Journal: Molecular Oncology

Article Title: Aberrant expression of nuclear prothymosin α contributes to epithelial‐mesenchymal transition in lung cancer

doi: 10.1002/1878-0261.70035

Figure Lengend Snippet: ProT inhibits TGF‐β‐induced EMT by downregulating the expression of EMT‐associated transcription factors. (A and B) Detection of Snail, Twist1, and ZEB1 mRNA expression by RT‐qPCR in A549 ( n = 3), A549/GFP ( n = 3), and A549/ProT ( n = 3) cells (A), as well as in A549 cells transduced with lentiviral vectors expressing shRNA specific to ProT (shProT) ( n = 3) or luciferase (shLuc) ( n = 3) (B) following treatment with TGF‐β (10 ng·mL −1 ) for 24 h. Relative expression levels are normalized to GAPDH , and ratios of control cells were arbitrarily set to 100. (C, D) Detection and quantification of EMT‐associated markers in mock ( n = 3)‐, GFP ( n = 3)‐, and ProT ( n = 3)‐transduced A549 (C) and H1299 (D) cells in the presence or absence of TGF‐β1 (10 ng·mL −1 ) for 48 h by immunoblotting. Expression of β‐actin served as the loading control. Representative immunoblots from three independent experiments and quantitative analysis of the indicated proteins are shown. Ratios between the intensity of the bands corresponding to the indicated protein and those corresponding to β‐actin were calculated, and ratios of control cells were arbitrarily set to 100. All quantification values and error bars shown were mean ± SEM, and P ‐values less than 0.05 were shown in the quantitative charts (* P < 0.05, ** P < 0.01, *** P < 0.001, two‐tailed unpaired t ‐test). GFP, green fluorescent protein; Luc, luciferase; ProT, prothymosin α; SEM, standard error of the mean; shRNA, short‐hairpin RNA; Snail, Zinc finger protein SNAI1; TGF‐β1, transforming growth factor‐β; Twist1, Twist‐related protein 1; ZEB1, Zinc finger E‐box‐binding homeobox 1.

Article Snippet: Primary antibodies included monoclonal antibodies against ProT (clone 2F11, ascites fluid) [ ], Smad7 (R&D, Bio‐Techne, Minneapolis, MI, USA), Snail (Abcam), Twist1 (Genetex), and ZEB1 (Novus).

Techniques: Expressing, Quantitative RT-PCR, Transduction, shRNA, Luciferase, Control, Western Blot, Two Tailed Test, Binding Assay

Journal: Molecular Oncology

Article Title: Aberrant expression of nuclear prothymosin α contributes to epithelial‐mesenchymal transition in lung cancer

doi: 10.1002/1878-0261.70035

Figure Lengend Snippet: Prot suppresses Snail, Twist1, and ZEB1 expression via promoting Smad7 acetylation. (A) Detection and quantification of Smad7 in A549/GFP ( n = 3) and A549/ProT ( n = 3) cells by immunoblotting. (B) Detection of Smad7 mRNA expression in A549/GFP ( n = 4) and A549/ProT ( n = 4) cells by RT‐qPCR. Relative expression levels are normalized to GAPDH , and ratios of control cells were arbitrarily set to 100. (C) Detection and quantification of ZEB1, Snail, and Twist1 by immunoblotting in H1299 cells transiently transfected with pcDNA3.1‐ProT ( n = 3), pLKO.1‐Flag‐Smad7 ( n = 3), or a control vector (pcDNA3.1) ( n = 3) for 24 h. Overexpression of ProT was confirmed in cells transfected with pcDNA3.1‐ProT by immunoblotting. (D) Detection of acetylated Smad7 in A549/GFP ( n = 3) and A549/ProT ( n = 3) cells transduced with Flag‐Smad7 and immunoprecipitated with anti‐Flag antibody, followed by immunoblotting for acetyl‐lysine. (E) Detection and quantification of ZEB1, Snail, and Twist1 by immunoblotting in A549/GFP ( n = 3) and A549/ProT ( n = 3) cells transduced with Flag‐Smad7(K64R/K70R), which contains lysine to arginine mutations at residues 64 and 70, following treatment with TGF‐β1 for 24 h. Overexpression of ProT and Smad7(K64R/K70R) was confirmed in cells transduced with ProT and/or mutant Smad7 by immunoblotting with antibodies against ProT or Flag. (A, C, D, E). Expression of β‐Actin serves as the loading control. Representative immunoblots from three independent experiments and quantitative analysis of the indicated proteins are shown. Ratios between the intensity of the bands corresponding to the indicated proteins and those corresponding to β‐Actin were calculated, and ratios of control cells were arbitrarily set to 100. The quantification values and error bars displayed were mean ± SEM, and P ‐values less than 0.05 were indicated in the quantitative charts. * P < 0.05, ** P < 0.01, *** P < 0.001, one‐way ANOVA (C) or two‐tailed unpaired t ‐test (A, D, E). Acetyl‐Lys, acetylated lysine; GFP, green fluorescent protein; IP, immunoprecipitation; ProT, prothymosin α; SEM, standard error of the mean; Smad7, mothers against decapentaplegic homolog 7; Snail, Zinc finger protein SNAI1; TGF‐β1, transforming growth factor‐β; Twist1, Twist‐related protein 1; ZEB1, Zinc finger E‐box‐binding homeobox 1.

