Journal: Molecular cancer

Article Title: Next-generation sequencing reveals novel differentially regulated mRNAs, lncRNAs, miRNAs, sdRNAs and a piRNA in pancreatic cancer.

doi: 10.1186/s12943-015-0358-5

Figure Lengend Snippet: Figure 3 Co-expression of miR-802, ZEB1, TCF4 and miR-21. A) Alignment of miR-802 to the predicted binding sites in the 3′ UTR of TCF4. B) Co-expression analysis of significantly upregulated transcription factors that harbour predicted miRNA binding sites in their 3′ UTRs for one of the ten most significantly upregulated miRNAs (miRNAs that have no seed sequence for a TF UTR not shown). Significant (p < 0.01) correlations are indicated by a dot, positive correlations are marked in blue, negative correlations in red. The more significant the correlation, the larger the dot size. Sequence complementarity between an UTR and a miRNA is indicated by an “S”. C) Expression of TCF4, ZEB1 and miR-21 across all control (C) and PDAC (P) samples (significant positive correlation) as well as the expression of miR-802 (significantly inversely correlated). The normalized expression for each gene/miRNA is given in log2-scale for each sample.

Article Snippet: Sections were incubated with the ZEB1 antibody (ZEB1: Atlas Antibodies #AMAb90510 (1:400)) at 4°C overnight followed by incubation with horseradish peroxidase-linked goat anti-mouse antibody, followed by a color-reaction with diaminebenzidine and counterstaining with Mayer’s hematoxylin.

Techniques: Expressing, Binding Assay, Sequencing, Control

Journal: Molecular cancer

Article Title: Next-generation sequencing reveals novel differentially regulated mRNAs, lncRNAs, miRNAs, sdRNAs and a piRNA in pancreatic cancer.

doi: 10.1186/s12943-015-0358-5

Figure Lengend Snippet: Figure 5 Immunohistochemistry of ZEB1 protein expression. Immunohistochemical detection of ZEB1 in human pancreatic tissue samples. Representative images of ZEB1 expression: Upper panels, ZEB1 is expressed in periacinar cells in normal pancreatic tissue samples. Lower panels, detection of ZEB1 in stromal cells, but not in epithelial tumor cells in PDAC samples.

Article Snippet: Sections were incubated with the ZEB1 antibody (ZEB1: Atlas Antibodies #AMAb90510 (1:400)) at 4°C overnight followed by incubation with horseradish peroxidase-linked goat anti-mouse antibody, followed by a color-reaction with diaminebenzidine and counterstaining with Mayer’s hematoxylin.

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining

Journal: Genes to Cells

Article Title: Roles of ZEB1 and ZEB2 in E‐cadherin expression and cell aggressiveness in head and neck cancer

doi: 10.1111/gtc.13167

Figure Lengend Snippet: Comparisons between C57BL/6 MOC1 and MOC2 cells. (a, b) mRNA levels of representative EMT‐TFs were determined by RT‐qPCR. Upon TGF‐β stimulation for the indicated times, normal mouse mammary epithelial NMuMG cells were used as controls to assess the expression levels of EMT‐TFs. The ratio of each mRNA to TBP in MOC1 cells was defined as 1. (c) Immunoblotting with the indicated antibodies was performed after infection with lentiviruses carrying negative control (NC), ZEB1, or ZEB2. The ratio of ZEB1, ZEB2, or E‐cadherin to α‐tubulin was validated by densitometric analysis and shown at the right panels. The ratio of each protein to α‐tubulin in MOC2 cells was indicated as 1. P, parental MOC1 cells (non‐infected cells). (d, e) Expression levels of ESRP1 and ESRP2 were determined by RT‐qPCR (d). Expression levels of the alternately spliced forms of FGFR1 and FGFR2 were examined by conventional PCR (e). A schematic illustration of the relationship between ZEB1/2, ESRP1/2, and FGFRs is shown at the top (e). NC, negative control (d, e). (f) ERK1/2 phosphorylation (phospho‐ERK1/2) in response to FGF2 or FGF7 was examined by immunoblot analysis. Cells were stimulated with the indicated concentrations of FGF2 or FGF7 in the presence of heparin for 30 min. α‐tubulin was used as a loading control (c, f). p values were determined by Student's t ‐test. * p < .01, ** p < .05. ns, not significant. Each value represents the mean ± SD of duplicate measurements from a representative experiment.

