Journal: Cell Death & Disease

Article Title: Exogenous WNT5A and WNT11 proteins rescue CITED2 dysfunction in mouse embryonic stem cells and zebrafish morphants

doi: 10.1038/s41419-019-1816-6

Figure Lengend Snippet: a Timeline depicting the protocol (embryoid bodies (EB) formation) used for differentiation C2 fl/fl [Cre]ESC from D0 to D4. The time of ethanol or 4HT treatment is indicated. Undifferentiated control ESC (Undiff.) were treated with ethanol for 2 days. b Principal Component Analysis (PCA) of the entire normalized array datasets. After normalization of the entire transcriptome dataset obtained from undifferentiated C2 fl/fl [Cre]ESC treated with ethanol (Undiff. D0/Ethanol) and differentiated for 4 days upon treatment with ethanol (D4/Ethanol) or 4HT (D4/4HT) for the first 48 h. Each sphere represents and individual sample. PC1 shows the main variability among the transcriptome differences and PC2 shows the second largest variability. c Top15 gene ontology biological process terms for the genes down-regulated by Cited2 depletion at D4 of differentiation determined using Enrichr. d Top10 KEGG pathway terms for the genes downregulated by Cited2 depletion at D4 of differentiation determined using Enrichr. e Expression of the epiblastic ( Fgf5 ) and mesoderm ( Brachyury, Mixl1, Mesp1, and Eomes ) markers from D1 to D6 of differentiation in cells generated from C2 fl/fl [Cre] ESC treated with ethanol or 4HT as described in a . Results are presented as the mean ± SEM of three independent biological experiments

Article Snippet: CITED2 protein was detected in zebrafish extracts using an anti-CITED2 rat monoclonal IgG 2A antibody (MAB5005, R&D System) used at 1:500 dilution as recommended by the manufacturer.

Techniques: Control, Expressing, Generated

Journal: Cell Death & Disease

Article Title: Exogenous WNT5A and WNT11 proteins rescue CITED2 dysfunction in mouse embryonic stem cells and zebrafish morphants

doi: 10.1038/s41419-019-1816-6

Figure Lengend Snippet: a Timeline depicting the protocol used for differentiation C2 fl/fl [Cre]ESC from D0 onward. The time of ethanol or 4HT treatment, as well as the supplementation with the conditioned media, and the days of beating activity assessment are indicated. b Percentage of colonies with contractile foci (top panel) counted at 8, 9 and 10 days after the initiation of differentiation in cell cultures derived from C2 fl/fl [Cre] ESC treated with ethanol or 4HT at D0 of differentiation, and simultaneously supplemented with conditioned medium either from control cells (Ethanol/CM-Ctl and 4HT/CM-Ctl, respectively) or from cells overexpressing CITED2 (Ethanol/CM-CITED2 and 4HT/CM-CITED2, respectively). The number of beating foci per beating colony is also indicated (bottom panel). c Relative expression of Cited2 , Brachyury , Mesp1 , Nkx2.5 , and Isl1 determined by qPCR at D4 of differentiation in cultures derived from C2fl/fl[Cre] ESC treated with 4HT either in the presence of and CM-Ctl or CM-CITED2 as described in b . The expression of the indicated genes is presented as the fold of expression in cells treated with CM-CITED2 over cells treated with CM-Ctl. The black bars indicate variations without reaching statistical significance, and gray bars indicate genes with statistical significance by Student’s t-test. NS, not significant. d Relative expression of the indicated genes encoding secreted proteins involved in cardiogenesis, determined by qPCR in E14/T ESC transfected with a plasmid expressing flag-tagged CITED2 (flagCITED2) or the control empty plasmid (control cells). The results are presented as in c . e Expression of the indicated genes encoding secreted proteins involved in cardiogenesis, in cells treated as described in c . The results are presented as in c . Results are presented as the mean ± SEM of three independent biological experiments

Article Snippet: CITED2 protein was detected in zebrafish extracts using an anti-CITED2 rat monoclonal IgG 2A antibody (MAB5005, R&D System) used at 1:500 dilution as recommended by the manufacturer.