Article Snippet: Primary antibodies included monoclonal antibodies against ProT (clone 2F11, ascites fluid) [ ], Smad7 (R&D, Bio‐Techne, Minneapolis, MI, USA), Snail (Abcam), Twist1 (Genetex), and ZEB1 (Novus).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Transfection, Plasmid Preparation, Over Expression, Transduction, Immunoprecipitation, Mutagenesis, Two Tailed Test, Binding Assay

Journal: Molecular Oncology

Article Title: Aberrant expression of nuclear prothymosin α contributes to epithelial‐mesenchymal transition in lung cancer

doi: 10.1002/1878-0261.70035

Figure Lengend Snippet: ProT promotes Smad7 nuclear localization and enhances Smad7‐mediated transcriptional repression of SNAI1 , TWIST1 , and ZEB1 genes. (A) Immunofluorescence staining of A549/GFP ( n = 3) and A549/ProT ( n = 3) cells with mouse anti‐human Smad7 antibody, followed by Alexa‐fluor 594‐conjugated goat anti‐mouse IgG after treatment with TGF‐β1 (10 ng·mL −1 ) for 3 h. Nuclei were counterstained with DAPI. Fluorescence signals for Smad7 (red) and the nucleus (blue) were examined by fluorescence microscopy. The merged column represents the superposition of the cells stained with anti‐Smad7 and DAPI. Scale bars shown on 400× images correspond to 20 μm. Quantification of fluorescence intensity of nuclear Smad7 was analyzed by the imagej software (National Institutes of Health, Bethesda, MD, USA), and values shown are nuclear : total Smad7 fluorescence ratios. (B) ChIP analysis of Smad2 binding to SNAI1 , TWIST1 , and ZEB1 promoters in A549/GFP ( n = 3) and A549/ProT ( n = 3) cells following treatment with TGF‐β1 for 3 h. (C) ChIP analysis of Smad7 binding to SNAI1 , TWIST1 , and ZEB1 promoters in A549/GFP ( n = 3) and A549/ProT ( n = 3) cells transduced with Flag‐Smad7 following treatment with TGF‐β1 for 3 h. (B, C) Representative PCR results from three independent experiments and quantitative analysis of the indicated genes are shown, and ratios of control cells were arbitrarily set to 100. The quantification values and error bars of (A–C) displayed were mean ± SEM, and P ‐values less than 0.05 were indicated in the quantitative charts (* P < 0.05, two‐tailed unpaired t ‐test). (D) ChIP analysis of Smad7, but not Smad7(K64R/K70R), binding to SNAI1 , TWIST1 , and ZEB1 promoters. 293T cells that had been transduced with lentiviral vectors encoding ProT ( n = 3) or GFP ( n = 3) were further transduced with Flag‐Smad7. Immunoprecipitation was done using anti‐Flag M2 magnetic beads. ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; ProT, prothymosin α; SEM, standard error of the mean; Smad, mothers against decapentaplegic homolog; Snail, Zinc finger protein SNAI1; Twist1, Twist‐related protein 1; ZEB1, Zinc finger E‐box‐binding homeobox 1.

Article Snippet: Primary antibodies included monoclonal antibodies against ProT (clone 2F11, ascites fluid) [ ], Smad7 (R&D, Bio‐Techne, Minneapolis, MI, USA), Snail (Abcam), Twist1 (Genetex), and ZEB1 (Novus).

Techniques: Immunofluorescence, Staining, Fluorescence, Microscopy, Software, Binding Assay, Transduction, Control, Two Tailed Test, Immunoprecipitation, Magnetic Beads, Chromatin Immunoprecipitation

Journal: Molecular Oncology

Article Title: Aberrant expression of nuclear prothymosin α contributes to epithelial‐mesenchymal transition in lung cancer

