53 (black arrows) in FFPE prostate cancer tissues (left panel). Immunohistochemical staining for high molecular weight cytokeratin (HMWCK) and p63 (middle and right panels, respectively) to identify loss of HMWCK and p63 in prostate cancer. NA normal associated tissue (black arrows). b Left panel, probes to ubiquitin C (UBC) as a positive control for RNA quality and right panel, probes to the bacterial gene DapB as a negative control. Nuclei were counterstained with hematoxylin. c Immunohistochemistry using the KJC8 antibody to detect p53β (black arrows) in FFPE prostate cancer tissues. d Absence of p53β staining in normal associated prostate epithelium. Nuclei were counterstained with hematoxylin

Article Snippet: Saos-2 cells were transfected using FuGENE6 (Promega, Fitchburg, WI, USA) with an IL-6 Luc reporter construct (SwitchGear Genomics, Carlsbald, CA, USA) at 1 μg per well of a 6-well plate (seeded at 2 × 105 cells) along with either an increasing amount of Δ133p53 expression construct or a short-hairpin targeting p63 (sh-p63) (Origene, Rockville, MD, USA).

Techniques: In Situ Hybridization, RNAscope, Immunohistochemical staining, Staining, High Molecular Weight, Ubiquitin Proteomics, Positive Control, Negative Control, Immunohistochemistry

Journal: Cell death & disease

Article Title: The Δ133p53β isoform promotes an immunosuppressive environment leading to aggressive prostate cancer.

doi: 10.1038/s41419-019-1861-1

Figure Lengend Snippet: Fig. 7 Δ133p53 isoforms regulate genes involved in immune cell activity and recruitment. a Venn diagram showing genes differentially regulated in Group A prostate cancers, containing p53/p63/p73 response elements in their promoters and are associated with Δ133TP53β mRNA expression (Spearman's correlation coefficient cutoff of ρ > 0.5). b Bar graph depicting a selected list of pathways (PantherdB) with >5 fold enrichment and FDR < 0.05. c Expression of selected genes in left panel: four clonal lines expressing Δ133p53α or Δ133p53β isoforms compared to control p53-null H1299 cells, right panel: 22Rv1 cells 48 h after knockdown of Δ133p53. Box (median ± 25th–75th percentile), and whiskers show the 10–90% CI. *p < 0.05, **p < 0.01 and ***p < 0.001, as determined by paired one-tailed t-test. d Transcriptional activation of the IL-6 promoter by Δ133p53 or by inhibiting TP63 (shp63). Cells were transiently transfected with 1.0 µg of IL-6 luciferase reporter plasmid and varying amounts of either Δ133p53 or shp63. Luciferase activity was determined and is normalized to cell number. Bars represent the mean and error bars are ± SD; n = 4 biological replicates. e 10.1/vector and 10.1/Δ122 cells treated with blocking antibody against either IL-6, CCL2 or both IL-6 (2.0 μg) and CCL2 (3.0 μg). Cells were allowed to migrate for 4 h then membranes were fixed, stained, imaged and quantified. Three technical replicate counts of cells per field were combined and are shown as mean ± SEM. Significance was determined as *p < 0.05, **p < 0.01, ***p < 0.005 using unpaired t-tests. ns not significant

Article Snippet: Saos-2 cells were transfected using FuGENE6 (Promega, Fitchburg, WI, USA) with an IL-6 Luc reporter construct (SwitchGear Genomics, Carlsbald, CA, USA) at 1 μg per well of a 6-well plate (seeded at 2 × 105 cells) along with either an increasing amount of Δ133p53 expression construct or a short-hairpin targeting p63 (sh-p63) (Origene, Rockville, MD, USA).

Techniques: Activity Assay, Expressing, Control, Knockdown, One-tailed Test, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Blocking Assay, Staining