Journal: Cell Death & Disease

Article Title: Pin1 modulates p63 α protein stability in regulation of cell survival, proliferation and tumor formation

doi: 10.1038/cddis.2013.468

Figure Lengend Snippet: p63 proteins physically interact with Pin1. ( a ) and ( b ) Pin1 interacts with TAp63 in vitro . H1299 cells transfected with either TAp63 α or TAp63 γ expression plasmid were lysed and subjected to GST pull-down assay with GST alone (lane 2) or GST–Pin1 (lane 3). 10 μ g of total protein from the same cell lysates were directly loaded as input controls (lane 1). Immunoblotting analysis (IB) was performed using an antibody specific for p63 (4A4, α -p63; top panels). Comparable amounts of GST and GST–Pin1 fusion proteins were shown by staining the membrane with Coomassie blue R-250 (bottom panels). ( c ) Pin1 interacts with ΔNp63 in vitro . Pull-down experiments were performed as described above. 10 μ g of total protein from lysate inputs (top panel) and pull-down products (bottom panel) were subjected to IB analysis with α -p63. ( d ) The WW domain of Pin1 mediates Pin1–p63 interaction, which can be abolished by W34A or Y23A point mutation in the WW domain of Pin1. GST pull-down experiments were performed with GST alone (lane 2), GST–Pin1 (lane 3), GST–PPIase (lane 4), GST–WW (lane 5), GST–Pin1 (W34A) (lane 6) or GST–Pin1 (Y23A) (lane 7). ( e ) Wild-type but not W34A mutant Pin1 can form a stable complex with TAp63 α . Lysates from H1299 cells transiently expressing HA-tagged wild-type or W34A mutant Pin1 were subjected to immunoprecipitation (IP) and IB using myc or HA antibody. ( f ) Endogenous proteins of ΔNp63 α and Pin1 form a stable complex in FaDu cells. 2 mg of FaDu cell lysate was subjected to IP with α -p63 (4A4) or IgG control. The IP products were subjected to IB with α -p63 or α -Pin1

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) analysis were performed as previously described., Antibodies used were specific for p63 (4A4 monoclonal antibody, Santa Cruz, Dallas, TX, USA, 1 : 200), myc (9E10 monoclonal antibody, Santa Cruz, 1 : 1000), Pin1 (rabbit polyclonal antibody, Cell Signaling, Danvers, MA, USA, 1 : 1000), actin (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), HA (mouse monoclonal antibody, Millipore, Billerica, MA, USA, 1 : 500), WWP1 (rabbit monoclonal antibody, Epitomics, Burlingame, CA, USA, 1 : 3000), Puma (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), and N-terminal cleaved PARP1 (rabbit polyclonal antibodies, Zenable, Chengdu, China, 1 : 8000).

Techniques: In Vitro, Transfection, Expressing, Plasmid Preparation, Pull Down Assay, Western Blot, Staining, Membrane, Mutagenesis, Immunoprecipitation, Control

Journal: Cell Death & Disease

Article Title: Pin1 modulates p63 α protein stability in regulation of cell survival, proliferation and tumor formation

