Journal: bioRxiv

Article Title: scRNA-Sequencing uncovers a TCF-4-dependent transcription factor network regulating commissure development

doi: 10.1101/2020.06.16.156083

Figure Lengend Snippet: A Scheme of the workflow used to recluster cells after GRN analysis and resulting tSNE-Plot of the Satb2 cluster. Regulons are binarized and reclustered accordingly. WT and KO cells segregated based on GRN activity with only minor overlap. B Differentially active regulons of the Satb2 cluster that may be possible interactors of TCF-4. C tSNE-Plots showing the regulon activity of Ctcf, Cux1, Foxg1, Pou3f3 (also known as Brn1 ), Smarca4 (also known as Brg1 ) and Sox11 in a continuous scale (left, red) or binarized (right, blue). The regulons are preferentially active in the WT cells with only a small number of KO cells in the ON-State. D Number and percentage of regulators associated with autism spectrum disorders (ASD), intellectual disability (ID) and schizophrenia in the Satb2 cluster. E Pie charts depicting the percentage of bHLH factors and differentially expressed regulators in the differentially active regulons. F Representative images of TCF-4 (white) and FOXG1, POU3F3 (BRN1), SMARCA4 (BRG1) and SOX11 (all in red) in E18.5 WT cortices. Note the expression in the same nuclei. Scale bar, 50 μm D Co-immunoprecipitation assay using anti-TCF-4 antibody conducted with HEK cell extract after overexpression of TCF-4 and CUX1, FOXG1, POU3F3 (BRN1), SMARCA4 (BRG1) and SOX11 in HEK cells. Upper panels: detection with anti-TCF-4 antibody. Lower panels: detection with anti-MYC, anti-FOXG1, anti-BRN1, anti-BRG1 or anti-SOX11 antibody. The blots presented are cropped. All proteins were co-immunoprecipitated with TCF-4, but not with an isotype control for IgG or Agarose A Beads alone except for BRN-1 which was precipitated to a small amount by the isotope control IgG. We could also show that TCF-4 was co-immunoprecipitated with anti-Myc antibody (precipitation of Myc-tagged Cux1). The interactions were confirmed in three independent biological replicates, (n = 3). E Co-immunoprecipitation assay conducted with E18.5 cortex lysates using anti-SOX11 antibody. Upper panel: detection with anti-TCF-4 antibody. Lower panel: detection with anti-SOX11 antibody. The blots presented are cropped. TCF-4 was co-immunoprecipitated with SOX11, but not with an isotype control for IgG and Agarose A Beads alone. The interaction was confirmed in three independent biological replicates (n = 3).

Article Snippet: At a confluency of 70-90% cells were transfected using JETPEI (Polyplus transfection, 101-10 N) with equal amounts of the expression vectors (7.5 μg/10 cm dish) of CAG-TCF-4-IRES-GFP and the predicted interaction partners [pCMV5 rBrn1( ); pBJ5-hBRG1 (pBJ5 hBRG1 was a gift from Jerry Crabtree (Addgene plasmid # 17873; http://n2t.net/addgene:17873 ; RRID:Addgene_17873))( ); pXJ42-p200 CUX1 (pXJ42-p200 CUX1 was a gift from Alain Nepveu (Addgene plasmid # 100813; http://n2t.net/addgene:100813 ; RRID:Addgene_100813))( ); CAG- Foxg1 -IRES-RFP; CAG- Sox11 -IRES-GFP( )) according to the manufacturer instruction.

Techniques: Activity Assay, Expressing, Co-Immunoprecipitation Assay, Over Expression, Immunoprecipitation, Control

Journal: Life sciences

Article Title: Neutrophil elastase-mediated proteolysis of the tumor suppressor p200 CUX1 promotes cell proliferation and inhibits cell differentiation in APL.

doi: 10.1016/j.lfs.2019.117229

Figure Lengend Snippet: Fig. 4. NE co-localizes and interacts with p200 CUX1. (A) Expression levels of

Article Snippet: Primary antibodies were purchased from the following sources: against human NE (no. ab131260) from Abcam (UK); against p200 CUX1 (no. 81557), P21 Waf1/Cip1 (no. 2947), P27Kip1 (no. 3686), and C/EBPα (no. 8178) from Cell Signaling Technology (CST, USA); against p110/p75 CUX1 (no. TA890108) from Origene Technologies (Origene, USA); against C/EBPβ (no. sc-137146) from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Expressing

