Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 1. Verification of the interaction between rat PPAR and CITED2 in vitro. CITED2 was radiolabeled with [35S]methionine and incubated with bacterially expressed PPAR-MBP fusion for 1 h at 4 °C in the presence of amylose resin. Resin was collected and washed three times with cold radioimmune precipitation assay buffer. Bound MBP was eluted from the resin using 10 mM maltose in radioimmune pre- cipitation assay buffer for 1 min at 4 °C. Eluate was resolved on a 12% Tris-glycine gel, dried, and subjected to autoradiography. The image is representative of two independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: In Vitro, Incubation, Autoradiography

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 2. CITED2 interacts with the D domain of rPPAR. COS-1 cells were transiently transfected with the GAL4-DBD fused to rPPAR and VP16 activation domain with or without fused CITED2. Cells were treated with 50 M Wy-14,643 (Wy), 200 M CLA mixture, or Me2SO (DMSO) for 6 h. Domains containing no ligand activation were treated with Me2SO. Luciferase activity was determined and corrected for transfection efficiency and extraction yield. Each domain and treatment group was corrected to their corresponding VP16 value (100%). *, p 0.01 comparing CITED2 bar to corresponding VP16 bar. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Activation Assay, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 3. CITED2 is a coregulator for PPAR. A, HepG2 cells were transiently transfected with expression vectors for rPPAR with CITED2 or empty vector control (pcDNA3) and a PPRE-driven lu- ciferase reporter. B, COS-1 cells were transiently transfected with rPPAR fused to the GAL4-DBD and CITED2 or empty vector control with GAL4-respon- sive reporter. Cells were treated with 50 M Wy-14,643 (Wy), 200 M CLA mixture, 100 M ciprofibrate, or Me2SO (DMSO) for 6 h. Luciferase activity was determined and corrected for transfection efficiency and extraction yield. All bars are corrected to untreated pcDNA3 level. *, p 0.05 compar- ing CITED2 bar to corresponding pcDNA3 bar. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 4. CITED2 acts as a dose-dependent coactivator of PPAR. A, COS-1 cells were transiently transfected with rPPAR fused to the GAL4-DBD and increasing amounts of CITED2. Cells were treated with 50 M Wy-14,643 for 6 h. Luciferase activity was deter- mined and corrected for transfection efficiency and extraction yield. Each treatment is corrected to luciferase activity with no CITED2 added (100%). Results show that CITED2 can act as a dose-dependent coactivator of PPAR in the presence or absence of ligand. *, p 0.05 comparing within a chemical treatment. Values in parentheses are relative luciferase units (rlu) for the accompanying data point. B, COS-1 cells were transiently transfected with rPPAR fused to the GAL4-DBD with or without CITED2. Cells were treated with 100 nM, 500 nM, 1 M, 5 M, 10 M, or 50 M Wy-14,643 for 6 h. Luciferase activity was determined and corrected for transfection efficiency and extraction yield. Each treatment is corrected to luciferase activity in the absence of Wy-14,643 (Me2SO (DMSO) at 100%). Graphs are representative of three independent experiments. CI, confidence interval.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 5. CITED2 acts as a coactivator for PPAR but not PPAR. A, HepG2 cells were transiently transfected with expression vectors for each PPAR subtype with CITED2 or empty vector control and a PPRE-driven luciferase reporter. B, COS-1 cells were transfected with GAL4-DBD-PPAR fusions for all three subtypes with and without exogenous CITED2. Transfected cells were treated for 6 h with 50 M Wy-14,643 (Wy), 50 M tetradecylthioacetic acid (TTA), 10 M prostag- landin J2 (PGJ2), or Me2SO (DMSO). Luciferase activity was deter- mined and corrected for transfection efficiency and extraction yield. Each treatment is corrected to the luciferase activity for Me2SO for each subtype. *, p 0.05 comparing CITED2 bar with corresponding pcDNA3 bar. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 6. CITED2 is ubiquitously expressed in mouse tissues. Total RNA from 10 different mouse tissues and the SV40-transformed mouse hepatocytes was examined for CITED2 mRNA using reverse transcription-PCR. Equivalent amounts of total RNA were tested using primers designed for the 5 -end of mouse CITED2 mRNA. The graph is representative of three independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Transformation Assay, Reverse Transcription

