Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: The molecular structure of SOX11. The SOX11 protein is composed of 441 amino acids. SOX11 contains two functional domains, the N-terminal HMG domain and the conserved TAD. SOX11, sex-determining region Y-related high-mobility-group box transcription factor 11; HMG, high mobility group; TAD, C-terminal transactivation domain.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Functional Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Overview of SOX11 function reported in diverse types of cancer.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Expressing, Over Expression, Biomarker Discovery, Migration, Methylation, DNA Methylation Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Overall carcinogenic actions of SOX11 on the hallmarks of tumor biology. SOX11 exerts tumor-stimulative effects through increasing cell proliferation, repressing cell differentiation, inducing angiogenesis, and promoting metastasis. BCL6, B-cell lymphoma 6; BNIP3, B-cell lymphoma 2 (BCL2)/adenovirus E1B 19 kDa protein-interacting protein 3; CIC, cancer-initiating cell; PAX5, Paired box protein 5; PDGFA, platelet-derived growth factor A; SETMAR, SET domain and mariner transposase fusion gene; SOX11, Sex-determining region Y-related high-mobility-group box transcription factor 11; TANK, TRAF family member-associated NF-κB activator.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Cell Differentiation, Derivative Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: SOX11: friend or foe in tumor prevention and carcinogenesis?

doi: 10.1177/1758835919853449

Figure Lengend Snippet: Different prognostic significance of SOX11 in tumor cases.

Article Snippet: MCL , 53 , IHC , Rabbit polyclonal (Atlas Antibodies AB) , SOX11-negative patients show worse prognosis and shorter OS. , Wang et al. .

Techniques: Expressing, Microarray, Over Expression

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 1. The IJM region is necessary for collagen-induced DDR2 activation. (a) Overall topology and alignment of the transmembrane (TM) domain and the intracellular juxtamembrane region (IJM) of DDR1a and DDR2. The IJM was divided into three regions: JM1, JM2 and JM3. (b) Schematic diagram of various DDR2 constructs used in our study. (c) HEK293T cells transiently transfected with plasmids encoding the full-length DDR2 and F-DJM1-JM2 mutant were stimulated by Type I collagen for 60 min. Tyrosine phosphorylation of the F-DJM1-JM2 mutant was inhibited compared to that of the full-length DDR2. (d) HEK293T cells were transfected with plasmids encoding full-length DDR2, F- DJM1 and F-DJM2 and were stimulated by collagen. The F-DJM2 mutant showed a significant decrease in tyrosine phosphorylation. **p < 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Activation Assay, Construct, Transfection, Mutagenesis, Phospho-proteomics

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 2. DDR2 dimerizes via the JM2 of the IJM region. (a and b) HEK293T cells were transiently cotransfected with plasmids encoding TM- JM1-JM2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blot analysis showed that the cytoplasmic domains of DDR2 bind to each other via the intact TM-JM1-JM2 domain and form homodimers. Asterisks indicate the expected size of TM-JM1-JM2. (c) HEK293T cells were transfected with plasmids encoding F-DJM1 and F-DJM2 mutants. A crosslinking assay showed that dimers of F-DJM1 were not changed compared to full-length DDR2, whereas dimers were significantly decreased for F-DJM2.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 3. JM2 has a dominant-negative effect on DDR2 activation. (a and b) HEK293T cells were cotransfected with plasmids encoding full- length DDR2-myc and TM-JM1-JM2-HA. Immunoprecipitation and Western blotting showed that the full-length DDR2 and TM-JM1-JM2 bind to each other to form heterodimers. The immunoprecipitates obtained with anti-IgG antibodies were used as a negative control. (c) H1299 cells transfected with plasmids encoding full-length DDR2 and TM-JM1-JM2 and HeLa cells were lysed, and the whole cell lysates (W) were separated into the plasma membrane (P) and cytosol (C) fractions. EGFR and a-tubulin were used as positive controls for the plasma mem- brane and cytosol fractions, respectively. Endogenous full-length DDR2 (HeLa cells), forced-expressed full-length DDR2 and TM-JM1-JM2 pro- teins were appropriately localized in the plasma membrane. (d) HEK293T cells were transfected with plasmids encoding a C-terminally myc- tagged full-length DDR2 and TM-JM1-JM2. Only under the permeabilized condition, full-length DDR2-myc and TM-JM1-JM2-myc were visual- ized, indicating that the C-termini of these proteins were located in the cytosol and not extracellular space. Full-length DDR2 was used as a positive control. Bar, 50 mm. (e) HEK293T cells were cotransfected with plasmids encoding full-length DDR2 (500 ng) and an increasing amount of TM-JM1-JM2 (100, 300 and 500 ng) as indicated and then stimulated with collagen. Tyrosine phosphorylation gradually decreased with an increasing amount of TM-JM1-JM2. (f) HeLa cells were transiently transfected with a plasmid encoding TM-JM1-JM2 and were then stimulated. Tyrosine phosphorylation was significantly decreased in endogenous DDR2. **p < 0.01, Student’s t-test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Dominant Negative Mutation, Activation Assay, Immunoprecipitation, Western Blot, Negative Control, Transfection, Clinical Proteomics, Membrane, Positive Control, Phospho-proteomics, Plasmid Preparation

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 4. JM2 regulates the collagen-binding affinity of DDR2. (a and b) HEK293T cells transiently transfected with various DDR2 constructs were harvested, and protein expression was verified by Western blot- ting (a). Collagen-binding affinities were reduced in F-DJM2, F-DJM1- JM2 and extra mutants but not the F-DJM1 mutant in a dose- dependent manner (b). Nontransfected (NC) and TM-JM1-JM2 samples were used as negative controls. **p< 0.01, Student’s t-test.

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Binding Assay, Transfection, Construct, Expressing, Western Blot, Mutagenesis

Journal: International journal of cancer

Article Title: The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression.

