Journal: Nature communications

Article Title: miR-205 acts as a tumour radiosensitizer by targeting ZEB1 and Ubc13.

doi: 10.1038/ncomms6671

Figure Lengend Snippet: Figure 1 | miR-205 increases radiosensitivity and is downregulated in radioresistant breast cancer cells. (a) Top: schematic representation of the generation of a radioresistant subline (SUM159-P2) from the parental SUM159 cells (SUM159-P0). Bottom: miRNA expression profiling of SUM159-P2 cells relative to SUM159-P0 cells using a quantitative PCR (qPCR)-based miRNA array. The expression values are shown in Supplementary Table 1. (b) TaqMan qPCR analysis of miR-205 in SUM159-P0 and SUM159-P2 cells. n ¼ 3 samples per group. (c) qPCR of miR-205 (top) and clonogenic survival assays (bottom) of mesenchymal-like and epithelial-like breast cancer cell lines. n ¼ 3 samples per group. (d) Clonogenic survival assays of SUM159-P2 cells transduced with miR-205. n ¼ 3 wells per group. (e) Clonogenic survival assays of SUM159-P0 cells transfected with the miR-205 inhibitor. n ¼ 3 wells per group. Inset in d,e: immunoblotting of ZEB1 and GAPDH. Data in b–e are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. The experiments were repeated three times.

Article Snippet: The following antibodies were used: antibodies to ZEB1 (1:1000, Bethyl Laboratories, A301-922A), gH2AX (1:1000, Cell Signaling Technology, 2577), K63-linkage specific polyubiquitin (1:1000, Cell signaling Technology, 12930), BRCA1 (1:1000, Bethyl Laboratories, A300-000A), Ubc13 (1:1000, Cell Signaling Technology, 4919), FLAG (1:5000, Sigma, F3165) and GAPDH (1:3000, Thermo, MA5-15738).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transduction, Transfection, Western Blot, Two Tailed Test

Journal: Nature communications

Article Title: miR-205 acts as a tumour radiosensitizer by targeting ZEB1 and Ubc13.

doi: 10.1038/ncomms6671

Figure Lengend Snippet: Figure 2 | Therapeutic effect of miR-205 mimics in mice. (a) Kaplan–Meier curves showing the distant relapse-free survival of patients with high (n ¼ 43) or low (n ¼ 38) expression of miR-205 in their breast tumours. The P value was determined by a log-rank test. (b) Clonogenic survival assays of SUM159- P2 cells incubated with 100 nM DOPC-encapsulated miR-205 mimics. n ¼ 3 wells per group. (c) Top: overview of the experimental scheme. Bottom: tumour size of mice bearing SUM159-P2 xenografts. Mice were treated with the vehicle or DOPC-encapsulated miR-205 mimics or scramble mimics. Tumours were locally irradiated with a 15-Gy single dose (radiation treatment). n ¼ 5 mice per group. General linear model multivariate analysis was performed to determine statistical significance. (d) Quantitative PCR of miR-205 in tumour tissues from the mice described in c. n ¼ 5 mice per group. (e) Immunoblotting of ZEB1 and GAPDH in tumour lysates. Data in b–d are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance in b,d was determined by a two-tailed, unpaired Student’s t-test. The experiments were repeated three times.

Article Snippet: The following antibodies were used: antibodies to ZEB1 (1:1000, Bethyl Laboratories, A301-922A), gH2AX (1:1000, Cell Signaling Technology, 2577), K63-linkage specific polyubiquitin (1:1000, Cell signaling Technology, 12930), BRCA1 (1:1000, Bethyl Laboratories, A300-000A), Ubc13 (1:1000, Cell Signaling Technology, 4919), FLAG (1:5000, Sigma, F3165) and GAPDH (1:3000, Thermo, MA5-15738).

Techniques: Expressing, Incubation, Irradiation, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Nature communications

Article Title: miR-205 acts as a tumour radiosensitizer by targeting ZEB1 and Ubc13.