doi: 10.1038/cddis.2013.468

Figure Lengend Snippet: Pin1 upregulates protein levels of p63 α , but not p63 γ , via increasing their half-lives. ( a ) Transient overexpression of wild-type but not W34A mutant Pin1, upregulates the protein level of TAp63 α in H1299 cells. 1 μ g HA-tagged wild-type or W34A mutant Pin1 expression plasmid, or vector control, was transfected together with 200 ng myc-tagged TAp63 α expression plasmid and 50 ng pEGFP-N1, into H1299 cells. 48 h after transfection, cells were lysed and subjected to IB. ( b ) Transient overexpression of wild-type but not W34A mutant Pin1, upregulates the protein level of ΔNp63 α in HEK293 cells. 1 μ g HA-tagged wild-type or W34A mutant Pin1 expression plasmid, or vector control, was transfected into a HEK293 cell line stably overexpressing ΔNp63 α HEK293:ΔNp63 α . Forty-eight hours after transfection, cells were lysed and subjected to IB analysis. ( c ) and ( d ) Stable expression of wild-type Pin1 upregulates protein levels of TAp63 α and ΔNp63 α , but not TAp63 γ or ΔNp63 γ . TA α , ΔN α , TA γ and ΔN γ isoforms of p63 expression plasmids (200 ng each) were respectively co-transfected with 50 ng pEGFP-N1 into H1299 cells stably expressing wild-type (H1299:Pin1) or W34A mutant [H1299:Pin1(W34A)] Pin1. Forty-eight hours after transfection, cells were lysed and subjected to IB analysis. ( e ) SiRNA-mediated knockdown of Pin1 downregulates protein levels of ΔNp63 α in HEK293 cells. SiPin1 or scrambled control (20 pmol each) was transiently transfected into HEK293 cells stably overexpressing ΔNp63 α . Forty-eight hours after transfection, cells were lysed and subjected to IB analysis. ( f ) and ( g ) Pin1 extends protein half-life of TAp63 α . H1299 cells were co-transfected with TAp63 α (200 ng) and pEGFP-N1 (50 ng), together with HA-Pin1 (400 ng) or its vector control, for 24 h and then treated with 50 mg/ml cycloheximide (CHX) for indicated durations followed by IB analysis ( f ). Protein levels of TAp63 α were normalized with Actin, and the protein half-life was quantified ( g ). ( h ) and ( i ) Pin1 extends protein half-life of ΔNp63 α . HEK293:ΔNp63 α cells were transfected with HA-Pin1 (1 μ g) or its vector control for 24 h and then treated with 50 mg/ml cycloheximide (CHX) for indicated durations followed by IB analysis ( h ). The half-life of ΔNp63 α proteins was quantified ( i )

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) analysis were performed as previously described., Antibodies used were specific for p63 (4A4 monoclonal antibody, Santa Cruz, Dallas, TX, USA, 1 : 200), myc (9E10 monoclonal antibody, Santa Cruz, 1 : 1000), Pin1 (rabbit polyclonal antibody, Cell Signaling, Danvers, MA, USA, 1 : 1000), actin (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), HA (mouse monoclonal antibody, Millipore, Billerica, MA, USA, 1 : 500), WWP1 (rabbit monoclonal antibody, Epitomics, Burlingame, CA, USA, 1 : 3000), Puma (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), and N-terminal cleaved PARP1 (rabbit polyclonal antibodies, Zenable, Chengdu, China, 1 : 8000).

Techniques: Over Expression, Mutagenesis, Expressing, Plasmid Preparation, Control, Transfection, Stable Transfection, Knockdown

Journal: Cell Death & Disease

Article Title: Pin1 modulates p63 α protein stability in regulation of cell survival, proliferation and tumor formation

doi: 10.1038/cddis.2013.468

Figure Lengend Snippet: T538A point mutation abrogates Pin1-mediated upregulation and siPin1-mediated downregulation of TAp63 α . ( a ) Schematic representation of putative Pin1-binding sites in different p63 isoforms. TAD, transactivation domain; DBD, DNA-binding domain; OLD, oligomerization domain; SAM, sterile alpha motif; TID, trans-inhibitory domain. Dots indicate putative Pin1-binding sites, which are Serine-Proline (SP) or Threonine-Proline (TP) motifs. A PPxY motif (framed letters) is adjacent to the T538-P in TAp63 α or the T444-P in ΔNp63 α . ( b ) T538A, but not other point mutations of putative Pin1-binding sites in the special C-terminus of TAp63 α , abrogates the Pin1-mediated upregulation of TAp63 α . S463A, T491A, T538A, or T619A mutant, or wild-type TAp63 α plasmid (200 ng each) was co-transfected pEGFP-N1 (50 ng) into H1299:Pin1(W34A) or H1299:Pin1 cells. Forty-eight hours after transfection, cells were lysed and subjected to immunoblotting analysis. The relative level of each mutant TAp63 α in Pin1(W34A) control was respectively set as 1.0. ( c ) SiRNA-mediated knockdown of Pin1 downregulates wild-type but not T538A mutant TAp63 α Pin1 siRNA (siPin1) or scrambled control (20 pmol each) was transiently co-transfected into H1299 cells with expression plasmid of wild-type (WT) or T538A mutant TAp63 α (1 μ g each), together with 50 ng pEGFP-N1. Forty-eight hours after transfection, cells were lysed and subjected to immunoblotting analysis. The relative level of wild-type or T538A mutant TAp63 α in scrambled control was respectively set as 1.0