doi: 10.1002/ijc.28901

Figure Lengend Snippet: Figure 5. Colony formation and proliferation of tumor cells are suppressed by overexpression of JM2. (a and b) Formalin-fixed tissue micro- array slides were used in immunohistochemistry experiments. DDR2 was overexpressed in bladder, testis, lung, kidney, prostate and stom- ach cancers. Bar, 50 mm. (c) Stable TM-JM1-JM2–expressing H1299 cells were stimulated by collagen and then harvested. Immunoprecipitation and Western blot analysis showed that tyrosine phosphorylation of DDR2 was decreased by TM-JM1-JM2 overexpres- sion (labeled JM1/2), but phosphorylation of DDR1 was unaffected. (d) A colony-forming assay of H1299 cells showed that the number and projected area of colonies were decreased by TM-JM1-JM2 overexpression. Bar, 100 mm. (e) Proliferation of H1299 cells was assessed by cell counting (left) and an MTT assay (right). Cell proliferation was inhibited by TM-JM1-JM2 overexpression. **p < 0.01, Student’s t-test; control, nontransfected cells; Mock, empty vector stably transfected cells; JM1/2, TM-JM1-JM2 stably transfected cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The antibodies used in our study were as follows: mouse anti-myc (sc-40; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-HA (sc-805; Santa Cruz Biotechnology), rabbit anti-DDR1 (sc-532; Santa Cruz Biotechnol- ogy), goat anti-DDR2 (sc-7555; Santa Cruz Biotechnology), anti-human DDR2 (AF2538; R&D Systems, Minneapolis, MN), anti-phosphotyrosine (clone 4G10; Upstate Biotechnology, Lake Placid, NY) and peroxidase- and fluoresceinconjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Over Expression, Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Phospho-proteomics, Labeling, Cell Counting, MTT Assay, Control, Plasmid Preparation, Stable Transfection, Transfection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) Immunohistochemistry showing reduced methylated H3K79 levels (H3K79me2) that reflect loss of DOT1L activity in damaged areas from osteoarthritic patients (OA) as compared to their corresponding preserved areas and to cartilage from non-OA patients. Images are representative of images from four different patients. Scale bar, 400 μm. ( b ) Heat maps of differential mRNA expression determined by quantitative PCR in chondrocytes treated with DOT1L inhibitor EPZ-5676 (EPZ) or vehicle (V) from passage 0 (P0) until P2, and from preserved versus damaged areas in OA cartilage. The colour code represents the mean expression level of six and four independent patient samples respectively. ( c ) Immunoblot analysis showing decreased methylated H3K79 levels in mouse articular chondrocytes after intra-articular injection of EPZ into C57Bl/6 wild-type mouse knees. The image is representative of one experiment with protein extracts pooled from two or three mice per condition. Unprocessed original scans of blots are shown in . ( d , e ) C57/Bl6 wild-type mouse knees were injected with EPZ (5 mg kg –1 ) or vehicle and killed after 2 or 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( d ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( e ). One experiment was performed with n =10 and 5. Representative images from the 4 week evaluation are shown. * P <0.05 (two-tailed t -test). Error bars indicate mean±s.e.m.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Immunohistochemistry, Methylation, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a ) KEGG pathway enrichment analysis of microarray data obtained from human articular chondrocytes treated with EPZ-5676 or vehicle. Nominal P values by EASE modified Fisher Exact test using the DAVID analysis tool (see Methods section) are shown. n =5 independent patient-derived cell cultures. ( b ) Co-immunoprecipitation (Co-IP) using an anti-DOT1L antibody showing interaction between DOT1L and β -catenin in human articular chondrocytes, that is increased upon Wnt activation by LiCl and disrupted upon DOT1L inhibition. The image is representative of three experiments. ( c ) TOP/FOP reporter assay in human articular chondrocytes after Wnt stimulation by LiCl and DOT1L inhibition by EPZ. Activity is compared to untreated cells (dotted line). n =3 biologically independent experiments. *** P <0.001 by one-way ANOVA. ( d , e ) LEF1 , TCF1 and c-MYC expression measured by quantitative PCR in chondrocytes treated with EPZ-5676 and LiCl ( d ) or in LiCl-treated chondrocytes transfected with siRNA directed against DOT1L or scrambled siRNA (siDOT1L or siSCR, respectively) ( e ). Data are from one experiment with three technical replicates. ( f ) Immunohistochemistry demonstrating increased TCF1 levels in the articular cartilage of C57/Bl6 wild-type mice after injection of EPZ-5676. The images are representative of three different animals. Scale bar, 200 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Microarray, Modification, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activation Assay, Inhibition, Reporter Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunohistochemistry, Injection

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: All experiments were performed in healthy human articular chondrocytes: treated as indicated with DOT1L inhibitor EPZ-5676, Wnt activator LiCl, SIRT1 antagonist EX527 or SIRT1 agonist SRT1720; or transfected with DOT1L or scrambled siRNA. All data are presented as mean±s.e.m. ( a ) Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analysis of DOT1L and methylated H3K79 and ( b ) acetylated H3K9 (H3K9Ac) and methylated H3K4 (H3K4me3) as markers of active transcription on the transcriptional start site (TSS) of Wnt target genes. Data are from two to five experiments. ( c ) Expression levels of TCF1 Wnt target gene measured by quantitative PCR in chondrocytes transfected with indicated specific or scrambled siRNA (siSCR). Data are from one experiment with technical triplicates. ( d ) TCF1 expression measured by quantitative PCR in the presence of SIRT1 agonist and antagonist. Data from two experiments each with technical triplicates. ( e ) Co-IP analysis using the indicated antibodies demonstrating the interaction of DOT1L and SIRT1. The image is a representative image of three biologically independent experiments. ( f ) SIRT1 activity relative to vehicle-treated cells (dotted line). Data are from three biologically independent experiments. * P <0.05, *** P <0.001 by one-way ANOVA. ( g ) ChIP-qPCR analysis of SIRT1, PPARGC1A, GCN5 and EP300 binding on the TCF1 promoter and ( h ) TCF1 expression after siRNA transfection with indicated specific or scrambled siRNA. Data from two biologically independent experiments.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Methylation, Expressing, Co-Immunoprecipitation Assay, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: ( a – c ) Inactivation of SIRT1 protects against DOT1L inhibitor-induced osteoarthritis: ( a ) C57/Bl6 wild-type mouse knees were injected with DOT1L inhibitor EPZ-5676 (5 mg kg −1 ) and SIRT1 inhibitor EX527 (1.25 mg kg –1 ), or vehicle (V) and killed after 4 weeks. Knees were sectioned and stained with Hematoxylin-Safranin O ( a ). Scale bar, 200 μm. Cartilage damage was scored (see Methods section) and is shown in ( b ). One experiment was performed with n =3 (vehicle), 8 (EPZ) and 10 (EPZ+EX527). * P <0.05 by one-way ANOVA. Error bars indicate mean±s.e.m. ( c ) Immunohistochemistry of TCF1 in the indicated groups. TCF1 levels are increased after EPZ treatment and normalized by additional EX527 treatment. The images are representative of three different animals. Scale bar, 200 μm. ( d , e ) Loss of DOT1L function causes severe growth retardation as demonstrated by skeletal staining ( d ) and histology of the growth plate ( e ) of 4-week-old Dot1l fl/fl ;Col2-Cre −/− (Cre-neg) and Dot1l fl/fl ;Col2-Cre +/− (Dot1l Cart-KO ) mice. ( f , g ) Increased TCF1 levels in Dot1l Cart-KO mice as shown by immunohistochemistry in the indicated mice strains in the articular cartilage ( f ) and growth plate ( g ). The images are representative of three different animals. Scale bar, 200 and 100 μm.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: DOT1L safeguards cartilage homeostasis and protects against osteoarthritis

doi: 10.1038/ncomms15889

Figure Lengend Snippet: Upon Wnt signalling activation, DOT1L-containing complexes bind Wnt target gene chromatin. DOT1L interacts with SIRT1 and inhibits its function, preventing Wnt pathway hyper-activation. When Wnt signalling is activated in the absence of DOT1L function, high SIRT1 activity mediates the recruitment of transcriptional activators to LEF1 and TCF1 genes. High Wnt signalling leads to deleterious downstream effects and loss of cartilage homeostasis.

Article Snippet: Antibody binding to the column was performed using 75 μg of either a mock antibody (donkey anti-goat IgG) as a control or DOT1L antibody (R&D Systems, MAB6546, clone 653613).

Techniques: Activation Assay, Activity Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Immunohistochemistry staining with antibody against phosphorylated Smad2 (pSmad2) and TGFβ receptor II (TβRII) showed increased nuclear signal for pSmad2 in the invasive ECdnT organotypic cultures. Scale bar 50 micron. (b) Analysis of immunohistochemistry staining for TβRII and pSmad2 in 83 ESCC cases in a tissue microarray shows no significant correlation. Fisher’s exact test, two tailed p= 0.3182. (c) Five paired normal adjacent and ESCC tissues (GSE17531) were analyzed for INHBA mRNA expression, which identified upregulation of INHBA in four ESCC samples. (d) Waterfall plot of a publically available dataset (GSE23400) represented upregulation of INHBA in the ESCC (grey bars) samples vs. normal (black bars).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Immunohistochemistry, Staining, Microarray, Two Tailed Test, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures with either fetal esophageal fibroblasts (FEF) or cancer-associated fibroblasts (CAF) embedded in the underlying matrix. Immunofluorescence staining with antibody against αSMA (green) and podoplanin (red) showed similar expression pattern in the cultures. Scale bar is 50 micron. (b) Activin A concentration in conditioned media from organotypic cultures is higher in invasive cultures as measured using indirect ELISA. * p=0.003, ** p= 0.005, *** p=0.03 (c) Stimulation of epithelial cells with Act A in monolayer plastic culture demonstrated phosphorylation of Smad. Neutralizing antibody against Activin (nAb) prevented the induction of pSmad2 by Act A. Following stimulation with Act A or with conditioned media from organotypic culture increased expression of vimentin was detected after 48 hours by Western Blot. The increase was reversed in the presence of neutralizing antibody (nAb). (d) Inhibition with the Act A antagonist, Follistatin, or a pan-TGFβ inhibitor A83-01 suppressed MMP-9 secretion in E, EC and ECdnT cells as measured by gelatin zymography. Upper bands reflect pro-MMP, lower bands activated, cleaved MMP (arrow).