Journal: Life sciences

Article Title: Neutrophil elastase-mediated proteolysis of the tumor suppressor p200 CUX1 promotes cell proliferation and inhibits cell differentiation in APL.

doi: 10.1016/j.lfs.2019.117229

Figure Lengend Snippet: Fig. 5. NE effectively hydrolyzes p200 CUX1. (A) NE overexpression decreased

Article Snippet: Primary antibodies were purchased from the following sources: against human NE (no. ab131260) from Abcam (UK); against p200 CUX1 (no. 81557), P21 Waf1/Cip1 (no. 2947), P27Kip1 (no. 3686), and C/EBPα (no. 8178) from Cell Signaling Technology (CST, USA); against p110/p75 CUX1 (no. TA890108) from Origene Technologies (Origene, USA); against C/EBPβ (no. sc-137146) from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Over Expression

Journal: Life sciences

Article Title: Neutrophil elastase-mediated proteolysis of the tumor suppressor p200 CUX1 promotes cell proliferation and inhibits cell differentiation in APL.

doi: 10.1016/j.lfs.2019.117229

Figure Lengend Snippet: Fig. 6. p200 CUX1 overexpression reverses the effects of NE on cell proliferation

Article Snippet: Primary antibodies were purchased from the following sources: against human NE (no. ab131260) from Abcam (UK); against p200 CUX1 (no. 81557), P21 Waf1/Cip1 (no. 2947), P27Kip1 (no. 3686), and C/EBPα (no. 8178) from Cell Signaling Technology (CST, USA); against p110/p75 CUX1 (no. TA890108) from Origene Technologies (Origene, USA); against C/EBPβ (no. sc-137146) from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Over Expression

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: Expression profiling was performed on microdissected tumor cells from 17 mammary tumors that developed in MMTV-p110 or p75 CUX1 transgenic mice, including 8 solid carcinomas (p110-416, p110-314, p110-360, p75-534, p75-150, p110-143, p75-129, p110-225), 4 adenosquamous carcinomas (p75-101, p75-94, p75-80, p75-47), 1 adenoma (p110-98), 2 adenomyoepitheliomas (p75-90, p110-374), and 1 adenocarcinoma (p75-216). The complete expression profiles are available on the Gene Expression Omnibus (GEO) repository under accession number GSE54804. The figure shows a heatmap of mammary tumors grouped according to an unsupervised hierarchical clustering of Wnt gene expression. Expression of Glis1 , as well as Vim , Cdh1 , Cdh2 , Snai1 , Snai2 , Ocln and Twist1 in each tumor is shown below. The cluster of “high-Wnt” tumors is boxed in yellow and the cluster of “low-Wnt” tumors is boxed in blue. Each column represents one tumor, with the transgene (p110 or p75 CUX1) and mouse ID number indicated below.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Expressing, Transgenic Assay, Gene Expression

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: Cell lines were established from two mammary tumors that developed in transgenic mice carrying a MMTV-p75 CUX1 transgene: an adenosquamous carcinoma from mouse #p75-80 and a solid carcinoma from mouse #p75-534. The two cell lines were analyzed for Wnt gene expression (A), nuclear β-catenin expression (B), and TCF/β-catenin transcriptional activity (C). (A) mRNA levels of Wnt ligand genes were measured by RT-qPCR. Results of triplicate experiments are shown. * P Value<0.05 on a Welch corrected T test. Und: Undetected. (B) Cells were analyzed by indirect immunofluorescence using antibodies specific for the active, non-phosphorylated form of β-Catenin. Histogram shows the relative mean nuclear signal for β-Catenin as measured using the ImageJ software. The value for p75-534 was set to 1. * P Value<0.05 on a Welch corrected T test. A representative image of each cell line is shown on the right. Scale bar: 20µm. (C) The TOP/FOP luciferase reporter assay was performed in the p75-534 and p75-80 mammary tumor cell lines. In addition, p75-80 cells were cotransfected with a vector expressing either sFRP1, sFRP2 or SOST or an empty vector. Where indicated, niclosamide was added to the cells at the time of transfection. Results of 3 independent transfections are shown. * P Value<0.05 on a Welch corrected T test. A schematic representation of the reporter constructs is shown.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Transgenic Assay, Gene Expression, Expressing, Activity Assay, Quantitative RT-PCR, Immunofluorescence, Software, Luciferase, Reporter Assay, Plasmid Preparation, Transfection, Construct