Article Snippet: Rabbit polyclonal anti‐ZEB1 (NBP1‐059870) and anti‐ZEB2 (NBP1‐82991) antibodies were obtained from Novus Biologicals (Littleton, CO).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Infection, Negative Control, Control

Journal: Genes to Cells

Article Title: Roles of ZEB1 and ZEB2 in E‐cadherin expression and cell aggressiveness in head and neck cancer

doi: 10.1111/gtc.13167

Figure Lengend Snippet: Knockdown of ZEB1/2 in human HNSCC cells. (a, b) After transfection with siRNAs, the expression levels of E‐cadherin and N‐cadherin in human HNSCC TSU and HOC313 cells were determined by RT‐qPCR (a) and immunoblot analysis (b). cont., control siRNA (a and b). α‐tubulin was used as a loading control (b). P values were determined by Tukey's multiple comparison test. * p < .01, ** p < .05. ns, not significant. Each value represents the mean ± SD of duplicate measurements from a representative experiment.

Article Snippet: Rabbit polyclonal anti‐ZEB1 (NBP1‐059870) and anti‐ZEB2 (NBP1‐82991) antibodies were obtained from Novus Biologicals (Littleton, CO).

Techniques: Knockdown, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, Comparison

Journal: Genes to Cells

Article Title: Roles of ZEB1 and ZEB2 in E‐cadherin expression and cell aggressiveness in head and neck cancer

doi: 10.1111/gtc.13167

Figure Lengend Snippet: Inhibition of cell proliferation by ZEB1/2. (a) Cell proliferation rates were determined with an MTT assay. Each value represents the mean ± SD of triplicate measurements from a representative experiment. Similar results were obtained in two independent experiments. (b) mRNA levels of representative CDK inhibitors were determined by RT‐qPCR. Each value represents the mean ± SD of duplicate measurements from a representative experiment. NC, negative control. (c) Two types of cells were seeded on a two‐well silicone insert. Approximately 24 h later, the insert was removed from the plate. After gap creation, the cells were monitored and visualized by phase contrast microscopy, followed by TRITC‐phalloidin staining or immunohistochemical analysis with the indicated antibodies. Yellow arrows show typical F‐Actin stress fiber formation in MOC1‐ZEB1 cells. (d) Cells were seeded on cell culture inserts. After 12 h, the cells that had migrated to the opposite side were stained and quantified. Each value represents the mean ± SD of triplicate measurements from a representative experiment. Similar results were obtained in two independent experiments. NC, negative control. (e) Chemoresistance assay in response to the indicated concentrations (Conc.) of cisplatin. Each value represents the mean ± SD of quadruple measurements from a representative experiment. Similar results were obtained in two independent experiments. P values were determined by Student's t ‐test (a and e) or by Tukey's multiple comparison test (b, d). * p < .01, ** p < .05.

Article Snippet: Rabbit polyclonal anti‐ZEB1 (NBP1‐059870) and anti‐ZEB2 (NBP1‐82991) antibodies were obtained from Novus Biologicals (Littleton, CO).

Techniques: Inhibition, MTT Assay, Quantitative RT-PCR, Negative Control, Microscopy, Staining, Immunohistochemical staining, Cell Culture, Comparison

Journal: Genes to Cells

Article Title: Roles of ZEB1 and ZEB2 in E‐cadherin expression and cell aggressiveness in head and neck cancer