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Immunofluorescence, Staining, Concentration Assay, Indirect ELISA, Phospho-proteomics, Western Blot, Inhibition, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Esophageal epithelial cells expressing wild-type full-length E-cadherin (E), dominant-negative mutant E-cadherin (EC) or dominant-negative mutant E-cadherin and TGFβ receptor II (ECdnT) were grown in organotypic cultures in the presence of recombinant Activin A (Act A), its antagonist Follistatin or a neutralizing antibody against Activin A (nAb); H&E staining. Stimulation with Act A inhibited invasion of E and EC cells, but failed to suppress ECdnT cell invasion. Follistatin increased cell invasion in all cell types, while the neutralizing antibody prevented invasion of E and EC cells, without an effect on ECdnT cells. (b) Immunohistochemistry staining with ki67-antibody showed no differences in cell proliferation. Scale bars are 50 micron. (c) Indirect ELISA with antibody against Act A measured increased levels after addition of recombinant Act A in fibroblasts (FEF) and ECdnT. Untreated ECdnT cells (Control) secreted higher baseline levels of Act A than FEF, which were reduced by Follistatin. (d) TGFβ1 concentration was increased in response to stimulation with Act A and overall baseline secretion was higher in control ECdnT cells than fibroblasts as measured by indirect ELISA. Follistatin inhibited TGFβ1 secretion.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Expressing, Dominant Negative Mutation, Recombinant, Staining, Immunohistochemistry, Indirect ELISA, Control, Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Activin A balance regulates epithelial invasiveness and tumorigenesis

doi: 10.1038/labinvest.2014.97

Figure Lengend Snippet: (a) Separating the cellular matrix and epithelium of the organotypic cultures growing ECdnT cells through a collagen I layer, dashed lines, prevented cell invasion in the absence (control) and presence of Act A (+Act A). When the cellular matrix of the organotypic culture was treated with puromycin to kill the embedded fibroblasts before the ECdnT cells were seeded, epithelial formation occurred but invasion was inhibited with and without Act A stimulation. (b) Treatment of ECdnT organotypic cultures with a pan-MMP inhibitor, GM6001, suppressed cell invasion, which was not restored in the presence of Act A. Untreated (no tx) control ECdnT cells in organotypic culture invaded into the underlying matrix. TGFβ1 treatment inhibited epithelial cell invasion. Scale bars are 50 micron. (c) Immunohistochemistry showed nuclear localization of phosphorylated Smad (pSmad2, red) in control and Act A stimulated conditions. Collagen IV, red, was disrupted in invasive cultures after Act A treatment. Loss of the fibroblasts (FEF), labeled green with antibody against vimentin (no staining in the lower panels), had no effect on the nuclear localization of pSmad2. The collagen IV layer was not disrupted in non-invasive cultures in the absence of FEFs.

Article Snippet: The following treatments were added to the organotypic cultures at the time of epithelial seeding and renewed with every media change: Five ng/ml recombinant human TGFβ1, 10ng/ml Activin A, 100 ng/ml Follistatin and 600 ng/ml neutralizing antibody against Activin A (all from R&D Systems, Minneapolis, MN), or 1 μM A83-01 (Tocris, Bristol, UK) and 1 μM GM6001 (Millipore EMD, Billerica, MA).

Techniques: Control, Immunohistochemistry, Labeling, Staining

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Antibody panels used for immunohistochemistry on HD tissue microarrays.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemistry, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Immunohistochemical profiling of HTT inclusion body ubiquitination and associated triage protein binding in the HD human middle temporal gyrus. Multiplexed immunohistochemical approaches were used to identify HTT inclusion bodies, ubiquitin species, and triage proteins in neurologically normal and HD human middle temporal gyrus tissue microarray cores. Example images from HD case, HC150, are shown. HTT inclusion body antibodies, EM48 ( A ), EPR ( B ), and MW1 ( C ), were used for labelling together with antibodies for pan-ubiquitin ( D ), K48- and K63-linked polyubiquitination ( E and F ), p62 ( G ), and ubiquilin 2 ( H ), with a Hoechst nuclear counterstain ( I ); scale bars = 20 μm.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Immunohistochemical staining, Ubiquitin Proteomics, Protein Binding, Microarray

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: HTT inclusion bodies are not frequently ubiquitinated, but when ubiquitinated, are predominantly ubiquitinated by K63-linked ubiquitin. Immunohistochemical labelling revealed that EM48, EPR, and/or MW1 HTT inclusion bodies were ubiquitinated by K48- and/or K63-linked ubiquitin ( A ); a representative image of K48- and K63-ubiquitinated HTT inclusion bodies from HD case, HC145, is shown; scale bars = 10 μm. The ubiquitination status of each HTT inclusion body was determined by labelling for pan-, K48-, and K63-linked ubiquitin, where positive labelling was identified if the maximum intensity was above manually determined thresholds. The percentage of EM48 + versus EM48- ( B ), EPR + versus EPR- ( C ), and MW1 + versus MW1- ( D ) HTT inclusion bodies that were ubiquitinated (either pan, K48-, and/or K63-linked) were compared using a Wilcoxon matched-pairs signed rank test. The percentage of ubiquitinated HTT inclusion bodies was determined for each EM48, EPR, and MW1 +/- phenotype per HD case ( E ), and compared between phenotypes using a mixed-effects analysis, with Geisser-Greenhouse correction and Tukey’s multiple comparisons test. The percentage of ubiquitinated HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin was compared using a Wilcoxon matched-pairs signed rank test ( F ). The percentage of ubiquitinated EM48 + versus EM48- ( G ), EPR + versus EPR- ( H ), and MW1 + versus MW1- ( I ) HTT inclusion bodies ubiquitinated by K48- versus K63-linked ubiquitin were compared using an ordinary two-way ANOVA with Tukey’s multiple comparisons test. The percentage of EM48, EPR, and MW1 +/- immunophenotypes HTT inclusion bodies identified as being ubiquitinated by K48- or K63-linked chains were compared using an ordinary two-way ANOVA with Sidak’s multiple comparisons test ( J ). Data are presented as truncated violin plots ( n = 20). Statistical significance of differences shown for B-D and F-J: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Statistical significance for E is shown in Supplementary Table 3.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Huntingtin inclusion bodies have distinct immunophenotypes and ubiquitination profiles in the Huntington’s disease human cerebral cortex

doi: 10.1038/s41598-025-00465-w

Figure Lengend Snippet: Summary of HTT inclusion body characteristics. Heatmap organised by HTT inclusion body phenotype, with each column representing a single case and each bar coloured according to that case’s value for the characteristic outlined by the row title ( A ). Schematic illustrating the general characteristics of each HTT inclusion body phenotype: (1) EPR + MW1 + inclusion bodies are more frequently located in the nucleus compared to other phenotypes, (2) HTT inclusion bodies that label for more than one epitope-specific antibody are more frequently ubiquitinated, and that ubiquitination occurs more frequently by K63- compared to K48-linked ubiquitin chains, (3) Ubiquitinated HTT inclusion bodies are more frequently tagged by ubiquilin 2 than p62 ( B ). Schematic summarising our hypothesis of HTT inclusion body immunophenotype, ubiquitination, and triage protein tagging with increasing HD severity ( C ); created in BioRender.

Article Snippet: K63-linked ubiquitin , Novus HWA4C4 , Mouse IgG2a , 1:300 , Goat anti- mouse IgG2a 647 , ThermoFisher A21241 , 3.

Techniques: Ubiquitin Proteomics

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: A. Slpi expression from the microarray data of BMDCs exposed to vehicle or ROBs-QP at day 5 and 7 and treated with 1 μg/mL of LPS for 6 hours (n=4, **P<0.01). B. Time course mRNA expression of Slpi mRNA measured by qPCR of BMDCs exposed to vehicle (black bars) or ROBs-QP (white bars) at day 5 and 7 and treated with 1 μg/mL of LPS. Fold change are expressed relative to vehicle at time 0. mRNA was extracted at indicated time points and bars represent the mean ± SEM of 3 independent experiments. (*P<0.05, **P<0.01).