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: CUX1 specific or scrambled siRNA were transfected in a panel of 6 human cancer cell lines from breast (A), ovarian (B) and lung (C) cancers. 3 days later total mRNA was isolated and quantitative RT-PCR was performed using HPRT1 mRNA as a control. The values represent fold difference in mRNA expression between cells treated with CUX1 or scrambled siRNA. (Und: undetected). * P Value<0.05 on a Welch corrected T test.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Transfection, Isolation, Quantitative RT-PCR, Control, Expressing

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: (A) HEK293 cells were infected with retroviruses to establish cells stably carrying a vector that expresses p110 CUX1 or nothing (vector). Nuclear and cytoplasmic extracts were analyzed by immunoblotting with the indicated antibodies. (B) The TOP or FOP luciferase reporters were introduced into HEK293 cells together with 500 ng of a vector expressing p110 CUX1 or an empty vector. Luciferase activity was measured 36 hours after transfection. Where indicated, a vector expressing either sFRP1, sFRP2, SOST or DKK1 was transfected as well, or niclosamide or IWP-2 was added to the cells at the time of transfection. Results of 3 independent transfections are shown. * P Value<0.05 on a Welch corrected T test. (C) Hs578t and MCF7 breast cancer cells were stably infected with either a lentivirus expressing p110 CUX1 or an empty lentivirus (vector). Nuclear and cytoplasmic extracts were analyzed by immunoblotting with the indicated antibodies. (D) The TOP or FOP luciferase reporters were introduced into Hs578t and MCF7 cells together with 500 ng of a vector expressing p110 CUX1 or an empty vector. Luciferase activity was measured 36 hours after transfection.* P Value<0.05 on a Welch corrected T test.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Infection, Stable Transfection, Plasmid Preparation, Western Blot, Luciferase, Expressing, Activity Assay, Transfection

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: (A) Heatmap of a human breast tumor dataset sorted according to WNT genes expression using the BreSAT algorithm. Expression of CUX1 , GLIS1 , VIM , TWIST1 , SNAI1 , CDH2 , SNAI2 , OCLN and CDH1 in each tumor is shown below. (B) CUX1 and GLIS1 expression in the top 25% and bottom 25% samples sorted according to WNT genes expression. * indicates p<0.05, ** <0.01, *** <0.001 on a Welch-corrected student's T test. (C) CDH1 , OCLN , CDH2 , SNAI1 , SNAI2 , TWIST1 and VIM expression in the top 25% and bottom 25% samples sorted according to Wnt genes expression. * indicates p<0.05, ** <0.01, *** <0.001 on a Welch-corrected student's T test.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Expressing

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: We established populations of MCF10A cells stably carrying lentiviral vectors that express nothing (vector), GLIS1, p110 CUX1, or both GLIS1 and p110 CUX1. (A) Wnt gene mRNA levels were measured by RT-qPCR. Und: Undetected. * P Value<0.05 on a Welch corrected T test when comparing the p110+Glis1 samples vs. each of the other 3. (B) β-Catenin protein levels were measured by immunofluorescence using antibodies specific for the active, non-phosphorylated form of the protein. Histogram shows the mean nuclear signal for β-Catenin for at least 50 cells per condition. * P Value<0.05 on a Welch corrected T test. A representative image of each condition is shown below. Scale bar: 20µm. (C) β-Catenin protein levels were measured by immunoblotting using antibodies specific for the active, non-phosphorylated form of the protein. γ-Tubulin is shown as a loading control. (D) MCF10A cells were transiently transfected with either the TOP of the FOP luciferase reporter, a vector expressing p110 CUX1 or an empty vector, and a vector expressing GLIS1 or the corresponding empty vector. Luciferase activity was measured in 3 independent transfections. * P Value<0.05 on a Welch corrected T test. (E) MCF10A cells ectopically expressing GLIS1, p110 CUX1 or both were imaged by time-lapse video microscopy and their speed of migration was quantified. The average of 3 independent experiments is shown with error bars representing standard error. ** P Value<0.01, *** <0.001 on 3 combined Welch corrected T tests using the Fisher method. (F) Time-lapse video microscopy was carried out as in E and distance migrated from the point of origin was quantified. (G) MCF10A cells overexpressing GLIS1 and p110 CUX1 alone or both were subjected to inverted Boyden chamber invasion assays. The percentage of cells able to migrate more than 20 µm into the matrix is shown for each line. The average of 3 independent experiments is shown. (H) Confocal Z-Stacks acquired 10 µm apart are aligned to demonstrate invasion of MCF10A-derivative cells into the matrix. Representative Z-stack for each condition is displayed. The leftmost image represents cells at the surface of the matrix (Depth = 0).