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) analysis were performed as previously described., Antibodies used were specific for p63 (4A4 monoclonal antibody, Santa Cruz, Dallas, TX, USA, 1 : 200), myc (9E10 monoclonal antibody, Santa Cruz, 1 : 1000), Pin1 (rabbit polyclonal antibody, Cell Signaling, Danvers, MA, USA, 1 : 1000), actin (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), HA (mouse monoclonal antibody, Millipore, Billerica, MA, USA, 1 : 500), WWP1 (rabbit monoclonal antibody, Epitomics, Burlingame, CA, USA, 1 : 3000), Puma (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), and N-terminal cleaved PARP1 (rabbit polyclonal antibodies, Zenable, Chengdu, China, 1 : 8000).

Techniques: Mutagenesis, Binding Assay, Sterility, Plasmid Preparation, Transfection, Western Blot, Control, Knockdown, Expressing

Journal: Cell Death & Disease

Article Title: Pin1 modulates p63 α protein stability in regulation of cell survival, proliferation and tumor formation

doi: 10.1038/cddis.2013.468

Figure Lengend Snippet: Pin1 inhibits proteasome- and WWP1- dependent degradation of p63 α proteins. ( a ) MG132 abrogates Pin1-mediated upregulation of TAp63 α H1299:Pin1 or H1299:Pin1(W34A) cells were co-transfected with TAp63 α (400 ng) and pEGFP-N1 (50 ng) for 24 h. Cells were then treated with 40 μ M MG132, which is a specific proteasome inhibitor, or its vehicle control (0.4% DMSO), for additional 18 h before collection. Cell lysates were subjected to immunoblotting analysis. ( b ) MG132 abrogates siPin1-mediated downregulation of TAp63 α (400 ng). Pin1 siRNA (siPin1) or scrambled control (20 pmol each) was transiently co-transfected into H1299 cells with expression plasmid of TAp63 α (1 μ g) and pEGFP-N1 (50 ng). Twenty-four hours later, cells were treated with 40 μ M MG132 or 0.4% DMSO for an additional 18 h. Then cells were collected and subjected to immunoblotting analysis. ( c ) WWP1 downregulates TAp63 α while wild-type Pin1 can rescue this downregulation. TAp63 α expression plasmid (400 ng) was co-transfected with WWP1 (200 ng) or (and) wild-type or W34A mutant HA-Pin1 (1 μ g) expression plasmids or their vector controls, together with pEGFP-N1 (50 ng). Forty-eight hours after transfection, cells were collected for immunoblotting analysis. ( d ) WWP1 downregulates ΔNp63 α while Pin1 can rescue this downregulation. WWP1 (200 ng) or (and) HA-Pin1 (1 μ g) expression plasmids or their vector controls were co-transfected into HEK293 cells stably overexpressing ΔNp63 α (HEK293:ΔNp63 α ). Forty-eight hours after transfection, cells were collected for immunoblotting analysis. ( e ) Knockdown of WWP1 upregulates TAp63 α and removes the siPin1-mediated downregulation of TAp63 α . SiPin1 and (or) siWWP1 (20 pmol each) were co-transfected into H1299 cells together with TAp63 α (500 ng) and pEGFP-N1 (50 ng). Forty-eight hours after transfection, cells were collected for immunoblotting analysis. ( f ) WWP1 downregulates T538A mutant TAp63 α but Pin1 cannot rescue this downregulation. T538A mutant TAp63 α expression plasmid (400 ng) was co-transfected with WWP1 (200 ng) or (and) HA-Pin1 (1 μ g) expression plasmids or its vector control, together with pEGFP-N1 (50 ng). Forty-eight hours after transfection, cells were collected for immunoblotting analysis. ( g ) Pin1 impairs the binding of WWP1 to wild-type but not T538A mutant TAp63 α . 0.5 μ g wild-type or T538A mutant Myc-TAp63 α plasmid was transfected into H1299:Pin1(W34A) or H1299:Pin1 cells, together with 0.5 μ g WWP1 expression plasmid. Twenty-four hours after transfection, cells were lysed and 1 mg total protein samples were respectively subjected to immunoprecipitation with myc antibody ( α -myc). The IP products were subjected to immunoblotting analysis