doi: 10.1002/1878-0261.70035

Figure Lengend Snippet: Nuclear ProT‐expressing lung tumors exhibit low metastatic potential in vivo and express low levels of Snail. (A) Tumor volumes in mice bearing A549/ProT or A549/GFP tumors. NOD/SCID mice were subcutaneously inoculated with A549/ProT ( n = 7) or A549/GFP ( n = 7) cells (4 × 10 6 ) at day 0, and tumor volumes were measured every 3–4 days. Mice were sacrificed when their tumor volumes exceeded 2000 mm 3 . Error bars indicated mean ± SEM. (B) The contingency of lung metastatic tumor was measured using Fisher's exact test ( left , P < 0.0001). The number of metastatic nodules was counted and assessed using the Mann–Whitney U ‐test ( right , P = 0.0210). (C and D) Gross appearance (upper panels) and histology (lower panels) of the lungs from A549/GFP (C) and A549/ProT (D) tumor‐bearing mice. Arrowheads indicate tumor nodules. Scale bars shown on 1×, 40×, and 100× images correspond to 5 mm, 200 μm, and 100 μm. (E) Immunohistochemical detection ( left ) and quantification ( right ) of ProT in non‐metastatic and metastatic (original and lung tumor parts) tumor tissues. Scale bars shown on 100× images correspond to 50 μm, and the boxed areas are magnified right on each panel. (F) Immunohistochemical detection ( left ) and quantification ( right ) of Snail, Twist1, and ZEB1 in non‐metastatic and metastatic (original and lung tumor parts) tumor tissues. Scale bars shown on 200× images correspond to 20 μm. The P ‐values of (B) were calculated using the Fisher's exact test or Mann–Whitney U ‐test. GFP, green fluorescent protein; IHC, immunohistochemistry; ProT, prothymosin α; SEM, standard error of the mean; Snail, Zinc finger protein SNAI1; Twist1, Twist‐related protein 1; ZEB1, Zinc finger E‐box‐binding homeobox 1.

Article Snippet: Primary antibodies included monoclonal antibodies against ProT (clone 2F11, ascites fluid) [ ], Smad7 (R&D, Bio‐Techne, Minneapolis, MI, USA), Snail (Abcam), Twist1 (Genetex), and ZEB1 (Novus).

Techniques: Expressing, In Vivo, MANN-WHITNEY, Immunohistochemical staining, Immunohistochemistry, Binding Assay

Journal: Molecular Oncology

Article Title: Aberrant expression of nuclear prothymosin α contributes to epithelial‐mesenchymal transition in lung cancer

doi: 10.1002/1878-0261.70035

Figure Lengend Snippet: Expression of Snail, Twist1, ZEB1, and Smad7 proteins and their correlations with patient survivals in lung cancer. (A–D) Immunohistochemical staining for Snail (A), Twist1 (B), ZEB1 (C), and Smad7 (D) in tumor specimens from 38 lung cancer patients, including 15 stage I, 3 stage II, 16 stage III, and 4 stage IV/metastasis (M) samples. Scale bars shown on 400× images correspond to 20 μm. Levels of immunointensity of Snail, Twist1, ZEB1, and Smad7 were analyzed by three randomly selected fields within the same specimen and quantified. Values shown are the relative immunointensity, with the level in stage I tumors arbitrarily set to 100. Quantification values and error bars of immunohistochemical staining displayed were mean ± SEM, and P ‐values less than 0.05 were indicated in the quantitative charts (* P < 0.05, *** P < 0.001, two‐tailed unpaired t ‐test). Immunointensity of the indicated proteins was categorized as low and high in tumor specimens of patients with lung cancer. Kaplan–Meier curves of OS, PFS, and DMFS in patients with high or low expression of Snail (A), Twist1 (B), ZEB1 (C), and Smad7 (D). Differences in survival intervals were analyzed by the log‐rank test, and P ‐values less than 0.05 were shown in the charts of Kaplan–Meier curves. DMFS, distant metastasis‐free survival; IHC, immunohistochemistry; M, metastasis; OS, overall survival; PFS, progression‐free survival; ProT, prothymosin α; SEM, standard error of the mean; Smad7, mothers against decapentaplegic homolog 7.

Article Snippet: Primary antibodies included monoclonal antibodies against ProT (clone 2F11, ascites fluid) [ ], Smad7 (R&D, Bio‐Techne, Minneapolis, MI, USA), Snail (Abcam), Twist1 (Genetex), and ZEB1 (Novus).

Techniques: Expressing, Immunohistochemical staining, Staining, Two Tailed Test, Immunohistochemistry

Journal: Molecular Oncology

Article Title: Aberrant expression of nuclear prothymosin α contributes to epithelial‐mesenchymal transition in lung cancer

doi: 10.1002/1878-0261.70035

Figure Lengend Snippet: Low expression of nuclear ProT is associated with the EMT process and poor prognosis in lung cancer. (A) A positive correlation ( P < 0.05; Pearson's correlation coefficient) between levels of nuclear ProT immunointensity and those of Smad7 immunointensity. (B) Kaplan–Meier curves of OS, PFS, and DMFS in patients with high or low expression of nuclear ProT and Smad7. (C–E) Negative correlations ( P < 0.05; Pearson's correlation coefficient) between levels of Snail (C), Twist1 (D), or ZEB1 (E) immunointensity and those of nuclear ProT or Smad7. (F–H) Kaplan–Meier curves of OS, PFS, and DMFS in patients with high or low expression of Snail (F), Twist1 (G), or ZEB1 (H), as well as nuclear ProT and Smad7. The P ‐values of (A, C, E, G) were analyzed by Pearson's correlation coefficient. Differences in survival intervals were analyzed by the log‐rank test and P ‐values less than 0.05 were shown in the charts of Kaplan–Meier curves (B, F, D, H). DMFS, distant metastasis‐free survival; OS, overall survival; PFS, progression‐free survival; ProT, prothymosin α; Smad7, mothers against decapentaplegic homolog 7.