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 7. Ameliorated CITED2 expression leads to decreased PPAR activity. Undifferentiated 3T3-L1 preadipocytes were tran- siently transfected with an RNAi for CITED2 or vehicle control (pSu- per), pM-PPAR, and pFR-luciferase reporter and treated with 50 M Wy-14,643 (Wy), 100 M CLA mixture, 100 M ciprofibrate, or Me2SO (DMSO). Luciferase activity was measured and corrected for transfec- tion efficiency and extraction yield. Luciferase values were standard- ized to uninhibited (CITED2/) untreated (Me2SO) cells (100%). Graphs are representative of two independent experiments. *, p 0.05 comparing CITED2- to pSuper-transfected cells.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Expressing, Activity Assay, Transfection, Control, Luciferase, Extraction

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 8. CITED2 and CITED2 sta- bly expressing hepatocytes. A, CITED2 inhibition was achieved using double- stranded RNAi molecules (pSUPER and CITED2). The inhibition was verified using Western blot. Lanes labeled pcDNA3 and CITED2 are MuSH wild type cells stably expressing exogenous CITED2. B, MuSH wild type cells that stably overexpress CITED2 or inhibited CITED2 were assayed for growth in re- sponse to Wy-14,643 (Wy). Cells were plated and treated with 50 M Wy-14,643 for 72 h and assayed for relative cell num- ber. Values were corrected for Me2SO (DMSO) control (100%) for the corre- sponding control cell line. The stable cell lines are pooled populations of trans- fected cells. Data represent two independ- ent experiments. neo, RNAi empty vector control. *, p 0.05 comparing untreated to treated cells within the same cell type; , p 0.05 comparing cell types within the same treatment group.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Expressing, Inhibition, Western Blot, Labeling, Stable Transfection, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 9. Regulation of gene expres- sion by peroxisome proliferators is affected by alterations in CITED2 ex- pression. A–E, MuSH wild type cells that were stably transfected with human CITED2 or CITED2 RNAi were treated with 50 M Wy-14,643, 100 M CLA, 100 M ciprofibrate (Cipro), or Me2SO (DMSO) for 6 h. Total RNA was isolated and used in real time reverse transcrip- tion-PCR for known PPAR-regulated genes: Angpl4 (A), HIF1 (B), FOXc2 (C), MKP-1 (D), and VEGF-D (E). The Me2SO level for each corresponding empty vector control was set to 100%. *, different than Me2SO-treated cells within the same cell line (p 0.05). F–J, data are identical to that presented in A–E with grouping based on treatment. *, different than con- trol stably transfected cells within the same treatment (p 0.05). The stable cell lines are pooled populations of trans- fected cells. Graphs are representative of two independent experiments.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Stable Transfection, Transfection, Isolation, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: Identification of the CREB-binding Protein/p300-interacting Protein CITED2 as a Peroxisome Proliferator-activated Receptor α Coregulator

doi: 10.1074/jbc.m401489200

Figure Lengend Snippet: FIG. 10. Analysis of altered gene expression in the CITED2 cells. Genes that were significantly regulated in gene expression microarrays (CITED2/pcDNA3, Table II) were examined using Pathway Assist (Version 2.01). The pathway was built by looking for common regulators of the genes shown in Table II, and the predominant cluster is depicted. The lines and arrows depict observations on regulation of gene expression (, increased expression; , decreased expression) from the literature. The effects of CITED2 overexpression on the amount of mRNA in the microarray experiments are shown (black, significantly increased; gray, significantly decreased; white, no significant effect observed). Following creation of this cluster, the proteins in the gradient filled ovals were included (PPAR, PPAR, Angptl4, and HIF1), and the connections were determined by Pathway Assist or by manually adding (PPAR and CITED2 interaction). EGF, epidermal growth factor; TGF, transforming growth factor; DCN, decorin; AQP5, aquaporin 5; TNF, tumor necrosis factor; IFG1R, insulin-like growth factor I receptor; IL2, interleukin 2; IGFBP2, insulin-like growth factor-binding protein 2; IFN, interferon; LTBP1, latent transforming growth factor--binding protein 1; EPS15, epidermal growth factor receptor pathway substrate 15; TGM2, transglutaminase 2; UCHL1, ubiquitin carboxyl-terminal hydrolase L1; FIGF, c-fos-induced growth factor; GHRL, growth hormone receptor, long form; IL13R, interleukin 13 receptor; GH1, growth hormone 1; ADCY8, adenylate cyclase 8; TSA, trichostatin A; MGP, matrix -carboxyglutamate protein.