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) analysis were performed as previously described., Antibodies used were specific for p63 (4A4 monoclonal antibody, Santa Cruz, Dallas, TX, USA, 1 : 200), myc (9E10 monoclonal antibody, Santa Cruz, 1 : 1000), Pin1 (rabbit polyclonal antibody, Cell Signaling, Danvers, MA, USA, 1 : 1000), actin (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), HA (mouse monoclonal antibody, Millipore, Billerica, MA, USA, 1 : 500), WWP1 (rabbit monoclonal antibody, Epitomics, Burlingame, CA, USA, 1 : 3000), Puma (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), and N-terminal cleaved PARP1 (rabbit polyclonal antibodies, Zenable, Chengdu, China, 1 : 8000).

Techniques: Transfection, Control, Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Stable Transfection, Knockdown, Binding Assay, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Pin1 modulates p63 α protein stability in regulation of cell survival, proliferation and tumor formation

doi: 10.1038/cddis.2013.468

Figure Lengend Snippet: Schematic model of a proposed mechanism showing how Pin1–p63 α pathway modulates cell survival/proliferation. TAp63 α and ΔNp63 α undergo a proteasomal degradation mediated by E3 ligase WWP1. However, Pin1 may mediate the cis-trans isomerization or physical occupation of pTP-PPxY motifs of p63 α proteins, leading to impairing their binding to WWP1. Consequently, TAp63 α or ΔNp63 α was stabilized. As a result, Pin1 inhibits cell survival/proliferation via stabilizing TAp63 α or promotes cell survival/proliferation via stabilizing ΔNp63 α

Article Snippet: Immunoprecipitation (IP) and immunoblotting (IB) analysis were performed as previously described., Antibodies used were specific for p63 (4A4 monoclonal antibody, Santa Cruz, Dallas, TX, USA, 1 : 200), myc (9E10 monoclonal antibody, Santa Cruz, 1 : 1000), Pin1 (rabbit polyclonal antibody, Cell Signaling, Danvers, MA, USA, 1 : 1000), actin (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), HA (mouse monoclonal antibody, Millipore, Billerica, MA, USA, 1 : 500), WWP1 (rabbit monoclonal antibody, Epitomics, Burlingame, CA, USA, 1 : 3000), Puma (rabbit polyclonal antibody, Santa Cruz, 1 : 1000), and N-terminal cleaved PARP1 (rabbit polyclonal antibodies, Zenable, Chengdu, China, 1 : 8000).

Techniques: Binding Assay

Journal: FEBS letters

Article Title: ING1b decreases cell proliferation through p53-dependent and -independent mechanisms.

doi: 10.1016/s0014-5793(03)01024-x

Figure Lengend Snippet: Fig. 7. ING1b can activate p53-related proteins. A: Activation of the transcriptional activities of p53-related proteins by ING1b. H1299 cells were transfected with plasmids expressing an MDM2 promoter-luciferase reporter and L-galactosidase. Control reactions without cotransfection of other plasmids are shown in experiment 1. Plasmids expressing p73K, p63K, and p53 were cotransfected with control plasmids (experiment 2) or ING1b (experiment 3) as indicated. Cell extracts were prepared, and the luciferase and L-galactosidase activities were determined. The lu- ciferase activities were normalized with the L-galactosidase activities to correct for transcriptional e⁄ciencies and plotted as a percentage of p53/p63K/p73K alone. The means and standard deviation of three independent experiments are shown. B: Puri¢cation of recombinant GST- ING1b. GST-ING1b was expressed in E. coli and puri¢ed as described in Section 2. The puri¢ed protein was applied onto SDS^PAGE and de- tected by Coomassie blue staining. The positions of molecular mass standards (in kDa) are indicated. C: ING1b can interact with p53 and p73K. Reticulocyte lysate-expressed p53 and p73K were incubated with either puri¢ed GST or GST-ING1b. The GST fusion proteins were cap- tured with GSH-agarose and unbound proteins were washed o¡. The coprecipitated p53 and p73K were detected by SDS^PAGE and Phos- phorImagery. D: ING1b can interact with p53 and p63K. H1299 cells were transfected with plasmids encoding p53 (lanes 1, 2, 5 and 6) or p63K (lanes 3, 4, 7 and 8) with control vector (odd-numbered lanes) or FLAG-ING1b (even-numbered lanes). Cell-free extracts were prepared at 24 h after transfection and 100 Wg was subjected to immunoprecipitation with anti-FLAG immune serum. The retained proteins were de- tected by immunoblotting using antibodies against p53 and p63.