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Expressing, Microarray

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: A. BMDCs were cultured from WT and Slpi-KO mice and treated with quercetin at day 5 and 7. BMDCs cultures were exposed to 1 μg/mL of LPS and the secretion of TNFα was determined by ELISA after 24 hours. Bars represent mean cytokine concentration ± SEM (n=4) for BMDCs from WT or Slpi-KO mice treated with and without quercetin (black and white bars, respectively, ***P<0.001). B. BMDCs were transfected with siRNA for Slpi and Lipofectamine® 3000 reagent (mock) at day 4, before the administration of quercetin. qPCR for Slpi demonstrated a good efficiency for siRNA transfection (left). Bars represent a mean fold change ± SEM (n=3) between LPS stimulated BMDCs +/− quercetin relative to LPS unstimulated cells +/− quercetin, respectively. **P<0.01. The secretion of TNFα was determined 24 hours after LPS stimulation by ELISA (right). Bars represent mean cytokine concentration ± SEM (n=3) for DCs treated with and without quercetin (black and white bars, respectively). ***P<0.001. C. BMDCs were treated with quercetin on day 5 and transfected with siRNA for Slpi at day 6 followed by quercetin administration at day 7 and a subsequent exposure to LPS on day 8. A good reduction in Slpi expression was observed by qPCR analysis (left). Bars represent a mean fold change ± SEM (n=3) between LPS stimulated BMDCs +/− quercetin relative to LPS unstimulated cells +/− quercetin, respectively. **P<0.01. The secretion of TNFα was determined 24 hours after LPS stimulation by ELISA (right). Bars represent mean cytokine concentration ± SEM (n=3) for DCs treated with and without quercetin (black and white bars, respectively). *P<0.05.

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Transfection, Expressing

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: A. Supernatant collected from WT BMDCs culture stimulated at day 5 and day 7 with quercetin was added 1:1 to an unrelated BMDCs culture (left). After 1 hour the recipient BMDCs were stimulated with 1μg/ml of LPS for 24 hours and the amount of TNFα was tested by ELISA relative to SN untreated BMDCs (panel B, white bar and black bar, respectively). *P<0.05. B. To discriminate the contribution of the endogenous and the extracellular form of Slpi, the SN collected from WT BMDCs plus quercetin was treated with an anti-Slpi antibody (BAF1735, R&D, panel A, right). Supernatant incubated with the antibody was treated with LPS as before and tested by ELISA for TNFα secretion (panel B, stripped bar). Bars represent mean cytokine concentration ± SEM (n=3) for BMDCs from WT mice.

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: Gavage of quercetin or vehicle was administered at day 0, 3, 5 and 7. Starting from day 4, 2%DSS was administered for 3 days in drinking water. At day 7 mice were sacrificed, the colon collected and the mRNA extracted. Slpi expression was measured by qPCR in the colon of vehicle (black bars) and quercetin (white bars) treated mice exposed or not to DSS. Bars represent mean expression ± SEM (n=3) for each treatment. ***P<0.001.

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Expressing

Journal: Oncotarget

Article Title: Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion

doi: 10.18632/oncotarget.12415

Figure Lengend Snippet: A-D. Representative immunohistochemistry images for SLPI expression in normal (panels A and B) and inflamed (panels C and D) human intestinal biopsies. FFPE sections were prepared at 20 μm thickness, stained for Slpi and visualized using BioCare polymer IHC kit. E. SLPI expression was measured on human normal and inflamed areas by qPCR relative to untreated non-inflamed biopsies. Bars represent mean ± SEM (n=7). **P<0.01; ***P<0.001. F. qPCR on human inflamed tissues treated with quercetin relative to inflamed untreated ones. Bars represent mean ± SEM (n=7). *P<0.05.

Article Snippet: Gene expression of Slpi and GAPDH was performed with TaqMan Gene Expression Assays (Thermo Fisher Scientific, MA, USA) - murine probes: Mm00441530_g1 and Mm00484668_m1, respectively; human probes: Hs00268204_m1 and Hs02758991_g1, respectively.

Techniques: Immunohistochemistry, Expressing, Staining, Polymer

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 2 DACH1 deletion PCa enhances AR signaling. A Interrogation of human PCa gene expression data [26], showing candidate genetic drivers ERG, ETV1/ETV4/FLI1, SPOP, FOXA1, and unknown. Samples with DACH1 homozygous (deep) genetic deletions (29/333) are shown as an additional subtype. The AR score (the average of the AR target gene expression) refers to a group of AR-responsive genes [26], and together with the expression Z-score of the AR target genes, are shown as colorimetric scales. The AR score-based gene names are shown. The androgen receptor (AR) activity, inferred by the induction of AR target genes, was increased in DACH1 homozygous (‘deep’) deletion PCa compared with normal (P = 2 × 10−5 by t-test) and ERG mutation groups (P = 0.003 by t-test). B AR mRNA and AR protein levels, shown for each DACH1 deletion sample, were not significantly different. C The iCluster [29], mRNA cluster, and SCNA (somatic copy-number alteration), and DNA methylation status are shown for the PCa classified by the corresponding gene deletion subtypes. D DACH1 homozygous deletions were enriched for iCluster 2 and 3 [29], mRNA cluster 2 (P = 0.0003 by Fisher exact test, SCNA (“more” somatic copy-number alteration, P = 0.0004 by Fisher exact test), but not for DNA methylation.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Gene Expression, Targeted Gene Expression, Expressing, Activity Assay, Mutagenesis, DNA Methylation Assay

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 3 Prostate-specific Dach1 gene deletion promotes prostate hyperplasia and dysplasia in OncoMice (15 weeks). A Schematic representation of transgenes integrated into mice. B Representative immunohistochemistry for Dach1, with data quantitated as mean ± standard error of the mean (SEM) for N = 20 (4 separate mice, with 5 views per mouse, in each group). C Blinded quantitative histology grading of prostate of multigenic mice at 15 weeks. Data are shown as mean ± SEM for N = 15 (5 separate mice, with 3 prostate areas [anterior, ventral, lateral] per mouse) in each group). H&E staining demonstrates the presence of a focal atypical intraductal proliferation in Dach1−/−prostate, compatible with prostatic intraepithelial neoplasia (PIN). Representative immunohistochemistry with results shown as mean ± SEM for Ki-67 (n = 20, 4 separate mice for each genotype, 5 views per mouse) (D), Beclin 1 (n = 9, 3 separate mice for each genotype, 3 views per mouse) (E); and AR (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 12 for Dach1fl/flmice, 3 separate mice, 2 views for one mouse and 5 views for other two mice) (F). Scale bars, 50 μm. A Student’s t test was performed for all comparisons.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Immunohistochemistry, Staining

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 4 Prostate-specific Dach1 gene deletion in TRAMP mice induces PIN lesions with increased TGFβ activity. Genome-wide expression analysis of TRAMP Dach1+/+ vs. Dach1−/−PIN lesions was analyzed for enrichment of known targets of upstream regulators using Ingenuity Pathway Analysis (IPA) and represented as (A) barplot was calculated by IPA activation Z-score labeled and as (B) bubble plot with size of the bubbles proportional to –log10 p values. C IHC was conducted for SMAD activation using SMAD2P, quantitated and shown as (D) mean ± SEM (n = 15 for Dach1wt/wt mice, 3 separate mice, 5 views per mouse) (n = 10 for Dach1fl/flmice, 2 separate mice, 5 views per mouse). E–G Western blot of either PCa cell lines for the presence of DACH1 (E, F) or (G) TGFβ-treated (10 ng/ml for 24 h) PC3 cells illustrating induction of nuclear vimentin and cytoplasmic cyclin D1. Protein loading controls are β-tubulin (a marker of cytoplasmic proteins) and Lamin B1 (a marker for nuclear protein enrichment). H Microarray-based gene expression analysis of PC3 cells stably expressing DACH1, showing restraint of genes mediating TGFβ signaling (shown with blue arrows), including reduction of TGFB2 and TGFBR2 [33].