Article Snippet: Primary antibodies included monoclonal antibodies against ProT (clone 2F11, ascites fluid) [ ], Smad7 (R&D, Bio‐Techne, Minneapolis, MI, USA), Snail (Abcam), Twist1 (Genetex), and ZEB1 (Novus).

Techniques: Expressing

Journal: Molecular Oncology

Article Title: Aberrant expression of nuclear prothymosin α contributes to epithelial‐mesenchymal transition in lung cancer

doi: 10.1002/1878-0261.70035

Figure Lengend Snippet: Proposed model for the inhibition of EMT by nuclear ProT through enhancing Smad7 acetylation and competing with the binding element of Smad2/3 on SNAI1 , TWIST1 , and ZEB1 promoters in lung cancer. Nuclear ProT expression decreases with lung cancer progression and correlates with poor prognosis. In the early stage of lung cancer, overexpression of nuclear ProT attenuates TGF‐β‐induced EMT, suggesting a regulatory role for ProT in TGF‐β signaling. ProT increases Smad7 acetylation, leading to Smad7‐mediated transcriptional repression of SNAI1 , TWIST1 , and ZEB1 . By increasing Smad7 binding to EMT gene promoters, ProT competes with Smad2 to repress Snail, Twist1, and ZEB1 expression. In the late/metastatic stage of lung cancer, the decrease of nuclear ProT loses its role in repressing EMT. The aberrant loss of nuclear ProT correlates with heightened EMT and poor clinical outcomes in lung cancer patients.

Article Snippet: Primary antibodies included monoclonal antibodies against ProT (clone 2F11, ascites fluid) [ ], Smad7 (R&D, Bio‐Techne, Minneapolis, MI, USA), Snail (Abcam), Twist1 (Genetex), and ZEB1 (Novus).

Techniques: Inhibition, Binding Assay, Expressing, Over Expression

Journal: Communications Biology

Article Title: Targeting endothelin 1 receptor-miR-200b/c-ZEB1 circuitry blunts metastatic progression in ovarian cancer

doi: 10.1038/s42003-020-01404-3

Figure Lengend Snippet: a Expression of ET A R, ZEB1, N-cadherin, Vimentin, and E-cadherin in ovarian cancer cell lines analyzed by Western blotting (WB). β-actin is used as loading control. b Correlation of the ET A R and ZEB1 expression in the indicated cell lines. The ratio of ET A R/β-actin and ZEB1/β-actin, evaluated by WB as shown in a , is presented as bar and line, respectively. Values are the mean ± SD ( n = 3). c Boxplot diagrams of EDNRA (ET A R) and ZEB1 gene expression in HG-SOC patients from TCGA, subdivided in four molecular subtypes. d Kaplan–Meier curves of overall survival (OS) of 352 patients from TCGA grouped in high EDNRA / ZEB1 levels (160 patients, z -score > 1.5, red line) and low EDNRA / ZEB1 expression levels (192 patients, z -score < 1.5, blue line). e Kaplan–Meier curves of progression-free survival (PFS) of 303 patients from TCGA subdivided in high levels of EDNRA / ZEB1 expression (135 patients, z -score > 1.5, red line) and low levels of EDNRA / ZEB1 (168 patients, z -score < 1.5, blue line).

Article Snippet: ZEB1 3′UTR reporter plasmid (SC-219122, Origene), ET A R 3′UTR reporter plasmid (SC-218505, Origene) and MutET A R 3′UTR plasmid, carrying a double mutation in positions 498–500 and 1726–1728, synthesized by Blue Heron Biotech (WA, USA).

Techniques: Expressing, Western Blot, Control, Gene Expression

Journal: Communications Biology

Article Title: Targeting endothelin 1 receptor-miR-200b/c-ZEB1 circuitry blunts metastatic progression in ovarian cancer