Article Snippet: Goat antibodies raised against a C-terminal peptide derived from ING1 (sc-7566), polyclonal antibodies against p21CIP1=WAF1 (sc-397), monoclonal antibodies 4A4 against p63 (sc8431), and monoclonal antibodies DO-1 against p53 (sc-126) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Transfection, Expressing, Luciferase, Control, Cotransfection, Standard Deviation, Recombinant, SDS Page, Staining, Incubation, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Histopathology

Article Title: Primary cutaneous NUT adnexal carcinoma: morphologic, genetic and methylation analysis of seven new cases with comparison to extracutaneous NUT carcinoma and NUTM1 ‐rearranged porocarcinoma

doi: 10.1111/his.15444

Figure Lengend Snippet: Immunohistochemical features of BRD4::NUTM1 adnexal carcinoma. Immunohistochemistry of the case showed diffuse positivity for EMA and SOX10. CEA confirmed the presence of ducts. P63 and p40 confined to the periphery of the tumour nests. Diffuse nuclear expression of NUT was demonstrated. YAP1 (C‐terminal) expression was preserved.

Article Snippet: Immunohistochemical staining for EMA (clone E29; Dako, Glostrup, Denmark), p40 (clone BC28; Eurobio, Les Ulis, France), p63 (clone 4A4; Biocare Medical, Pacheco, CA, USA), SOX10 (clone SP267; Cell Marque, Rocklin, CA, USA), CEA (clone CEA 31; Cell Marque), EpCAM (Clone BerEP4; Dako), NUT (clone C52B1; Cell Signalling Technology, Danvers, MA, USA), YAP1 (clone D8H1\u00D7; Cell Signalling Technology) was performed using a BenchMark XT Platform (Roche Diagnostics GmbH, Penzberg, Germany).

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing

Journal: Cancer Research

Article Title: miR-31 Displays Subtype Specificity in Lung Cancer

doi: 10.1158/0008-5472.can-20-2769

Figure Lengend Snippet: Figure 1. miR-31 is overexpressed in specific histologic subtypes of lung cancer. A, IHC of patient tumors from each histologic subtype with markers of each (10) and ISH of miR-31 (100) depicting overexpression of miR-31 in lung adenocarcinoma (LUAD), SQCC, ADSQ, and LCNEC, but not in SCLC. TTF-1 is a marker of LUAD, p63 of SQCC, and synaptophysin (SYN) of neuroendocrine carcinomas. B and C, miR-31 levels determined with Taqman qRT-PCR. Expression normalized to endogenous control RNU6B. B, Archived patient tissue from 2016–2019 of normal, lung adenocarcinoma, SQCC, ADSQ, LCNEC, SCLC, and atypical carcinoid (AC) lung tumors. C, Cell lines. Mean miR-31 expression (error bars SEM: , P < 0.05; Student t test). Number (n) samples indicated.

Article Snippet: IHC was performed by Vanderbilt University Medical Center TPSR: anti-p63 antibody [4A4] SKU: CM163A, TTF-1 Leica PA0364, SYP Leica PA0299, CSK ProteinTech 17720–1 -AP, ATM Invitrogen MA5–32063, EPHB4 Cell Signaling Technology14960

Techniques: Over Expression, Marker, Quantitative RT-PCR, Expressing, Control

Journal: NAR Cancer

Article Title: An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma

doi: 10.1093/narcan/zcad038

Figure Lengend Snippet: Protein interaction network involving EGFR and TGF-β signaling pathways embedded in the p63 signature network. ( A ) Venn diagram showing the 291 genes that overlap between the p63 targets and p63 consensus signature. ( B ) The 291 genes were used as input to perform an interaction network analysis on the STRING database . ( C ) Approximately 21% of the 291 targets used as input in the STRING database were part of an interconnected network of protein–protein interactions that link the TGF-β1 signaling and EGFR signaling. The bubbles are color coded to reflect the corresponding log 2 fold change values.