Techniques: Activity Assay, Derivative Assay, Control, Expressing, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Exogenous WNT5A and WNT11 proteins rescue CITED2 dysfunction in mouse embryonic stem cells and zebrafish morphants

doi: 10.1038/s41419-019-1816-6

Figure Lengend Snippet: a Conditioned media obtained from E14/T ESC transfected with the flagCITED2 expressing vector (CM-CITED2) or the control plasmid (CM-Ctl) immunoprecipitated (IP) with anti-WNT5A and anti-WNT11 antibodies. Ponceau S stained blots on 10% of the input material used for immunoprecipitation show loading controls. b Time course of Wnt5a and Wnt11 expression determined by qPCR in samples prepared as described in Fig. . c Percentage of colonies with contractile foci counted at D10 of differentiation in cell cultures derived from C2 fl/fl [Cre] ESC treated with ethanol or 4HT at D0 of differentiation, or with 4HT in differentiation medium supplemented with conditioned medium either from control cells (4HT/CM-Ctl) or from cells overexpressing CITED2 (4HT/CM-CITED2), or 4HT/CM-CITED2 medium depleted from WNT5A, WNT11 depletion by immunoprecipitation as described in a , or no depletion using PBS 1× as vehicle (NIL). d Percentage of colonies with contractile foci counted at D8 of differentiation in cell cultures derived from C2 fl/fl [Cre] ESC treated with ethanol or 4HT at the onset of differentiation (vehicle), in the presence of 100 ng/ml of recombinant WNT5A (rWnt5a) or WNT11 (rWnt11) proteins or simultaneously with rWnt5a and rWnt11 (50 ng/ml of each). e Percentage of colonies with beating foci derived from C2 Δ/Δ [LA11] ESC at D10 of differentiation, in presence of rWnt5a or/and rWnt11, or PBS 1x as described in d . f Relative expression of Cited2 , Brachyury , Mesp1 , Isl1 , Nkx2.5 , and Tbx5 determined by qPCR at D4 of differentiation in cultures derived from C2 fl/fl [Cre] ESC treated as indicated in d . Gene expression in ethanol/vehicle conditions was set to 1. Bars represent mean ± SEM of three independent experiments

Article Snippet: CITED2 protein was detected in zebrafish extracts using an anti-CITED2 rat monoclonal IgG 2A antibody (MAB5005, R&D System) used at 1:500 dilution as recommended by the manufacturer.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Staining, Derivative Assay, Recombinant, Gene Expression

Journal: Cell Death & Disease

Article Title: Exogenous WNT5A and WNT11 proteins rescue CITED2 dysfunction in mouse embryonic stem cells and zebrafish morphants

doi: 10.1038/s41419-019-1816-6

Figure Lengend Snippet: a Schematic representation of the experimental steps and analysis performed with zebrafish embryos. The timings of development are indicated in hours post fertilization (hpf). b Percentage of embryos which are normal or dead at 24 hpf, after injection of 5 ng (0.7 pmol) of control morpholino (Control MO), anti- cited2 morpholinos targeting either the transcriptional start site (AUG MO; 5 ng) or the splicing site in the exon 1 (SPLICING MO; 5 ng), simultaneously with AUG MO and SPLICING MO (AUG+SPLICING MO, 2.5 ng of each morpholino), as well as non-injected embryos (Non-injected). Statistical significance was determined against control embryos using Student’s t test. c Brightfield images of live embryos showing the representative morphological features of zebrafish embryos considered as normal (top panel) or delayed in development (bottom panel) at 20 hpf. d Percentage of embryos which are normal or delayed in the developmental process at 20 hpf, after injection of morpholinos as described in b , in the presence of 400 pg of recombinant CITED2 protein (8R-CITED2), rWnt5a and rWnt11 alone (5 pg) or in combination (rWnt5a/11) in a final amount of 5 pg (2.5 pg each) or 10 pg (5 pg each), or no treatment (NIL). In panels, b and d n represents the number of embryos analyzed in each condition in at least 3 independent experiments

Article Snippet: CITED2 protein was detected in zebrafish extracts using an anti-CITED2 rat monoclonal IgG 2A antibody (MAB5005, R&D System) used at 1:500 dilution as recommended by the manufacturer.

Techniques: Injection, Control, Recombinant

Journal: Cell Death & Disease

Article Title: Exogenous WNT5A and WNT11 proteins rescue CITED2 dysfunction in mouse embryonic stem cells and zebrafish morphants

doi: 10.1038/s41419-019-1816-6

Figure Lengend Snippet: a Heart rate of embryos treated at presented in Fig. measured at 48 hpf at 28 °C. Each dot represents the value obtain for a single embryo. The mean and standard deviations for each condition are also presented. Statistical significance was determined against control embryos using Student’s t test. b Brightfield images of live embryos showing a representative morphology at 48 hpf of control embryos and embryos affected with cardiac anomalies, such as pericardial effusion, linear heart tube, enlarged atrium and/or ventricle. c Percentage of embryos which are normal, dead or presenting cardiac anomalies at 72 hpf, after injection of morpholinos as described in Fig. , with the exception that 500 pg of CITED2 recombinant protein were injected. The death rate presented is the cumulative death observed in each condition from 6 to 72 hpf. n represents the number of embryos analyzed in each condition in at least 3 independent experiments

Article Snippet: CITED2 protein was detected in zebrafish extracts using an anti-CITED2 rat monoclonal IgG 2A antibody (MAB5005, R&D System) used at 1:500 dilution as recommended by the manufacturer.