doi: 10.1038/ncomms6671

Figure Lengend Snippet: Figure 3 | miR-205 regulates DNA damage repair and radiosensitivity through ZEB1 and Ubc13. (a) gH2AX and 4’,6-diamidino-2-phenylindole (DAPI) staining of SUM159-P2 cells transfected with miR-205 or scramble control oligonucleotides, 24 h after 6-Gy IR. Scale bar, 20 mm. (b) HR repair assays of U2OS_DR-GFP cells transfected with miR-205 alone or in combination with ZEB1. n ¼ 3 wells per group. (c) Immunoblotting of ZEB1, Ubc13, BRCA1 and GAPDH in SUM159-P0 cells transfected with miR-205 mimics or the miR-205 inhibitor. (d) Luciferase activity of the wild-type (UTR-WT) or mutant (UTR-mut.) Ubc13 30 UTR reporter gene in 293T cells transfected with the miR-205-expressing or empty vector. n ¼ 3 wells per group. (e) Immunoblotting of K63-linked polyubiquitin chains, Ubc13 and GAPDH in SUM159-P0 and SUM159-P2 cells. (f) Clonogenic survival assays of SUM159-P2 cells transfected with Ubc13 siRNA. n ¼ 3 wells per group. (g) Top: clonogenic survival assays of miR-205-transduced SUM159-P2 cells with or without ectopic expression of ZEB1 and Ubc13. Bottom: immunoblotting of ZEB1, Ubc13 and GAPDH. n ¼ 3 wells per group. (h) HR repair assays of U2OS_DR-GFP cells transfected with miR-205 alone or in combination with ZEB1 or Ubc13, or both. n ¼ 3 wells per group. Data in b,d,f,g,h are the mean of biological replicates from a representative experiment, and error bars indicate s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. The experiments were repeated three times.

Article Snippet: The following antibodies were used: antibodies to ZEB1 (1:1000, Bethyl Laboratories, A301-922A), gH2AX (1:1000, Cell Signaling Technology, 2577), K63-linkage specific polyubiquitin (1:1000, Cell signaling Technology, 12930), BRCA1 (1:1000, Bethyl Laboratories, A300-000A), Ubc13 (1:1000, Cell Signaling Technology, 4919), FLAG (1:5000, Sigma, F3165) and GAPDH (1:3000, Thermo, MA5-15738).

Techniques: Staining, Transfection, Control, Western Blot, Luciferase, Activity Assay, Mutagenesis, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: A non-canonical pathway regulates ER stress signaling and blocks ER stress-induced apoptosis and heart failure

doi: 10.1038/s41467-017-00171-w

Figure Lengend Snippet: Mechanism for AGGF1 regulation of miR-183-5p expression. a ChIP–qPCR analysis for the interaction between ZEB1 and miR-183-5p promoter DNA ( n = 4/group, ** P < 0.01). b Schematic diagram showing that the miR-183-5p promoter-luciferase reporter with two ZEB1-binding motifs. c Luciferase activity ( n = 6/group, ** P < 0.01). d Real-time RT-PCR analysis for miR-183-5p expression in H9C2 cells after AGGF1 treatment with or without ZEB1 overexpression ( n = 3/group, ** P < 0.01). e Western blot analysis for the ZEB1 level in H9C2 cells after AGGF1 treatment ( n = 5/group, * P < 0.05). f Western blot analysis for activation of ERK1/2 in H9C2 cells after AGGF1 treatment ( n = 5/group, ** P < 0.01). g Real-time RT-PCR analysis for ERK1 and ERK2 expression by ERK siRNA ( n = 3/group, ** P < 0.01). h Western blot analysis for the ZEB1 level by ERK siRNA ( n = 5/group, * P < 0.05). i Schematic model for a non-canonical ER stress signaling pathway mediated by AGGF1. Data are shown as the mean ± s.d. from at least three independent experiments. Statistical analysis was carried out by a Student’s two-tailed t -test

Article Snippet: Protein–DNA complexes were immunoprecipitated with IgG or an anti-ZEB1 antibody (2 μg; Proteintech).

Techniques: Expressing, ChIP-qPCR, Luciferase, Binding Assay, Activity Assay, Quantitative RT-PCR, Over Expression, Western Blot, Activation Assay, Two Tailed Test

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: Molecular Cancer

Article Title: ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH

doi: 10.1186/s12943-015-0357-6

Figure Lengend Snippet: CA9 is upregulated by ZEB1 in tongue cancer cells. (A) A schematic representation of ZEB1 binding sites with the E-box sequence (CACCTG) in the 3kb putative CA9 promoter. The first base of the 3kb strand is defined as ‘1’. (B) Chromatin immunoprecipitation assays identified ZEB1 binding sites within the putative CA9 promoter. Primers specific for sites B, C and E yielded PCR reaction products from ZEB1–DNA immunoprecipitates. The input represents DNA directly after lysis. The PCR reaction product for immunoprecipitates obtained using the RNA Polymerase antibody represents the positive control. (C and D) Changes in CA9 mRNA and protein expression following the inhibition of ZEB1 in tongue cancer cell lines were evaluated by qRT-PCR and western blotting, respectively. (E) Luciferase activity driven by the putative CA9 promoter was higher in Tca8113/PYM cells (which exhibit endogenous ZEB1 overexpression) than in Tca8113 and SCC-25 cells. (F) Reporter assays revealed changes in luciferase activity after inhibition of ZEB1 expression in tongue cancer cells. (G) ZEB1 promoted luciferase activity driven by the putative CA9 promoter in HEK283T cells. * p < 0.01.