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Activity Assay, Genome Wide, Expressing, Activation Assay, Labeling, Western Blot, Marker, Protein Enrichment, Microarray, Gene Expression, Stable Transfection

Journal: Oncogene

Article Title: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses.

doi: 10.1038/s41388-023-02668-9

Figure Lengend Snippet: Fig. 6 DACH1 facilitates the recruitment of, and co-accumulates with, Ku70/Ku80 proteins at sites of DNA damage. A Co-accumulation of Ku-70/Ku-80 at laser micro irradiation-induced DSBs sites in Dach1+/+ 3T3 cells. B, C 24 h after transfection, the accumulation of DACH1 and Ku70/Ku80 in Dach1−/−3T3 cells transfected with EGFP or EGFP-tagged DACH1 and red fluorescent protein (RFP)-tagged Ku70 or RFP-tagged Ku80 expression vectors were treated with laser micro-irradiation (403 nm) to induce DSBs. Time is shown after micro-irradiation. Accumulation of the transfected proteins was indicated by EGFP (green) or RFP (red) fluorescence at laser-irradiated sites. Co-accumulation was visualized in yellow merged images. Time is shown in minutes and -fold increase in foci intensity is shown as mean ± SEM for N = 5 separate cells.

Article Snippet: For detection of DACH1 protein, antigen retrieval was done in Tris/EDTA buffer at pH 9 for 30min at 97 °C, followed by 30min incubation with rabbit polyclonal DACH1 antibody (Cat. #10914-1-AP, Proteintech, Rosemont, IL; dilution 1:1,000) [33], HRP-conjugated polymer (Envision FLEX, Cat#GV80011-2, Agilent), and DAB chromogen deposition.

Techniques: Irradiation, Transfection, Expressing

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

doi: 10.1016/j.jcmgh.2022.07.005

Figure Lengend Snippet: Figure 8. Negative corre- lation between ecto- enzymes and CCND1 expression in CRC. (A) IHC staining of human normal colon (n ¼ 40) and colon adenocarcinoma (n ¼ 64) specimens in a tissue microarray for CCND1 and CD163 (a macrophage marker). (B) Relative intensities of CCND1 staining in the lamina propria of a normal and cancerous colon. **P < .01 (unpaired t test). (C) Correlation analysis of NT5E and CCND1 expres- sion in the TCGA colorectal adenocarcinoma dataset. ***P < .001 (Student t test).

Article Snippet: For the fluorescence-activated cell sorting analysis, cells were stained with APC,-anti human CD163 (clone: GHI/61), PD-anti human CTLA4 (clone: L3D10), Alexa 647-anti human IDO1 (clone: 2E2/IDO1), PE-anti human DR4 (clone: DJR1), FITC-anti human CD47 (clone: REA220, FITC-anti human MICA&B (clone: 6D4), PE-anti human PD-L1 (clone: MIH2), PD-anti human CD69 (clone: FNM50), FITCanti human CD2 (clone: RPA-2.10), FITC-anti human CD20 (clone: 2H7), APC-anti mouse F4/80 (clone: BM8), Brilliant Violet 421TM-anti mouse CD11b (clone: M1/70), Brilliant Violet 570TM-anti mouse CD45 (clone: 104), PE-anti-human CD45 (clone: HI30), monoclonal antibodies (Biolegend; San Diego, CA, USA), FITC-anti-human CD40 (clone: REA733), PE-anti human CD80 (clone: 2D10), PE/vio770-anti human CD206 (clone: DCN228), PE-anti human CD62E (clone: REA280), PE-anti human CD192 (clone: REA264), APC-anti human I-CAM (clone: REA266), APC-anti human HLA-DR, DP, DQ (clone: REA332), APC-anti human CCL2 (clone: REA485), APC-anti human CD14 (clone: HI30), Vioblue-anti human CD31 (clone: TUK4), FTIC-anti-human CD86 (clone: FM95), PE-anti human CD73 (clone: AD2), PE-anti mouse PD-L1 (clone: 60533), PE-anti mouse CD39, and PE-anti human CD39 (clone: REA739) monoclonal antibodies (Miltenyi Biotec; Bergisch Gladbach, Germany), and PE/CF594anti human CD3 (clone: UCHT1), PE-anti human CD44 (clone: G44-26), PE-anti human CD183 (clone: 150503), and PD-1 (clone: REA739) monoclonal antibodies (BD Biosciences; San Jose, CA, USA) following the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemistry, Microarray, Marker, Staining

Journal: Nature Communications

Article Title: Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice

doi: 10.1038/s41467-023-39586-z

Figure Lengend Snippet: a Schematic of cyp27a1 conversion of cholesterol to 27HC in a macrophage. 27HC content ( b , n = 8, 6) and cyp27a1 expression ( c , n = 5) were evaluated in aortas from apoE +/+ and apoE −/− mice following receipt of standard chow or atherogenic diet, respectively. d C yp27a1 expression in circulating monocytes (n = 8) from apoE +/+ and apoE -/- mice administered diets as in ( b ). e , f Cyp27a1 expression was assessed in human atherosclerotic versus control arteries in two independent cohorts. Cohorts I ( e ) and II ( f ) contained n = 4 and 198, and 32 and 32 samples respectively. FPKM is fragments per kilobase of transcript per million mapped reads. Box-and-whisker plots are provided; central line denotes the median value, edges represent the upper and lower quartiles, and whiskers indicate minimum and maximum values. In ( b ), ( c ) and ( d ) values are expressed relative to findings in apoE +/+ . g–r Atherosclerosis and related parameters in apoE -/- background cyp27a1 fl/fl and cyp27a1 ΔMAC mice. Representative lipid-stained en face images of aortas ( g ) and lesion areas ( h ; percent of total surface area, n = 10 and 9). Representative lipid and haematoxylin-stained aortic root sections ( i ), and lesion areas ( j , n = 11 and 9). Representative immunohistochemistry images of Mac2 staining in aortic root ( k ; nuclei DAPI stained) and quantification of Mac2 positive staining expressed per unit lesion area ( l ; n = 8). m Monocyte recruitment to atherosclerotic lesions was evaluated by YG bead incorporation into circulating monocytes and quantification of beads per aortic root section 24 h later. N = 8. Immunohistochemical determinations of endothelial ICAM-1 ( n , p ) and VCAM-1 abundance ( o , q ) with representative images ( n , o ) and quantification ( p , q , n = 9 and 8). Arrows indicate endothelium. r Inflammatory gene expression in the aorta (n = 4). s Representative still images of leukocyte-endothelial cell adhesion in apoE -/- background cyp27a1 fl/fl and cyp27a1 ∆MAC mice. t Summary data for leukocyte velocity (n = 4). Scale bar equals 100 um ( i ) or 50 um ( k , n , o ). Except in ( e ) and ( f) , data are mean ± SEM. P values by two-sided Student’s t-test ( b – f , h , j , l , m , p , q , t ) or two-sided Mann-Whitney U-test ( r ) are shown. Source data are provided as a Source Data file.

Article Snippet: HAEC were infected with the adenovirus (MOI 200) in EBM2 medium with 2% FBS for 5 h, and EBM2 medium with 10% FBS was then added for 72 h. Subsequently the cells were treated with vehicle, E2 (10 −8 M) or 27HC (10uM) for 10 min, proteins were crosslinked using 1% formaldehyde (8 min) followed by quenching with 2 M glycine, cells were washed, proteins were extracted, and ERα was immunoprecipitated with anti-ERα antibody (HC-20, Santa Cruz Biotechnology, sc-543, 1:50).