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence, Western Blot, Control, Transfection, Luciferase, Expressing, Activity Assay, Microscopy, Migration

Journal: Biology Open

Article Title: Autocrine Activation of the Wnt/β-Catenin Pathway by CUX1 and GLIS1 in Breast Cancers

doi: 10.1242/bio.20148193

Figure Lengend Snippet: We established populations of Hs578t cells stably carrying lentiviral vectors that express nothing (vector), GLIS1, p110 CUX1, or both GLIS1 and p110 CUX1. (A) Wnt gene mRNA levels were measured by RT-qPCR. Und: Undetected. * P Value<0.05 on a Welch corrected T test. (B) β-Catenin protein levels were measured by indirect immunofluorescence using antibodies specific for the active, non-phosphorylated form of the protein. Histogram shows the mean nuclear signal for β-Catenin for at least 50 cells per condition. * P Value<0.05 on a Welch corrected T test. A representative image of each condition is shown below. Scale bar: 20µm. (C) Hs578t cells were transiently transfected with either the TOP of the FOP luciferase reporter, and suboptimal amounts (50 ng) of effector plasmids coding for nothing (−), p110 CUX1 or GLIS1. Luciferase activity was measured in 3 independent transfections. * P Value<0.05 on a Welch corrected T test. (D) Hs578t cells ectopically expressing GLIS1, p110 CUX1 or both were imaged by time-lapse video microscopy and their speed of migration was quantified. The average of 3 independent experiments is shown with error bars representing standard error. ** P Value<0.01, *** <0.001 on a Welch corrected T test. (E) Time-lapse video microscopy was carried out as in D and distance migrated from the point of origin was quantified.

Article Snippet: For TOP/FOP reporter assay, Cells plated at 8×10 4 per well in 12-well plates were cotransfected with 0.25 μg of either the TOP 8× or FOP 8× reporter plasmids (Addgene) and 0.5 μg of either CUX1 p110 or empty vector as effector.

Techniques: Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence, Transfection, Luciferase, Activity Assay, Expressing, Microscopy, Migration

Journal: Bone Research

Article Title: The WNT1 G177C mutation specifically affects skeletal integrity in a mouse model of osteogenesis imperfecta type XV

doi: 10.1038/s41413-021-00170-0

Figure Lengend Snippet: Wnt1 G177C/G177C mice display a bone-specific phenotype. a Representative images and quantification of alizarin red staining of ST2 cells transfected with empty vector (pLNCX) and expression plasmids for wild-type ( Wnt1 ) or mutant WNT1 ( G177C ) after 15 days of osteogenic differentiation. n = 3 samples per group. b Expression analysis by qRT–PCR of the indicated genes in ST2 cells transiently transfected with the same plasmids 5 days after transfection. n = 3 samples per group. c Representative Nissl staining of cortex, hippocampus, and cerebellum sections from 4-week-old Wnt1 +/+ and Wnt1 G177C/G177C mice. d Representative immunofluorescent images of the hippocampus in the same mice. Red: Anti-doublecortin (DCX); Blue: DAPI. e Quantification of the cortical Cux1-positive cell layer thickness and of the doublecortin-positive neuroblasts in the subgranular zone of the hippocampus. n = 3 animals per genotype. f Representative contact radiographs of spontaneous fractures in 24-week-old Wnt1 G177C/G177C mice. g Number of spontaneous fractures observed per animal in female and male Wnt1 +/+ , Wnt1 +/G177C , and Wnt1 G177C/G177C mice at 12 and 24 weeks of age. Each symbol represents an individual mouse. h Histological images of a tibial fracture callus from a 24-week-old Wnt1 G177C/G177C mouse. Top left: von Kossa/van Gieson stain; bottom left: Masson-Goldner stain, squares indicate areas magnified on the right side as indicated by the numbers. Top right: Masson-Goldner stain; bottom right: toluidine blue stain. i Representative images of tibial bone marrow from nonfractured 24-week-old wild-type or Wnt1 G177C/G177C mice and quantification of the number of bone marrow adipocytes per marrow area (N.Ad/M.Ar). The red rectangle on the symbolic tibia in the top left corner indicates the region depicted in the representative images. Arrows and boxes indicate regions with high numbers of adipocytes. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test ( a , b ) or Student’s t -test ( e , i ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1. Error bars indicate standard deviation