Article Snippet: Proteins were then electro-transferred to Immunoblot polyvinylidene difluoride membranes (Bio-Rad), which were incubated with the following antibodies: p63 4A4, FST (sc-365003 [Santa Cruz Biotechnology] or EPR10903 [Abcam]), pAKT ser-473 (66444-I-Ig; Proteintech), pAKT Thr-308 (29163-1-AP; Proteintech), pSTAT3 (Epitomics), STAT3 (2281-1; Epitomics), AKT1 (10176-2-AP; Proteintech), pERK1/2 (sc-7883; Santa Cruz Biotechnology), ERK1/2 (sc-66192-1-Ig [Proteintech] or sc-514302 [Santa Cruz Biotechnology]), GAPDH (MAB374; EMD Millipore), pSMAD2 (GTX13364; GeneTex), SMAD2 (GTX111075; GeneTex), pSMAD1/5 (41D10; Cell Signaling), and SMAD1 (D49D7; Cell Signaling).

Techniques: Protein-Protein interactions

Journal: NAR Cancer

Article Title: An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma

doi: 10.1093/narcan/zcad038

Figure Lengend Snippet: p63-driven gene signature. ( A ) Western blot showing robust depletion of p63 in A253 and SCC25 cells treated with p63-targeting shRNA constructs. ( B ) Schematic showing the analysis of the RNA-seq data generated from the p63 knockdown experiments. ( C ) Venn diagram showing the numbers of p63-regulated DEGs in both A253 and SCC25 cells. 555 genes (red) were upregulated, and 795 genes (blue) were downregulated in both cells. 140 genes (black) were not consistently upregulated or downregulated in both cells. ( D ) WikiPathway analyses of the genes that were upregulated following p63 ( E ) WikiPathway analyses of the genes that were downregulated after p63 depletion. ( F ) (Top) Schematic showing how both TCGA-HNSCC patient data and in vitro knockdown data were combined to generate the consensus signature. (Bottom left) Heat map showing the expression profile in the 430-gene consensus signature in the TCGA data set. (Bottom right) The pattern of expression of this signature is conserved in an independent preclinical HNSCC data set reported by Huang et al. . ( G ) Biological processes enriched in the 430-gene consensus signature, including Rho and ERK1/2 signaling cascades, cell migration and cellular response to drugs.

Article Snippet: Proteins were then electro-transferred to Immunoblot polyvinylidene difluoride membranes (Bio-Rad), which were incubated with the following antibodies: p63 4A4, FST (sc-365003 [Santa Cruz Biotechnology] or EPR10903 [Abcam]), pAKT ser-473 (66444-I-Ig; Proteintech), pAKT Thr-308 (29163-1-AP; Proteintech), pSTAT3 (Epitomics), STAT3 (2281-1; Epitomics), AKT1 (10176-2-AP; Proteintech), pERK1/2 (sc-7883; Santa Cruz Biotechnology), ERK1/2 (sc-66192-1-Ig [Proteintech] or sc-514302 [Santa Cruz Biotechnology]), GAPDH (MAB374; EMD Millipore), pSMAD2 (GTX13364; GeneTex), SMAD2 (GTX111075; GeneTex), pSMAD1/5 (41D10; Cell Signaling), and SMAD1 (D49D7; Cell Signaling).

Techniques: Western Blot, shRNA, Construct, RNA Sequencing, Generated, Knockdown, In Vitro, Expressing, Migration

Journal: NAR Cancer

Article Title: An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma

doi: 10.1093/narcan/zcad038

Figure Lengend Snippet: Overview of the super enhancer landscape in A253 and SCC25 cells. ( A ) Super enhancer profiles of A253 and SCC25 cells according to H3K27Ac with gene assignment. ( B ) Pie charts detailing the distribution of p63 binding in regular enhancer versus super enhancer regions. ( C ) Common super enhancer-associated genes in A253 and SCC25 cells. ( D ) Genomic maps showing the super enhancers associated with TP63, FOSL1 and EGFR in both A253 and SCC25 cells.