doi: 10.1111/gtc.13167

Figure Lengend Snippet: Tumor‐promoting effects of ZEB1/2 in immunocompetent mice (a) The luciferase activity of cells infected with lentiviruses carrying the luciferase reporter (Luc) was measured with the IVIS Lumina imaging system. (b–d). After the cells were injected intravenously into the tail veins of syngeneic C57BL/6 mice ( n = 7), survival rates were analyzed using the Kaplan–Meier method (b). Beginning 14 days after the injection, the emission intensity was measured using the IVIS Lumina imaging system about once every 3 days. Representative image data at 24 days (i), 33 days (ii) and 40 days (iii) are shown (c). Lungs were extracted from mice that died from cancer and from mice that were sacrificed due to severe debility, followed by macroscopic analysis (left in d), determination of the total number of nodules, and measurement of lung weight (right in d). Yellow arrowheads show nodules that were observed macroscopically. Green and blue arrowheads show abnormalities due to massive hemorrhage and consolidation, respectively (d). P values were determined by Tukey's multiple comparison test (d). Typical histological findings following HE staining are shown (f). T, tumor.

Article Snippet: Rabbit polyclonal anti‐ZEB1 (NBP1‐059870) and anti‐ZEB2 (NBP1‐82991) antibodies were obtained from Novus Biologicals (Littleton, CO).

Techniques: Luciferase, Activity Assay, Infection, Imaging, Injection, Comparison, Staining

Journal: Cells

Article Title: Optimization of In-Situ Exosome Enrichment Methodology On-a-Chip to Mimic Tumor Microenvironment Induces Cancer Stemness in Glioblastoma Tumor Model.

doi: 10.3390/cells14090676

Figure Lengend Snippet: Figure 5. The RT-qPCR analysis. The mRNA expression levels of the BAX (as an apoptotic marker), Bcl2 (as an anti-apoptotic marker), Ki67 (as a proliferation marker), OCT-4 and SOX2 (as stemness gene), NF-κB (as an inflammation factor and coactivator of CSC and EMT related genes), E-cadherin, N-cadherin, Vimentin, SNAIL and ZEB1 (as EMT marker), β-catenin, STAT3, Notch1, TGFβ, EGF and SMAD (as crucial regulator genes in stemness and EMT related genes) and β-actin (as an internal gene), were detected by Real-time PCR in µBR and traditional cell culture on days 0 and 7. ** p-values < 0.01, *** p-values < 0.001 and **** p-values < 0.0001.

Article Snippet: The HIF1 antibody (Cat# H-3091K) was purchased from Abnova (Taipei, Taiwan), and ZEB1 (Cat# GWB-KXYPE) from Aviva Systems Biology (San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Expressing, Marker, Real-time Polymerase Chain Reaction, Cell Culture

Journal: Cells

Article Title: Optimization of In-Situ Exosome Enrichment Methodology On-a-Chip to Mimic Tumor Microenvironment Induces Cancer Stemness in Glioblastoma Tumor Model.

doi: 10.3390/cells14090676

Figure Lengend Snippet: Figure 6. Enzyme-linked immunosorbent assay (ELISA). The expression level of key proteins in Glioblastoma cell line (U87) culture under optimized condition in µBR vs. Traditional cell culture supernatant were measured by ELISA. Notch1 (main player of important pathway in triggering EMT), HIF1 (Hypoxia-Inducible Factor 1 is known for its involvement in promoting aggressiveness in cancer cells, regulates cellular response to hypoxia and can enhance invasion potential by inducing various target genes related to angiogenesis, epithelial-mesenchymal transition (EMT), and extracellular matrix remodeling), ZEB1 (as EMT marker, related to dedifferentiation of the glioblastoma cells to mesenchyme which convert cells to a motile type and its expression is associated with higher grades of malignancy, tumor progression and invasion), SOX2 (as stemness marker has been linked to enhanced invasiveness in various cancer types, potentially through EMT induction and interaction with other signaling pathways), β-catenin (a component of the cell-cell adhesion complex and of the canonical Wnt pathway, regulates proliferation, adhesion, and migration in different cell types, as crucial regulator in stemness and EMT related genes), E-cadherin (an epithelial marker), N-cadherin (cancer epithelial marker), Vimentin (mesenchymal marker), Snail (as EMT marker constitutes a master switch that directly represses the epithelial phenotype). ** p-values < 0.01, and **** p-values < 0.0001.