doi: 10.1038/s42003-020-01404-3

Figure Lengend Snippet: a Venn diagram of miRNAs whose expression is associated with a good prognosis in ovarian cancer patients (MiROvaR signature ) showing the overlap ( n = 3) between miRNA predicted to target the ET A R 3′UTR ( n = 5, red circle) and miRNA predicted to target the ZEB1 3′UTR ( n = 7, green circle). b Expression levels of ET A R and miR-200b or miR-200c in the indicated cell lines. The ratio of ET A R/β-actin, evaluated by WB and miR-200b/c expression, evaluated by qRT-PCR and normalized to U6, are shown as bar and line, respectively. Values are the mean ± SD (n = 3). c ET A R mRNA levels in HEY and SKOV3 cells transfected for 48 h with mimic-miRNA control (miR-Ctr), mimic-miR-200b (miR-200b) or mimic-miR-200c (miR-200c) evaluated by qRT-PCR and normalized to cyclophilin-A. Values are the mean ± SD ( n = 3; * p < 0.001 vs. Ctr). d Lysates from HEY and SKOV3 cells transfected as in c are analyzed by WB for ET A R expression. β-actin is used as loading control. e Luciferase activity in HEY cells co-transfected for 48 h with miR-Ctr, miR-200b, or miR-200c together with the reporter plasmid containing the 3’UTR region of ET A R (ET A R 3′UTR) or its triple mutant (mut/ΔET A R 3′UTR). Values are the mean ± SD expressed as fold induction ( n = 3; * p < 0.001 vs. Ctr). f WB for ET A R expression in the lysates from HEY cells 48 h transfected with miRNA control, miR-200b or miR-200c inhibitors (anti-miR-Ctr, anti-miR-200b, and anti-miR-200c). β-actin is used as loading control. g Cell viability of HEY cells, transfected as in c and f , or with siRNA control (si-Ctr), or si-ET A R, and treated or not with macitentan (MAC) for 48 h. Values are the mean ± SD ( n = 4; * p < 0.001 vs the respective Ctr). h Lysates of HEY cells transfected as in c together with a plasmid control (Mock), a wt ET A R expression plasmid, or with an ET A R expression plasmid w/o the 3′UTR as indicated are analyzed by WB for ET A R expression. β-actin is used as loading control. i Cell vitality of HEY cells transfected as in h . Values are the mean ± SD expressed as fold induction ( n = 4; * p < 0.001 vs. Mock; ** p < 0.001 vs. wt ET A R).

Article Snippet: ZEB1 3′UTR reporter plasmid (SC-219122, Origene), ET A R 3′UTR reporter plasmid (SC-218505, Origene) and MutET A R 3′UTR plasmid, carrying a double mutation in positions 498–500 and 1726–1728, synthesized by Blue Heron Biotech (WA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis

Journal: Communications Biology

Article Title: Targeting endothelin 1 receptor-miR-200b/c-ZEB1 circuitry blunts metastatic progression in ovarian cancer

doi: 10.1038/s42003-020-01404-3

Figure Lengend Snippet: a Expression levels of ZEB1 and miR-200b or miR-200c in the indicated cell lines. The ratio of ZEB1/β-actin, evaluated by WB and miR-200b/c expression, evaluated by qRT-PCR, are shown as bar and line, respectively. Values are the mean ± SD ( n = 3). b ZEB1 mRNA levels in HEY and SKOV3 cells transfected for 48 h with mimic-miRNA control (miR-Ctr), mimic-miR-200b (miR-200b), or mimic-miR-200c (miR-200c) evaluated by qRT-PCR and normalized to cyclophilin-A. Values are the mean ± SD ( n = 3; * p < 0.001 vs. Ctr). c Lysates from HEY and SKOV3 cells transfected as in b are analyzed by WB for ZEB1 expression. β-actin is used as loading control. d Luciferase activity in HEY cells co-transfected for 48 h with miR-Ctr, miR-200b, or miR-200c together with the reporter plasmid containing the 3′UTR region of ZEB1. Values are the mean ± SD ( n = 3; * p < 0.001 vs. Ctr). e WB for ZEB1 expression in the lysates from HEY cells transfected for 48 h with miRNA control, miR-200b, or miR-200c inhibitors (anti-miR-Ctr, anti-miR-200b, and anti-miR-200c). β-actin is used as loading control.

Article Snippet: ZEB1 3′UTR reporter plasmid (SC-219122, Origene), ET A R 3′UTR reporter plasmid (SC-218505, Origene) and MutET A R 3′UTR plasmid, carrying a double mutation in positions 498–500 and 1726–1728, synthesized by Blue Heron Biotech (WA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Luciferase, Activity Assay, Plasmid Preparation

Journal: Communications Biology

Article Title: Targeting endothelin 1 receptor-miR-200b/c-ZEB1 circuitry blunts metastatic progression in ovarian cancer