Article Snippet: Proteins were then electro-transferred to Immunoblot polyvinylidene difluoride membranes (Bio-Rad), which were incubated with the following antibodies: p63 4A4, FST (sc-365003 [Santa Cruz Biotechnology] or EPR10903 [Abcam]), pAKT ser-473 (66444-I-Ig; Proteintech), pAKT Thr-308 (29163-1-AP; Proteintech), pSTAT3 (Epitomics), STAT3 (2281-1; Epitomics), AKT1 (10176-2-AP; Proteintech), pERK1/2 (sc-7883; Santa Cruz Biotechnology), ERK1/2 (sc-66192-1-Ig [Proteintech] or sc-514302 [Santa Cruz Biotechnology]), GAPDH (MAB374; EMD Millipore), pSMAD2 (GTX13364; GeneTex), SMAD2 (GTX111075; GeneTex), pSMAD1/5 (41D10; Cell Signaling), and SMAD1 (D49D7; Cell Signaling).

Techniques: Binding Assay

Journal: NAR Cancer

Article Title: An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma

doi: 10.1093/narcan/zcad038

Figure Lengend Snippet: EGF signaling drives FST expression via ERK signaling in HNSCC. ( A ) Western blots showing the effects of 4-h stimulation of SCC25 (left) and A253 (right) cells with 50ng of indicated growth factors. Although the downstream signaling cascades of these growth factors were activated in both cell lines, only EGF treatment upregulated FST expression. ( B ) Treatment of A253 and SCC25 cells with 30 ng EGF for 4 h resulted in activation of ERK1/2 but not AKT or STAT3. ( C ) Depletion of p63 prevented EGF-mediated FST expression in A253 and SCC25 cells. ( D ) Inhibition of ERK phosphorylation via Selumetinib (ADZ6244) reduced basal levels of FST expression. ( E ) Inhibition of ERK activation by ADZ6244 blocks the EGF-mediated increase in FST expression in both A253 and SCC25 cells.

Article Snippet: Proteins were then electro-transferred to Immunoblot polyvinylidene difluoride membranes (Bio-Rad), which were incubated with the following antibodies: p63 4A4, FST (sc-365003 [Santa Cruz Biotechnology] or EPR10903 [Abcam]), pAKT ser-473 (66444-I-Ig; Proteintech), pAKT Thr-308 (29163-1-AP; Proteintech), pSTAT3 (Epitomics), STAT3 (2281-1; Epitomics), AKT1 (10176-2-AP; Proteintech), pERK1/2 (sc-7883; Santa Cruz Biotechnology), ERK1/2 (sc-66192-1-Ig [Proteintech] or sc-514302 [Santa Cruz Biotechnology]), GAPDH (MAB374; EMD Millipore), pSMAD2 (GTX13364; GeneTex), SMAD2 (GTX111075; GeneTex), pSMAD1/5 (41D10; Cell Signaling), and SMAD1 (D49D7; Cell Signaling).

Techniques: Expressing, Western Blot, Activation Assay, Inhibition, Phospho-proteomics

Journal: NAR Cancer

Article Title: An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma

doi: 10.1093/narcan/zcad038

Figure Lengend Snippet: Epithelial cells are the primary producers of FST in the tumor microenvironment. ( A ) UMAP plot showing the identities of the various cellular clusters identified in the HNSCC single-cell RNA-seq data reported by Puram et al. . ( B ) Expression of FST by cells in the different clusters showing predominantly epithelial cell-specific expression. ( C ) UMAP plot showing that expression of TP63 is predominantly in epithelial cells. ( D ) EGFR expression in the different cellular clusters.

Article Snippet: Proteins were then electro-transferred to Immunoblot polyvinylidene difluoride membranes (Bio-Rad), which were incubated with the following antibodies: p63 4A4, FST (sc-365003 [Santa Cruz Biotechnology] or EPR10903 [Abcam]), pAKT ser-473 (66444-I-Ig; Proteintech), pAKT Thr-308 (29163-1-AP; Proteintech), pSTAT3 (Epitomics), STAT3 (2281-1; Epitomics), AKT1 (10176-2-AP; Proteintech), pERK1/2 (sc-7883; Santa Cruz Biotechnology), ERK1/2 (sc-66192-1-Ig [Proteintech] or sc-514302 [Santa Cruz Biotechnology]), GAPDH (MAB374; EMD Millipore), pSMAD2 (GTX13364; GeneTex), SMAD2 (GTX111075; GeneTex), pSMAD1/5 (41D10; Cell Signaling), and SMAD1 (D49D7; Cell Signaling).