Article Snippet: The transfection of the pLEX-ZEB1 vector, the pLEX-CA9 vector and related controls was carried out using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. siRNAs targeting ZEB1 or CA9 and siRNA controls were purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Sequencing, Chromatin Immunoprecipitation, Lysis, Positive Control, Expressing, Inhibition, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Over Expression

Journal: Molecular Cancer

Article Title: ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH

doi: 10.1186/s12943-015-0357-6

Figure Lengend Snippet: The ZEB1–CA9 axis regulates chemosensitivity in tongue cancer cells. (A and B) MTS cell proliferation assays showed that ZEB1 overexpression promoted resistance in Tca8113 and SSC-25 cells in response to PYM and cDDP, and that knockdown of CA9 abolished this effect. (C) Knockdown of ZEB1 enhanced the sensitivity of Tca8113/PYM cells to PYM and cDDP, while overexpression of CA9 attenuated this effect. * p < 0.01.

Article Snippet: The transfection of the pLEX-ZEB1 vector, the pLEX-CA9 vector and related controls was carried out using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. siRNAs targeting ZEB1 or CA9 and siRNA controls were purchased from Santa Cruz Biotechnology.

Techniques: Over Expression, Knockdown

Journal: Molecular Cancer

Article Title: ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH

doi: 10.1186/s12943-015-0357-6

Figure Lengend Snippet: The ZEB1–CA9 axis prevents pHi decrease induced by chemotherapy in tongue cancer cells. (A, C and E) The change in pHi found for each given cell line in response to PYM or cDDP treatment, as determined using the BCECF-AM pH fluorescence probe in conjunction with confocal microscopy. (B, D and F) Changes in apoptosis in response to PYM or cDDP treatment for each given cell line, as determined by Hoechst staining evaluated by fluorescence microscopy. * p < 0.01.

Article Snippet: The transfection of the pLEX-ZEB1 vector, the pLEX-CA9 vector and related controls was carried out using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. siRNAs targeting ZEB1 or CA9 and siRNA controls were purchased from Santa Cruz Biotechnology.

Techniques: Fluorescence, Confocal Microscopy, Staining, Microscopy

Journal: Molecular Cancer

Article Title: ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH

doi: 10.1186/s12943-015-0357-6

Figure Lengend Snippet: ZEB1–CA9 prevents chemotherapy-induced caspase-3 activation. (A and B) PYM and cDDP induced caspase-3 activation in Tca8113 and SCC-25 cell lines, respectively, as measured by reporter assays and western blotting. Overexpression of ZEB1 prevented caspase-3 activation in response to chemotherapy, while knockdown of CA9 impaired the effects of ZEB1 overexpression. (C) Chemotherapy had no significant effect on caspase-3 activation in Tca8113/PYM cells. Knockdown of ZEB1 enhanced caspase-3 activation induced by chemotherapy, and overexpression of CA9 attenuated this effect. vs. no treatment, * p < 0.01.

Article Snippet: The transfection of the pLEX-ZEB1 vector, the pLEX-CA9 vector and related controls was carried out using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. siRNAs targeting ZEB1 or CA9 and siRNA controls were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Western Blot, Over Expression, Knockdown

Journal: Molecular Cancer

Article Title: ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH

doi: 10.1186/s12943-015-0357-6

Figure Lengend Snippet: ZEB1 expression correlates with that of CA9 in tongue cancer and is associated with poor clinical prognosis. (A) Representative images of ZEB1 and CA9 protein expression in tongue cancer tissues. ZEB1 protein expression was positively correlated with that of CA9 (left). The level of CA9 protein expression in tongue cancer tissues that exhibit different levels of ZEB1 protein expression (right). (B) Kaplan–Meier analysis estimated overall survival according to the ZEB1 protein level, CA9 protein level, and both ZEB1 and CA9 protein levels.

Article Snippet: The transfection of the pLEX-ZEB1 vector, the pLEX-CA9 vector and related controls was carried out using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. siRNAs targeting ZEB1 or CA9 and siRNA controls were purchased from Santa Cruz Biotechnology.

Techniques: Expressing