Techniques: Expressing, Control, Whisker Assay, Staining, Immunohistochemistry, Immunohistochemical staining, Gene Expression, MANN-WHITNEY

Journal: Nature Communications

Article Title: Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice

doi: 10.1038/s41467-023-39586-z

Figure Lengend Snippet: a Representative still images of leukocyte-endothelial cell adhesion evaluated by intravital microscopy in ERα fl/fl and ERα ∆EC mice injected with vehicle versus 27HC (20 mg/kg BW) daily for 3 days. b Summary data for leukocyte velocity in ( a ), n = 11-12. c – f ApoE -/- background ERα fl/fl and ERα ΔEC mice were placed on an atherogenic diet for 8 weeks and administered vehicle versus 27HC, and samples were obtained. Representative lipid-stained en face images of aortas ( c ), and lesion areas ( d , percent of total surface area). N = 13 and 10 for ERα fl/fl given vehicle versus 27HC, respectively, and n = 10 and 12 for ERα ΔEC given vehicle versus 27HC, respectively. Representative lipid and haematoxylin-stained aortic root sections ( e ), and lesion areas ( f , n = 10). g – o , Atherosclerosis and related parameters in bone marrow transplant experiments performed employing apoE -/- background cyp27a1 fl/fl versus cyp27a1 ΔMAC donors and apoE -/- background ERα fl/fl versus ERα ΔEC recipients. g Schematic of donors and recipients. Representative lipid-stained en face images of aortas ( h ) and lesion areas ( i , n = 11-12). Representative lipid and haematoxylin-stained aortic root sections ( j ), and lesion areas ( k , n = 11-12). Representative immunohistochemistry images of Mac2 staining in aortic root ( l ; nuclei DAPI stained) and quantification of Mac2 positive staining ( m ; expressed per unit lesion area, n = 8−10). Immunohistochemical determinations of endothelial ICAM-1 and VCAM-1 abundance with representative images ( n ) and quantification ( o , n = 8-9). Arrows indicate endothelium. Scale bar equals 100 um ( e , j ) or 50 um ( l , n ). Data are mean ± SEM. In ( b ) and ( d ), P values shown are by Krusakal-Wallis with Dunn’s post hoc testing. In all other graphs ( f , i , k , m , o ), P values shown are by ANOVA with Tukey’s post-hoc testing. Source data are provided as a Source Data file.

Article Snippet: HAEC were infected with the adenovirus (MOI 200) in EBM2 medium with 2% FBS for 5 h, and EBM2 medium with 10% FBS was then added for 72 h. Subsequently the cells were treated with vehicle, E2 (10 −8 M) or 27HC (10uM) for 10 min, proteins were crosslinked using 1% formaldehyde (8 min) followed by quenching with 2 M glycine, cells were washed, proteins were extracted, and ERα was immunoprecipitated with anti-ERα antibody (HC-20, Santa Cruz Biotechnology, sc-543, 1:50).

Techniques: Intravital Microscopy, Injection, Staining, Immunohistochemistry, Immunohistochemical staining

Journal: Nature Communications

Article Title: Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice

doi: 10.1038/s41467-023-39586-z

Figure Lengend Snippet: a – c 27HC activation of NF-κB activity in HAEC via ERα. a , NF-κB luciferase activity was measured in HAEC treated with TNFα (10 ng/ml), E2 (10 −8 M),TNFα plus E2, or 27HC (20uM) for 6 h (n = 8). b , c Following transduction with control shRNA versus shRNA targeting ERα ( b , findings for 3 samples per group are shown), the experiments described in ( a ) were performed ( c , n = 8). d Venn diagrams of proteins identified by LC-MS/MS to have increased or decreased association with ERα upon E2 versus 27HC treatment in HAEC. Diagrams display intersects for common directional changes with the two ligands (upper panel), and opposing directional changes with the two ligands (lower two panels). e Co-immunoprecipitation (IP) of endogenous ERα and septin 11 in HAEC treated with vehicle versus E2 (left panel), or with vehicle versus 27HC (right panel) for 30 min. f HAEC were treated with 27HC, and IP of endogenous ERα was performed and immunoblotting was done for ERα and septin 11 (upper panel), or IP of septin 11 was performed followed by immunoblotting. Septin 11 expression in human atherosclerotic versus control arteries in two independent cohorts. Cohorts I ( g ) and II ( h ), which were analyzed by RNAseq and microarray, respectively, contained n = 4 and 201, and 32 and 32 samples respectively. FPKM is fragments per kilobase of transcript per million mapped reads. Box-and-whisker plots are provided in which the central line denotes the median value, the edges represent the upper and lower quartiles, and whiskers indicate the minimum and maximum values. Effect of vehicle versus 27HC on NF-κB activity in HAEC expressing or lacking ERα ( i , n = 8) or septin 11 ( j , n = 4). In ( a ), ( c ), ( i ) and ( j ), NF-κB activity is expressed relative to values with vehicle treatment. Data are mean ± SEM. In ( a ) and ( c ), P values shown are by Krusakal-Wallis with Dunn’s post hoc testing, and in ( g ) and ( h ) two-sided Student’s t tests were used. In ( i ) and ( j ), P values by ANOVA with Tukey’s post-hoc testing are shown. The findings in ( e ) and ( f ) were confirmed in two independent experiments. Source data are provided as a Source Data file.

Article Snippet: HAEC were infected with the adenovirus (MOI 200) in EBM2 medium with 2% FBS for 5 h, and EBM2 medium with 10% FBS was then added for 72 h. Subsequently the cells were treated with vehicle, E2 (10 −8 M) or 27HC (10uM) for 10 min, proteins were crosslinked using 1% formaldehyde (8 min) followed by quenching with 2 M glycine, cells were washed, proteins were extracted, and ERα was immunoprecipitated with anti-ERα antibody (HC-20, Santa Cruz Biotechnology, sc-543, 1:50).

Techniques: Activation Assay, Activity Assay, Luciferase, Transduction, Control, shRNA, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot, Expressing, Microarray, Whisker Assay

Journal: Nature Communications

Article Title: Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice

doi: 10.1038/s41467-023-39586-z

Figure Lengend Snippet: a HAEC were treated with 27HC and immunoblotting was performed to detect Jnk Thr183/Tyr185 phosphorylation and total Jnk. b Following transfection of HAEC with control shRNA or shRNA targeting Jnk1 (upper panel, findings for 2 samples/group are shown), the effects of vehicle versus 27HC on NF-κB activity were determined (lower panel, n = 8). Following transduction of HAEC with control shRNA or shRNA targeting ERα ( c ) or septin 11 ( d ), HAEC were treated with 27HC, and immunoblotting was performed to detect activating phosphorylation of MLK3, MKK7 and Jnk, and total MLK3, MKK7 and Jnk. HAEC were treated with 27HC for 0 to 10 min, MKK7 was IP’d and immunoblotting was performed for MKK7 and GADD45β ( e ), or septin 11 was IP’d and immunoblotting was done for MKK7 and septin 11 ( f ). g In pull-down experiments a complex between HA-tagged bound MKK7 and GADD45β was formed. Wild-type septin 11 (WT) or septin 11 deletion mutants lacking AA184-192 (septin 11-∆G4) or AA38-304 (septin 11-∆GTP) were added, and protein associations were evaluated by immunoblotting. h , i Requirement for septin 11 interaction with MKK7 in 27HC action was evaluated in HAEC in which endogenous septin 11 was knocked down by shRNA, and reconstitution was performed with either wild-type septin 11, septin 11-∆G4, or septin 11-∆GTP. ( h ) Cells were treated with vehicle or 27HC, and immunoblotting was performed to detect activating phosphorylation of MKK7 and Jnk, and total MKK7 and Jnk. ( i ) In the same study groups as in ( h ), NF-κB activity was evaluated in cells treated with vehicle or 27HC. n = 8. In ( b ) and ( i ), NF-κB activity is expressed relative to values with vehicle treatment. Data are mean ± SEM. In ( b ) and ( i ), P values by ANOVA with Tukey’s post-hoc testing are shown. The findings in ( a ) and ( c )–( h ) were confirmed in two independent experiments. Source data are provided as a Source Data file.

Article Snippet: HAEC were infected with the adenovirus (MOI 200) in EBM2 medium with 2% FBS for 5 h, and EBM2 medium with 10% FBS was then added for 72 h. Subsequently the cells were treated with vehicle, E2 (10 −8 M) or 27HC (10uM) for 10 min, proteins were crosslinked using 1% formaldehyde (8 min) followed by quenching with 2 M glycine, cells were washed, proteins were extracted, and ERα was immunoprecipitated with anti-ERα antibody (HC-20, Santa Cruz Biotechnology, sc-543, 1:50).