Article Snippet: For immunohistochemical analysis of the brain, polyclonal rabbit anti-CUX1 (1:100, Cat# ABE217, Merck KGaA, Darmstadt, Germany) or polyclonal rabbit anti-doublecortin (anti-DCX) (1:400; Cat#326003, Synaptic Systems GmbH, Germany) antibodies were utilized.

Techniques: Staining, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Quantitative RT-PCR, Standard Deviation

Journal: bioRxiv

Article Title: Chondroitin and dermatan sulfate exposure induces a wound healing state in fibroblasts through Cux1-mediated SerpinB2 transcriptional repression

doi: 10.1101/2023.08.01.551410

Figure Lengend Snippet: (A) Enrichment of transcription factor binding sites in the promoters of the genes in CS/DS/pML vs pDF. (B) mRNA expression levels in DF vs ML with and without surfen treatment in hCUX1. Mean relative to control cells and standard deviation as error bars were plotted, n=3. (C) Western blot for Cux1 and ß-actin in DF and ML with and without surfen treatment. (D) Immunofluorescence images for Dapi and Cux1 in DF and ML with and without surfen treatment. (E) Quantification of positive cells (%) from images in D. (F) Quantification of Cathepsin L activity in DF with and without CS/DS treatment and ML with and without surfen treatment. (G) Procathepsin L docking simulation against CS. Green and blue regions in procathepsin L represent processed and mature regions, respectively. P-values were obtained using two-tailed t-test (**** represents p<0,0001, *** represents p<0,001, * represents p<0,05 and ns means no significative differences).

Article Snippet: The primary antibodies anti-Cux1 (Abnova, #H00001523-M01) and anti-Actin (SIGMA-ALDRICH, #A2066) were diluted in blocking solution (1:500).

Techniques: Binding Assay, Expressing, Control, Standard Deviation, Western Blot, Immunofluorescence, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Chondroitin and dermatan sulfate exposure induces a wound healing state in fibroblasts through Cux1-mediated SerpinB2 transcriptional repression

doi: 10.1101/2023.08.01.551410

Figure Lengend Snippet: (A) Dot plot representing chIP peaks (read depth) for Cux1 in ML cells (x-axis) compared to ML cells treated with surfen (B) Distribution of Cux1 peaks in ML cells in different types of genomic regions. (C) Motif discovery in Cux1 regions from chIP-seq, including P-values from this detection (D) Position of Cux1 peaks towards TSS (from 50kb upstream to 10kb downstream). In colors, we include all peaks (blue) or peaks with associated Cux1 motif (red) or Smad2 motif (green). (E) Average fold change expression in TSA-treated ML cells compared to untreated cells in genes with Cux1 peaks present in regions near their TSS (from -60kb to +10kb). (F) Gene ontology enrichment of genes with Cux1 peaks near their TSS and significative upregulated response after TSA treatment (G) Quantification of Acetyl H4 levels in ML cells with and without surfen treatment from chIP in hFGFR4 and hSERPINB2. (H) mRNA expression levels in ML cells with different Histone Deacetylase (HDAC) inhibitors treatments; hFGFR4 (left) and hSERPINB2 (right). Mean expression is relative to untreated cells and standard deviation as error bars were plotted, n=3. (C) reChIP with either Cux1 and GADPH or Cux1 and Smad2/3 antibodies in the FGFR4 region, using input and igG as controls. P-values were obtained using two-tailed t-test (*** represents p<0,001, ** represents p<0,01, * represents p<0,05 and ns means no significative differences).

Article Snippet: The primary antibodies anti-Cux1 (Abnova, #H00001523-M01) and anti-Actin (SIGMA-ALDRICH, #A2066) were diluted in blocking solution (1:500).

Techniques: ChIP-sequencing, Expressing, Histone Deacetylase Assay, Standard Deviation, Two Tailed Test