Article Snippet: The HIF1 antibody (Cat# H-3091K) was purchased from Abnova (Taipei, Taiwan), and ZEB1 (Cat# GWB-KXYPE) from Aviva Systems Biology (San Diego, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Marker, Protein-Protein interactions, Migration

Journal: Oncogenesis

Article Title: miR-200b restoration and DNA methyltransferase inhibitor block lung metastasis of mesenchymal-phenotype hepatocellular carcinoma

doi: 10.1038/oncsis.2012.15

Figure Lengend Snippet: Transient delivery of pre-miR-200a or pre-miR-200b in mesenchymal cells partially reversed EMT. ( a ) Mesenchymal (M) cells demonstrate loss of four miR-200 family members including miR-200a, miR-200b, miR-200c and miR-429 compared with parent epithelial (E) cells ( n =3, * P <0.05). ( b ) Zeb1 and Zeb2 were significantly downregulated and E-cadherin was significantly upregulated in precursor miR-200-transfected mesenchymal cells compared with cells transfected with NC pre-miRNA ( n =3, * P <0.05). ( c ) Protein expression of E-cadherin was significantly upregulated after miR-200 transfection in mesenchymal cells. ( d ) Compared with NC miRNA, the transient expression of miR-200a or miR-200b significantly inhibited tumor cell migration on wound-healing assay ( n =3, NC 149±21, miR-200a 54±10 and miR-200b 26±8, * P <0.05 compared with NC).

Article Snippet: The polyclonal E-cadherin antibody for western blot, immunohistochemistry and immunofluorescence, and polyclonal Zeb1 antibody for western blot were purchased from Cell Signaling (Cat# 3195s and 3396s, respectively, Danvers, MA, USA), and Zeb1 antibody for immunohistochemistry and Zeb2 antibody for immunohistochemistry and western blot were purchased from Novus Biologicals (Littleton, CO, USA) with catalog numbers NBP1-05987 and NBP1-67782, respectively.

Techniques: Transfection, Expressing, Migration, Wound Healing Assay

Journal: Oncogenesis

Article Title: miR-200b restoration and DNA methyltransferase inhibitor block lung metastasis of mesenchymal-phenotype hepatocellular carcinoma

doi: 10.1038/oncsis.2012.15

Figure Lengend Snippet: Stable expression of miR-200b reverses EMT. ( a ) In mesenchymal cells, miR-200b expression results in downregulation of Zeb1 and Zeb2 , and upregulation of E-cadherin compared with NC miRNA (miR-NC, n =3/group, * P <0.01). ( b ) The protein level of E-cadherin was upregulated in miR-200b expressing mesenchymal cells compared with control miR-NC cells, but remained below the level of parent epithelial cells ( n =3, representative image shown). ( c ) Green fluorescence protein (GFP) fluorescence demonstrates that mesenchymal cells undergo morphology change to larger cells with more cell–cell contact after miR-200b upregulation ( n =10, representative image shown). ( d ) Compared with miR-NC control mesenchymal cells, invasion is significantly inhibited after miR-200b expression (expressed as mean number of cells invading into lower chamber, n =3, * P <0.05). ( e ) Mesenchymal cells expressing miR-NC or miR-200b spread to multiple abdominal organs following orthotopic transplant in nude mice, as demonstrated by in vivo imaging ( n =8–9 mice/group, representative images shown). ( f ) Histology demonstrates mesenchymal cells with miR-200b expression invade the pancreas (P) and colon (C) (L, liver; T, tumor). Metastatic lesions (arrow) are detectable in the lungs of both miR-NC and miR-200b groups (miR-NC, n =9, lung metastasis 22.2% (2/9), miR-200b, n =8, lung metastasis 37.5% (3/8), H&E stain).

Article Snippet: The polyclonal E-cadherin antibody for western blot, immunohistochemistry and immunofluorescence, and polyclonal Zeb1 antibody for western blot were purchased from Cell Signaling (Cat# 3195s and 3396s, respectively, Danvers, MA, USA), and Zeb1 antibody for immunohistochemistry and Zeb2 antibody for immunohistochemistry and western blot were purchased from Novus Biologicals (Littleton, CO, USA) with catalog numbers NBP1-05987 and NBP1-67782, respectively.

Techniques: Expressing, Control, Fluorescence, In Vivo Imaging, Staining