Techniques: Western Blot, Phospho-proteomics, Transfection, Control, shRNA, Activity Assay, Transduction

Journal: Nature Communications

Article Title: Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice

doi: 10.1038/s41467-023-39586-z

Figure Lengend Snippet: a–d ApoE -/- mice were placed on an atherogenic diet for 8 weeks, receiving vehicle or the cyp27a1 inhibitor GW273297X (GW) during the last 4 weeks, and atherosclerotic lesions were evaluated. Representative lipid-stained en face images of aortas ( a ) and lesion areas ( b ; percent of total surface area, n = 14). Representative lipid and haematoxylin-stained aortic root sections ( c ), and lesion areas ( d , n = 10). e – h In parallel experiments, apoE -/- mice received vehicle, cyp27a1 inhibitor GW, or GW plus exogenous 27HC during the last 4 weeks of an 8 week period on an atherogenic diet. Representative lipid-stained en face images of aortas ( e ) and lesion areas ( f , n = 9-13). Representative lipid and haematoxylin-stained aortic root sections ( g ), and lesion areas ( h , n = 8-9). i – l The effect of vehicle versus GW was determined in apoE -/- background cyp27a1 fl/fl and cyp27a1 ΔMAC mice. Representative lipid-stained en face images of aortas ( i ) and lesion areas ( j , n = 9-12). Representative lipid and haematoxylin-stained aortic root sections ( k ), and lesion areas ( l , n = 9-12). Effect of cyp27a1 inhibitor on abundance of endothelial ICAM-1 ( m, n ) and VCAM-1 abundance ( o , p ) with representative images ( m , o ) and quantification ( n , p , n = 8-9). q Effect of cyp27a1 inhibitor on monocyte recruitment, evaluated by YG bead incorporation into circulating monocytes and quantification of beads per aortic root section 24 h later. The quantitation was corrected for monocyte labeling efficiency. N = 7-8. r , s Effect of cyp27a1 inhibitor on lesion macrophage accumulation. Representative immunohistochemistry images of Mac2 staining in aortic root ( r ; nuclei DAPI stained) and quantification of Mac2 positive staining ( s ; expressed per unit lesion area, n = 8-9). Scale bar equals 100 um ( c , g , k ) or 50 um ( m , o , r ). Data are mean ± SEM, P values by two-sided Student’s t test ( b , d , n , p , q and s ), by Krusakal-Wallis with Dunn’s post hoc testing ( f ), or by ANOVA with Tukey post-hoc testing ( h , j and l ) are shown. Source data are provided as a Source Data file.

Article Snippet: HAEC were infected with the adenovirus (MOI 200) in EBM2 medium with 2% FBS for 5 h, and EBM2 medium with 10% FBS was then added for 72 h. Subsequently the cells were treated with vehicle, E2 (10 −8 M) or 27HC (10uM) for 10 min, proteins were crosslinked using 1% formaldehyde (8 min) followed by quenching with 2 M glycine, cells were washed, proteins were extracted, and ERα was immunoprecipitated with anti-ERα antibody (HC-20, Santa Cruz Biotechnology, sc-543, 1:50).

Techniques: Staining, Quantitation Assay, Labeling, Immunohistochemistry

Journal: Journal of Breast Cancer

Article Title: The Presence of EpCAM - /CD49f + Cells in Breast Cancer Is Associated with a Poor Clinical Outcome

doi: 10.4048/jbc.2015.18.3.242

Figure Lengend Snippet: Dual-immunohistochemistry (IHC) and immunofluorescence (IF) staining on tissue microarray (TMA) slides. Dual-IHC and IF staining with primary antibodies of EpCAM and CD49f were performed on TMA tissue. (A) Patients only contain EpCAM-positive cancer cells (membrane); (B) Patient show only CD49f-positive cancer cells (cytoplasm); (C) Case express none or both (D) biomarkers. In IF staining (E-P), green signal shows the expression of CD49f, red signal shows EpCAM with DAPI staining for nuclei. (E-H) One case that tumor cell expression only EpCAM on the membrane (white arrows); (I-L) One case that tumor cell express only CD49f (red arrow); (M-P) One case with tumor cells express both (yellow arrow) (for both IHC and IF, magnification is actually ×400).

Article Snippet: IHC was performed using antibodies against EpCAM (TA310957, 1:300; OriGene, Rockville, USA) and CD49f (TA506627, 1:150; OriGene) with an Autostainer with the EnVision G|2 Double Stain System ((DAKO company, Code K5361, Glostrup, Denmark) Rabbit/Mouse (DAB+/Permanent Red) according to the manufacturer's instructions.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Microarray, Membrane, Expressing

Journal: Journal of Breast Cancer

Article Title: The Presence of EpCAM - /CD49f + Cells in Breast Cancer Is Associated with a Poor Clinical Outcome

doi: 10.4048/jbc.2015.18.3.242

Figure Lengend Snippet: Prevalence of EpCAM - /CD49f + with histopathologic characteristics. Analyzing of 150 breast cancer samples stratified to tumor size (A), node status (B), tumor grade (C; low=grade I and II, high=grade III), recurrence (D), distant metastasis (E), and estrogen receptor (ER) expression (F). Tumor containing EpCAM - /CD49f + are associated with higher tumor grade (C; p =0.031) and distant metastasis (E; p =0.048). Chi-square exact and Fisher exact test were performed.

Article Snippet: IHC was performed using antibodies against EpCAM (TA310957, 1:300; OriGene, Rockville, USA) and CD49f (TA506627, 1:150; OriGene) with an Autostainer with the EnVision G|2 Double Stain System ((DAKO company, Code K5361, Glostrup, Denmark) Rabbit/Mouse (DAB+/Permanent Red) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Journal of Breast Cancer

Article Title: The Presence of EpCAM - /CD49f + Cells in Breast Cancer Is Associated with a Poor Clinical Outcome

doi: 10.4048/jbc.2015.18.3.242

Figure Lengend Snippet: Univariate survival analysis of EpCAM - /CD49f + with disease-free survival (DFS) and overall survival (OS). All the patients' tumor samples were stained with EpCAM and CD49f. (A, B) The present of EpCAM - /CD49f + tumor cells have significant negative association with both DFS and OS (EpCAM - /CD49f + : p DFS =0.009; p OS =0.001).

Article Snippet: IHC was performed using antibodies against EpCAM (TA310957, 1:300; OriGene, Rockville, USA) and CD49f (TA506627, 1:150; OriGene) with an Autostainer with the EnVision G|2 Double Stain System ((DAKO company, Code K5361, Glostrup, Denmark) Rabbit/Mouse (DAB+/Permanent Red) according to the manufacturer's instructions.

Techniques: Staining

Journal: Oncology Letters

Article Title: Cadherin 17 is related to recurrence and poor prognosis of cytokeratin 19-positive hepatocellular carcinoma

doi: 10.3892/ol.2017.7320

Figure Lengend Snippet: Heat map presentation of correlations between CK19 and various cadherins expressions after Affymetrix U133A oligonucleotide microarray analysis. Red colors represented positive correlation and blue colors represented negative correlation. The significance of correlation was determined by Pearson correlation coefficient R2. For CDH17, the R2 was 0.867, P<0.001.

Article Snippet: HCC, hepatocellular carcinoma; CK19, cytokeratin 19; CDH17, cadherin 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 Over-expression assays to study causal relationships. (A and B) Over-expression study by transfecting HepG2 cells with FLAG ® -CDH17 [pCMV6-Entry (OriGene™) with CDH17 gene incorporated] or FLAG ® -vector [pCMV6-Entry (OriGene™)].

Techniques: Microarray

Journal: Oncology Letters

Article Title: Cadherin 17 is related to recurrence and poor prognosis of cytokeratin 19-positive hepatocellular carcinoma

doi: 10.3892/ol.2017.7320

Figure Lengend Snippet: Immunohistochemical microphotograph of primary HBV-HCC. Two identical slides of tissue were sectioned from each tumor sample and then prepared for IHC study as described. The left column probed for CK19 and right column probed for CDH17. The expression of CK19 seemed to coincide with that of CDH17 in these HBV-HCC. The vast majority of the CK19(+) tumor cells in these HCC also had strong CDH17 expression. The Spearman's correlation coefficient between CK19 and CDH17 was 0.414, P<0.001. Magnifications, ×100. HBV, hepatitis B virus; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; CK19, cytokeratin 19; CDH17, cadherin 17.