doi: 10.1038/s42003-020-01404-3

Figure Lengend Snippet: a , b Expression of miR-200b/c and ET A R, ZEB1 proteins in HEY cells transfected for 72 h with siRNA control (si-Ctr) or si-ZEB1 as determined by qRT-PCR ( a ) and WB ( b ). U6 and β-actin are used to normalize miRNA and protein expression, respectively. Values are the mean ± SD ( n = 3; * p < 0.0001 vs. Ctr). c , d Luciferase activity ( c ) in HEY cells co-transfected for 48 h with the indicated mimic-miRNAs, in the presence or in the absence of ZEB1 plasmid together with the ET A R 3′UTR reporter plasmid. qRT-PCR ( d ) of the miR-200b/c expression in cells transfected as above and normalized to U6. Values are the mean ± SD expressed as fold induction ( n = 3; * p < 0.01 vs. Ctr; ** p < 0.001 vs. ZEB1-transfected cells). e Expression of ET A R and ZEB1 proteins in lysates from cells treated as in c analyzed by WB. β-actin is used as loading control. f , g Luciferase activity ( f ) in HEY cells co-transfected for 48 h with the indicated anti-miRNAs, in the presence or absence of si-ZEB1 together with the ET A R 3′UTR reporter plasmid. qRT-PCR ( g ) of the miR-200b/c expression, in cells transfected as above and normalized to U6. Values are the mean ± SD expressed as fold induction ( n = 3; * p < 0.01 vs. Ctr; ** p < 0.001 vs. si-ZEB1-transfected cells). h Luciferase activity in HEY cells co-transfected for 48 h with si-Ctr or si-ZEB1 together with the reporter plasmid containing the 3’UTR region of ET A R (ET A R 3′UTR) or its triple mutant (mut/ΔET A R 3′UTR). Values are the mean ± SD expressed as fold induction ( n = 3; *, p < 0.001 vs. ET A R 3′UTR-transfected Ctr cells). i Schematic model of the ET A R/ZEB1 and miR-200b/c feedback circuit.

Article Snippet: ZEB1 3′UTR reporter plasmid (SC-219122, Origene), ET A R 3′UTR reporter plasmid (SC-218505, Origene) and MutET A R 3′UTR plasmid, carrying a double mutation in positions 498–500 and 1726–1728, synthesized by Blue Heron Biotech (WA, USA).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Luciferase, Activity Assay, Plasmid Preparation, Mutagenesis

Journal: Communications Biology

Article Title: Targeting endothelin 1 receptor-miR-200b/c-ZEB1 circuitry blunts metastatic progression in ovarian cancer

doi: 10.1038/s42003-020-01404-3

Figure Lengend Snippet: a Expression of ZEB1 and ET A R proteins in HEY cells transfected for 48 h with siRNA control (si-Ctr) or si-ET A R or treated with MAC and stimulated or not for 48 h with ET-1 analyzed by WB. β-actin is used as loading control. b Luciferase activity in HEY cells co-transfected for 48 h with si-Ctr, si-ET A R, and the ZEB1 promoter reporter plasmid and stimulated with ET-1 and/or MAC for 24 h.Values are the mean ± SD expressed as fold induction ( n = 3; * p < 0.0001 vs. si-Ctr; ** p < 0.0001 vs. ET-1 stimulated si-Ctr). c qRT-PCR for miR-200b/c expression in HEY cells transfected with si-Ctr or si-ZEB1 and stimulated with ET-1 for 72 h in the absence or in the presence of MAC. U6 is used to normalize. Values are the mean ± SD ( n = 3; * p < 0.001 vs. unstimulated Ctr; ** p < 0.001 vs. ET-1-stimulated Ctr). d Pri-miR-200b/c expression in cells transfected and treated as in c for 48 h is evaluated by qRT-PCR and normalized to cyclophilin-A. Values are the mean ± SD ( n = 3; *, p < 0.01 vs. unstimulated Ctr; ** p < 0.001 vs. ET-1-stimulated Ctr). e Schematic representation of the ZEB1 binding sites contained in the miR-200b/c promoter reporter plasmids. f Luciferase activity in HEY cells co-transfected for 48 h with si-Ctr, si-ET A R, or si-ZEB1 and the reporter plasmids for miR-200b/c promoters and stimulated or not with ET-1 and/or MAC for 24 h. Values are the mean ± SD expressed as fold induction ( n = 3; * p < 0.001 vs. unstimulated Ctr; ** p < 0.001 vs. ET-1-stimulated Ctr).

Article Snippet: ZEB1 3′UTR reporter plasmid (SC-219122, Origene), ET A R 3′UTR reporter plasmid (SC-218505, Origene) and MutET A R 3′UTR plasmid, carrying a double mutation in positions 498–500 and 1726–1728, synthesized by Blue Heron Biotech (WA, USA).