Techniques: RNA Sequencing, Expressing

Journal: NAR Cancer

Article Title: An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma

doi: 10.1093/narcan/zcad038

Figure Lengend Snippet: FST , EGFR and TP63 expression correlates with immune cell infiltration. FST , EGFR and TP63 expression correlated negatively with tumor suppressive T-lymphocyte infiltration ( A ) and positively with tumor-promoting myeloid suppressor cells ( B ) in HNSCC tissues. ( C ) Kaplan–Meier plot showing overall survival of cohorts of patients with high and low FST expression levels.

Article Snippet: Proteins were then electro-transferred to Immunoblot polyvinylidene difluoride membranes (Bio-Rad), which were incubated with the following antibodies: p63 4A4, FST (sc-365003 [Santa Cruz Biotechnology] or EPR10903 [Abcam]), pAKT ser-473 (66444-I-Ig; Proteintech), pAKT Thr-308 (29163-1-AP; Proteintech), pSTAT3 (Epitomics), STAT3 (2281-1; Epitomics), AKT1 (10176-2-AP; Proteintech), pERK1/2 (sc-7883; Santa Cruz Biotechnology), ERK1/2 (sc-66192-1-Ig [Proteintech] or sc-514302 [Santa Cruz Biotechnology]), GAPDH (MAB374; EMD Millipore), pSMAD2 (GTX13364; GeneTex), SMAD2 (GTX111075; GeneTex), pSMAD1/5 (41D10; Cell Signaling), and SMAD1 (D49D7; Cell Signaling).

Techniques: Expressing

Journal: Discover Oncology

Article Title: Spatial transcriptomics of urothelial carcinoma with basal/squamous differentiation identifies Galectin-7 as a specific marker of squamous lineage commitment

doi: 10.1007/s12672-026-04927-z

Figure Lengend Snippet: LGALS7 is selectively expressed in Ba/Sq differentiation and promotes squamous carcinoma cell proliferation. A The H&E image from Fig. B is shown again, with a red box indicating the region analyzed in this figure. Spatial transcriptomic maps display TP63 and LGALS7 expression patterns within the boxed region, corresponding to the UC–Ba/Sq interface. B Cluster-level expression of TP63 and LGALS7. C Immunohistochemistry (IHC) of the cystectomy specimen showing diffuse p63 positivity in both urothelial carcinoma (UC) and Ba/Sq-differentiated tumor components, and selective Galectin-7 expression restricted to Ba/Sq-differentiated tumor nests. Scale bars, 1 mm. D Western blot showing Galectin-7 expression across cancer cell lines (left), and immunofluorescence (IF) of SCaBER cells showing cytoplasmic localization (right). E Proliferation assay of SCaBER cells following LGALS7 knockdown

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against p63 (mouse monoclonal, clone 4A4, ready-to-use; Nichirei Biosciences) and Galectin-7 (rabbit monoclonal, clone EPR4287, 1:1500; Abcam).

Techniques: Expressing, Immunohistochemistry, Western Blot, Immunofluorescence, Proliferation Assay, Knockdown

Journal: Discover Oncology

Article Title: Spatial transcriptomics of urothelial carcinoma with basal/squamous differentiation identifies Galectin-7 as a specific marker of squamous lineage commitment

doi: 10.1007/s12672-026-04927-z

Figure Lengend Snippet: Diagnostic and prognostic value of Galectin-7 in urothelial carcinoma. A Immunohistochemistry (IHC) for p63 and Galectin-7 in UC with Ba/Sq features ( n = 28) and UC without Ba/Sq ( n = 64). B Immunohistochemistry (IHC) of three representative UC with Ba/Sq differentiation cases showing diffuse p63 positivity in both urothelial carcinoma (UC) and Ba/Sq-differentiated tumor components, and selective Galectin-7 expression restricted to Ba/Sq-differentiated tumor nests. Scale bars, 100 μm. C Summary of key clinicopathological parameters associated with Galectin-7 (G7) expression. Detailed clinicopathological data are provided in Supplementary Table 1. Kaplan–Meier analysis of progression-free survival stratified by Galectin-7 expression

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against p63 (mouse monoclonal, clone 4A4, ready-to-use; Nichirei Biosciences) and Galectin-7 (rabbit monoclonal, clone EPR4287, 1:1500; Abcam).

Techniques: Diagnostic Assay, Immunohistochemistry, Expressing