Article Snippet: HCC, hepatocellular carcinoma; CK19, cytokeratin 19; CDH17, cadherin 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 Over-expression assays to study causal relationships. (A and B) Over-expression study by transfecting HepG2 cells with FLAG ® -CDH17 [pCMV6-Entry (OriGene™) with CDH17 gene incorporated] or FLAG ® -vector [pCMV6-Entry (OriGene™)].

Techniques: Immunohistochemical staining, Expressing, Immunohistochemistry

Journal: Oncology Letters

Article Title: Cadherin 17 is related to recurrence and poor prognosis of cytokeratin 19-positive hepatocellular carcinoma

doi: 10.3892/ol.2017.7320

Figure Lengend Snippet: The relationship between clinicopathological variables and CK19 expression in hepatocellular carcinoma (n=114).

Article Snippet: HCC, hepatocellular carcinoma; CK19, cytokeratin 19; CDH17, cadherin 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 Over-expression assays to study causal relationships. (A and B) Over-expression study by transfecting HepG2 cells with FLAG ® -CDH17 [pCMV6-Entry (OriGene™) with CDH17 gene incorporated] or FLAG ® -vector [pCMV6-Entry (OriGene™)].

Techniques: Expressing

Journal: Oncology Letters

Article Title: Cadherin 17 is related to recurrence and poor prognosis of cytokeratin 19-positive hepatocellular carcinoma

doi: 10.3892/ol.2017.7320

Figure Lengend Snippet: Relationship between clinicopathological variables and CDH17 expression in hepatocellular carcinoma.

Article Snippet: HCC, hepatocellular carcinoma; CK19, cytokeratin 19; CDH17, cadherin 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 Over-expression assays to study causal relationships. (A and B) Over-expression study by transfecting HepG2 cells with FLAG ® -CDH17 [pCMV6-Entry (OriGene™) with CDH17 gene incorporated] or FLAG ® -vector [pCMV6-Entry (OriGene™)].

Techniques: Expressing

Journal: Oncology Letters

Article Title: Cadherin 17 is related to recurrence and poor prognosis of cytokeratin 19-positive hepatocellular carcinoma

doi: 10.3892/ol.2017.7320

Figure Lengend Snippet: Kaplan-Meier survival curves. (A and B) HCC with CK19 expression had a significantly poorer prognosis in terms of (A) DFS and (B) OS; (C) HCC with CDH17 expression had a significantly worse DFS. (D) However, the OS was comparable to those without CDH17 expression; (E) HCC with dual CK19 and CDH17 expressions had a worst prognosis in terms of DFS. (F) The OS was comparable to those with either single positive or dual negative HCC. HCC, hepatocellular carcinoma; CK19, cytokeratin 19; DFS, disease-free survival; OS, overall survival; CDH17, cadherin 17.

Article Snippet: HCC, hepatocellular carcinoma; CK19, cytokeratin 19; CDH17, cadherin 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 Over-expression assays to study causal relationships. (A and B) Over-expression study by transfecting HepG2 cells with FLAG ® -CDH17 [pCMV6-Entry (OriGene™) with CDH17 gene incorporated] or FLAG ® -vector [pCMV6-Entry (OriGene™)].

Techniques: Expressing

Journal: Oncology Letters

Article Title: Cadherin 17 is related to recurrence and poor prognosis of cytokeratin 19-positive hepatocellular carcinoma

doi: 10.3892/ol.2017.7320

Figure Lengend Snippet: Cox regression multivariate analyses of factors associated with disease-free survival in hepatocellular carcinoma after hepatectomy.

Article Snippet: HCC, hepatocellular carcinoma; CK19, cytokeratin 19; CDH17, cadherin 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 Over-expression assays to study causal relationships. (A and B) Over-expression study by transfecting HepG2 cells with FLAG ® -CDH17 [pCMV6-Entry (OriGene™) with CDH17 gene incorporated] or FLAG ® -vector [pCMV6-Entry (OriGene™)].

Techniques: Expressing

Journal: Oncology Letters

Article Title: Cadherin 17 is related to recurrence and poor prognosis of cytokeratin 19-positive hepatocellular carcinoma

doi: 10.3892/ol.2017.7320

Figure Lengend Snippet: (A) Western blot analysis of various HCC cell lines. Primary antibodies were that of CK19 and CDH17 and a dilution of 1:100,000 and 1:2,000, respectively, were adopted. GAPDH was used as internal control. Hep3B, Huh7, and PLC5 were CK19(+) HCC cell lines, while HepG2 was CK19(−) HCC cell line. On the other hand, Hep3B and Huh7 were CDH17(+) HCC cell lines, while HepG2 and PLC5 were CDH17(−) HCC cell lines. (B and C) The study of EGF-treated HepG2 cells (EGF, 30 ng/ml for 5 days). Western blot analysis confirmed that CK19 can be induced by EGF treatment. In the meanwhile, the expression of CDH17 was also enhanced after EGF treatment (B). Real-time qPCR analysis of EGF-treated HepG2 cells demonstrated that both CK19 and CDH17 transcripts were significantly elevated (P<0.001 and <0.001, respectively) (C). In the other words, EGF can induce both CK19 and CDH17 expressions. HCC, hepatocellular carcinoma; CK19, cytokeratin 19; CDH17, cadherin 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor.

Article Snippet: HCC, hepatocellular carcinoma; CK19, cytokeratin 19; CDH17, cadherin 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 Over-expression assays to study causal relationships. (A and B) Over-expression study by transfecting HepG2 cells with FLAG ® -CDH17 [pCMV6-Entry (OriGene™) with CDH17 gene incorporated] or FLAG ® -vector [pCMV6-Entry (OriGene™)].

Techniques: Western Blot, Expressing

Journal: Oncology Letters

Article Title: Cadherin 17 is related to recurrence and poor prognosis of cytokeratin 19-positive hepatocellular carcinoma

doi: 10.3892/ol.2017.7320

Figure Lengend Snippet: Over-expression assays to study causal relationships. (A and B) Over-expression study by transfecting HepG2 cells with FLAG®-CDH17 [pCMV6-Entry (OriGene™) with CDH17 gene incorporated] or FLAG®-vector [pCMV6-Entry (OriGene™)]. Western blot analysis showed that the expression of both CDH17 and CK19 increased significantly after transfecting HepG2 with FLAG®-CDH17 (A). Real-time qPCR revealed that the transcripts of both CDH17 and CK19 were significantly increased in HepG2 FLAG®-CDH17 cells (P<0.001 and 0.03, respectively) (B). (C and D) Over-expression study by transfecting HepG2 cells with FLAG®-CK19 [pCMV-3Tag-8 (Invitrogen™) with CK19 gene incorporated] or FLAG®-vector [pCMV-3Tag-8 (Invitrogen™)]. Western blot analysis showed that the expression of CK19 increased significantly after transfecting with FLAG®-CK19. However, the expression of CDH17 remained unchanged after CK19 over-expression (C). Real-time qPCR revealed that the transcripts of CK19 were significantly increased in HepG2 FLAG®-CK19 cells (P<0.001). However, the transcripts of CDH17 were not altered by CK19 over-expression (P=0.1217) (D). In other words, CDH17 was not downstream to CK19. It was upstream to CK19 and, like CK19, was regulated by EGF. The experiment of real-time qPCR was conducted in triplicates. CDH17, cadherin 17; CK19, cytokeratin 19; EGF, epidermal growth factor.

Article Snippet: HCC, hepatocellular carcinoma; CK19, cytokeratin 19; CDH17, cadherin 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EGF, epidermal growth factor. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 5. caption a7 Over-expression assays to study causal relationships. (A and B) Over-expression study by transfecting HepG2 cells with FLAG ® -CDH17 [pCMV6-Entry (OriGene™) with CDH17 gene incorporated] or FLAG ® -vector [pCMV6-Entry (OriGene™)].

Techniques: Over Expression, Plasmid Preparation, Western Blot, Expressing