Techniques: Expressing, Transfection, Control, Luciferase, Activity Assay, Plasmid Preparation, Quantitative RT-PCR, Binding Assay

Journal: Communications Biology

Article Title: Targeting endothelin 1 receptor-miR-200b/c-ZEB1 circuitry blunts metastatic progression in ovarian cancer

doi: 10.1038/s42003-020-01404-3

Figure Lengend Snippet: a Conditioned media from SKOV3 cells transfected with siRNA control (si-Ctr), si-ZEB1, or mimic-miR-200b/c (miR-200b/c), or treated with MAC, stimulated or not with ET-1 for 48 h are analyzed by gelatin zymography to detect the active forms of MMP-2/9. b Expression of E-cadherin, N-cadherin, Vimentin, and ZEB1 proteins is analyzed by WB in SKOV3 cells transfected and stimulated as in a . β-actin is used as loading control. c E-cadherin, ZEB1, N-cadherin, and Vimentin gene expression in SKOV3 cells transfected with si-Ctr, si-ZEB1, si-ET A R, or mimic-miR-200b/c (miR-200b/c), and treated as in b is analyzed by qRT-PCR and normalized to cyclophilin-A. Values are the mean ± SD ( n = 3; * p < 0.02 vs. si-Ctr; ** p < 0.004 vs. ET-1 stimulated si-Ctr). d Luciferase activity in SKOV3 cells transfected as in c and co-transfected with the E-cadherin promoter reporter plasmid and stimulated with ET-1 and/or MAC for 48 h. Values are the mean ± SD expressed as fold induction ( n = 3; * p < 0.002 vs. si-Ctr; ** p < 0.004 vs. ET-1 stimulated si-Ctr). e Assay of tubule-like structure formation in SKOV3 cells transfected for 48 h as in c and overnight stimulated or not with ET-1. Original magnification 20×. (Scale bar: 100 μm). Graphs represent the quantification of the number of nodes and the tube length. Columns show the mean ± SD ( n = 3; * p < 0.02 vs. unstimulated Ctr; ** p < 0.001 vs. ET-1-stimulated Ctr). f Chemoinvasion assay in SKOV3 cells transfected as in c and overnight stimulated or not with ET-1. Images represent the crystal violet-stained invasive cells. Magnification ×10. Graph represents the number of invading cells. Columns show the mean ± SD ( n = 3; * p < 0.001 vs. unstimulated Ctr; ** p < 0.001 vs. ET-1-stimulated Ctr).

Article Snippet: ZEB1 3′UTR reporter plasmid (SC-219122, Origene), ET A R 3′UTR reporter plasmid (SC-218505, Origene) and MutET A R 3′UTR plasmid, carrying a double mutation in positions 498–500 and 1726–1728, synthesized by Blue Heron Biotech (WA, USA).

Techniques: Transfection, Control, Zymography, Expressing, Gene Expression, Quantitative RT-PCR, Luciferase, Activity Assay, Plasmid Preparation, Staining

Journal: Communications Biology

Article Title: Targeting endothelin 1 receptor-miR-200b/c-ZEB1 circuitry blunts metastatic progression in ovarian cancer

doi: 10.1038/s42003-020-01404-3

Figure Lengend Snippet: a , b Female nude mice are i.p. injected with SKOV3, OVCAR-3, A2780, or A2780 CIS cells and treated with vehicle (Ctr) or MAC (30 mg/kg oral daily) for 5 weeks. Graph represents the number of visible metastasis. Columns show the mean ± SD (* p < 0.001 vs. Ctr). c WB analysis of the indicated proteins in lysates from i.p. nodules of three representative (M1-M3) SKOV3, OVCAR-3, A2780, and A2780 CIS xenografts treated as in a . β-actin is used as loading control. d ZEB1, Vimentin, and E-cadherin gene expression in i.p. nodules of SKOV3, OVCAR-3, A2780, and A2780 CIS xenografts treated as in a is analyzed by qRT-PCR and normalized to cyclophilin-A. Values are the mean ± SD (* p < 0.004 vs. Ctr). e miR-200b/c expression in i.p. nodules of SKOV3, OVCAR-3, A2780, and A2780 CIS xenografts treated as in a is analyzed by qRT-PCR. U6 is used to normalize. Values are the mean ± SD (* p < 0.006 vs. Ctr). f The binding of ZEB1 on miR-200b promoter region and on a region -1900 bp upstream the miR-200b TSS site (lacking ZEB1 binding sites, negative control for non-specific enrichment) is analyzed by ChIP assay followed by PCR in SKOV3 xenografts treated as in a . Anti-IgG mouse (IgGM) Ab is used as control for all ChIP reactions. g Schematic model of a regulatory circuit established by ET A R-miR-200b/c-ZEB1 to control metastatic progression that is activated by ET-1 and inhibited by macitentan.

Article Snippet: ZEB1 3′UTR reporter plasmid (SC-219122, Origene), ET A R 3′UTR reporter plasmid (SC-218505, Origene) and MutET A R 3′UTR plasmid, carrying a double mutation in positions 498–500 and 1726–1728, synthesized by Blue Heron Biotech (WA, USA).