Techniques: Control, Injection, Recombinant

Journal: Oncology Letters

Article Title: CITED2 attenuates macrophage recruitment concordant with the downregulation of CCL20 in breast cancer cells

doi: 10.3892/ol.2017.7420

Figure Lengend Snippet: Effect of silencing CITED2 on macrophage infiltration in vivo and recruitment in vitro . (A) Representative images of immunohistochemical analysis revealed staining for the macrophage marker F4/80, as indicated by 3,3-diaminobenzamindine staining (brown) and visualized using a light microscope (magnification, ×200). The adjacent histograms represent the average number of macrophages/mm 2 . *P<0.05 compared with the scramble group. (B) Representative images revealed macrophage recruitment in response to conditioned media obtained from scramble and shCITED2-expressing breast cancer cells at 6 and 20 h, respectively, using an in vitro Transwell migration assay (magnification, ×400). The adjacent histograms represent quantification of the average number of macrophages recruited at each time point. *P<0.05; **P<0.01 vs. scramble CM. CITED2, Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2; sh, short hairpin; CM, conditioned media.

Article Snippet: ChIP was performed on nuclear cell lysates using anti-mouse CITED2 (cat. no. AF5005; dilution, 1:40; R&D Systems, Inc.) and anti-mouse IgG (cat. no. 515-005-003; dilution, 1:100; Jackson ImmunoResearch Europe, Ltd., Newmarket, UK) antibodies, according to the manufacturer's protocol of the SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: In Vivo, In Vitro, Immunohistochemical staining, Staining, Marker, Light Microscopy, Expressing, Transwell Migration Assay

Journal: Oncology Letters

Article Title: CITED2 attenuates macrophage recruitment concordant with the downregulation of CCL20 in breast cancer cells

doi: 10.3892/ol.2017.7420

Figure Lengend Snippet: Effect of CITED2 silencing on the expression of macrophage chemotactic factors. mRNA expression in (A) orthotopic xenograft tumors and (B) cells in culture, as determined by reverse transcription-quantitative polymerase chain reaction. (C) Protein expression in the two groups, as determined by ELISA. (D) Localization of CITED2 to the CCL20 promoter, as assessed by a chromatin immunoprecipitation assay using anti-CITED2 or IgG antibodies (control) in MDA-MB-231 and MDA-MB-468 cells. *P<0.05; **P<0.01; ***P<0.001 vs. scramble group (A-C) and vs. IgG (D). CITED2, Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2; sh, short hairpin; CCL, C-C motif chemokine ligand; Ig, immunoglobulin; IL, interleukin; CX3CL, C-X3-C motif chemokine ligand.

Article Snippet: ChIP was performed on nuclear cell lysates using anti-mouse CITED2 (cat. no. AF5005; dilution, 1:40; R&D Systems, Inc.) and anti-mouse IgG (cat. no. 515-005-003; dilution, 1:100; Jackson ImmunoResearch Europe, Ltd., Newmarket, UK) antibodies, according to the manufacturer's protocol of the SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Chromatin Immunoprecipitation, Control

Journal: Oncotarget

Article Title: Down-regulation of CITED2 attenuates breast tumor growth, vessel formation and TGF-β-induced expression of VEGFA

doi: 10.18632/oncotarget.14048

Figure Lengend Snippet: ( A – B ) Wild-type MDA-MB-231 and MDA-MB-468 cells were treated with or without 2.5 ng/ml TGF-β for 24 hours. (A) mRNA expression of VEGFA was determined by qRT-PCR. (B) Protein expression of VEGFA was determined by western blot analysis performed on equal amounts of protein obtained from serum-free conditioned media. Protein bands obtained from low (5 seconds) and high (20 seconds) exposure times are presented. ( C ) Localization of CITED2 to the VEGFA promoter as assessed by ChIP assay using anti-CITED2 or IgG antibodies (control) in wild-type MDA-MB-231 cells upon treatment with or without 2.5 ng/ml of TGF-β for 24 hours. ( D ) scramble and shCITED2-expressing MDA-MB-231 cells were treated with or without 2.5 ng/ml TGF-β for 24 hours and western blot analysis of VEGFA isoform expression was performed on equal amounts of protein obtained from serum-free conditioned media. (* P < 0.05, ** P < 0.01).

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed on nuclear cell lysates utilizing anti-sheep CITED2 (R&D Systems) and anti-sheep IgG (Jackson ImmunoResearch) antibodies and following the SimpleChIP Enyzmatic Chromatin IP Kit (Cell Signaling Technology) protocol based on manufacturer's instructions.

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