Techniques: Injection, Control, Gene Expression, Quantitative RT-PCR, Expressing, Binding Assay, Negative Control

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Inhibition of ZEB1 by miR-200 characterizes Helicobacter pylori-positive gastric diffuse large B-cell lymphoma with a less aggressive behavior.

doi: 10.1038/modpathol.2013.229

Figure Lengend Snippet: Figure 1 Inhibition of ZEB1 by miRNA-200 characterized H. pylori-positive gastric diffuse large B-cell lymphomas. (a) H. pylori-positive gastric diffuse large B-cell lymphomas have higher levels of miR-200 a, b, and c. y axis: miR-200 expression levels measured by the NanoString technology; x axis: miR-200, a, b, or c in nine HP-negative (white) or nine HP-positive (gray) gastric diffuse large B-cell lymphomas. (b) H. pylori-positive gastric diffuse large B-cell lymphomas have lower levels of ZEB1. y axis: ZEB1 expression levels measured by microarrays; x axis: ZEB1 in nine H. pylori-negative (white) or nine H. pylori-positive (gray) gastric diffuse large B-cell lymphomas. (c) Inverse correlations between ZEB1 and miR-200 in gastric diffuse large B-cell lymphomas. y axis: ZEB1 levels; x axis: miR-200 a, b, or c. Gray: nine H. pylori-negative gastric diffuse large B-cell lymphomas; black: nine H. pylori-positive gastric diffuse large B-cell lymphomas. Correlation coefficients at 0.60 at P ¼ 0.008 for miR-200a, 0.71 at Po0.001 for miR-200b, and 0.63 at P ¼ 0.005 for miR-200c, respectively.

Article Snippet: Immunohistochemistry for ZEB1 and BCL6 Immunoperoxidase stains on formalin-fixed, paraffin-embedded tissue sections with a ZEB1 antibody (1:100, rabbit polyclonal, Novus, Littleton, CO, USA) and a BCL6 antibody (1:100, PG-B6P, mouse monoclonal, Abcam, Cambridge, MA, USA).

Techniques: Inhibition, Expressing

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Inhibition of ZEB1 by miR-200 characterizes Helicobacter pylori-positive gastric diffuse large B-cell lymphoma with a less aggressive behavior.

doi: 10.1038/modpathol.2013.229

Figure Lengend Snippet: Figure 2 Hierarchical clustering of nine H. pylori-positive and nine H. pylori-negative gastric diffuse large B-cell lymphomas. (a) Hierarchical clustering with the whole panel of eight miRNAs and 30 mRNAs generated cluster I with eight H. pylori-positive gastric diffuse large B-cell lymphomas, and cluster II with nine H. pylori- negative gastric diffuse large B-cell lymphomas plus one H. pylori- positive gastric diffuse large B-cell lymphomas (8:0 vs 1:9, Po0.001). (b) Hierarchical clustering with miR-200 and ZEB1 generated cluster I with eight H. pylori-positive gastric diffuse large B-cell lymphomas and two H. pylori-negative gastric diffuse large B-cell lymphomas, and cluster II with seven H. pylori-negative gastric diffuse large B-cell lymphomas and one H. pylori-positive gastric diffuse large B-cell lymphomas (8:2 vs 1:7, P ¼ 0.007).

Article Snippet: Immunohistochemistry for ZEB1 and BCL6 Immunoperoxidase stains on formalin-fixed, paraffin-embedded tissue sections with a ZEB1 antibody (1:100, rabbit polyclonal, Novus, Littleton, CO, USA) and a BCL6 antibody (1:100, PG-B6P, mouse monoclonal, Abcam, Cambridge, MA, USA).

Techniques: Generated

Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

Article Title: Inhibition of ZEB1 by miR-200 characterizes Helicobacter pylori-positive gastric diffuse large B-cell lymphoma with a less aggressive behavior.

doi: 10.1038/modpathol.2013.229

Figure Lengend Snippet: Figure 4 Immunohistochemistry for ZEB1 and BCL6 and qRT-PCRs for miR-200 in gastric diffuse large B-cell lymphomas. (a) Immunoperoxidase stains for ZEB1 and BCL6 in H. pylori-positive and H. pylori-negative gastric diffuse large B-cell lymphomas ( 1000). An Olympus BX60 microscope with 10/0.22 ocular lens and 100/1.35 objective lens was used (Olympus). (b, c) Distributions for the percentages of ZEB1-positive (b) or BCL6-positive (c) cells in gastric diffuse large B-cell lymphomas. y axis: percentages of positive cells; x axis: H. pylori-negative vs H. pylori-positive gastric diffuse large B-cell lymphomas. (d) qRT-PCRs for miR- 200 a, b, or c in gastric diffuse large B-cell lymphomas. y axis: dCt; x axis: H. pylori-negative vs H. pylori-positive gastric diffuse large B-cell lymphomas.

Article Snippet: Immunohistochemistry for ZEB1 and BCL6 Immunoperoxidase stains on formalin-fixed, paraffin-embedded tissue sections with a ZEB1 antibody (1:100, rabbit polyclonal, Novus, Littleton, CO, USA) and a BCL6 antibody (1:100, PG-B6P, mouse monoclonal, Abcam, Cambridge, MA, USA).

Techniques: Immunohistochemistry, Microscopy