Article Snippet: Immunoblotting was performed using a mouse anti-CITED2 antibody (Novus Biologicals, Littleton, CO) in TBS , 0.5% dry milk.

Techniques: Gene Expression, Expressing, Over Expression, Microarray, Binding Assay, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Schematic showing the rat placentation site. Invaded trophoblast cells are depicted in green. b . Relative expression of Cited2 transcripts in the ectoplacental cone ( EPC ), whole placenta ( P ), junctional zone ( JZ ), and labyrinth zone ( LZ ) of the rat placenta during gestation. Values depicted were normalized to EPC 9.5 samples. c . Relative expression of Cited2 transcript in postnatal day 1 ( PND1 ) rat neonatal tissues and gd 14.5 JZ tissue. d . Relative expression of Cited2 transcripts within the uterine-placental interface during gestation. e . In situ hybridization showing Cited2 transcript distribution (top left) and Cited2 and Prl7b1 (invasive trophoblast marker) transcript co-localization in rat gestation day ( gd ) 18.5 placentation site (bottom left). Higher magnification images of the area outlined by a yellow rectangle (bottom left) are shown to the right. Scale bar=500 μm (left panels), scale bar=100 μm (right panels). Uterine-placental interface ( UPI ), spiral artery ( SpA ). The histograms presented in panels b, c, and d represent means ± SEM, n=5-10, 3-6 pregnancies. One-way ANOVA, Tukey’s post hoc test, * p < 0.05, ** p < 0.01, **** p<0.0001.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Expressing, In Situ Hybridization, Marker

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Schematic of the Cited2 wild type and null alleles. A 1477 bp deletion was generated using CRISPR /Cas9 genome editing. The deletion included the entire coding sequence of the Cited2 gene. b . Western blot for CITED2 protein in rat junctional zone tissue samples from Cited2 +/- x Cited2 +/- breeding on gd 18.5. c . Fetal and placental weights from Cited2 +/- x Cited2 +/- breeding for the rat. Values represent mean ± SEM, n=17-35, unpaired t-test, **p<0.01, ***p<0.001, **** p<0.0001.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Generated, CRISPR, Sequencing, Western Blot

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Immunohistological analysis of vimentin in wild type ( WT, +/+ ) and null (-/-) placentas from Cited2 +/- x Cited2 +/- breeding on gd 18.5. Scale bar=1000 μm. UPI uterine-placental interface, JZ junctional zone, LZ labyrinth zone. b . JZ and LZ weights from Cited2 +/- x Cited2 +/- breeding on gd 14.5 and gd 18.5. c . Simplified schematic depicting the strategy for achieving trophoblast specific CITED2 knockdown in vivo. d . Relative expression of Cited2 transcripts in control ( CTRL ) and CITED2 shRNA-exposed gd 14.5 JZ. e . JZ, LZ and fetal weights from control and gd 14.5 trophoblast specific Cited2 knockdown. CTRL, control knockdown, KD, Cited2 knockdown. Shown are mean values ± SEM, n=12-20, unpaired t-test, **p<0.01, **** p<000.1.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Knockdown, In Vivo, Expressing, Control, shRNA

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Significantly upregulated and downregulated transcripts from RNA-seq analysis of rat 14.5 junctional zone tissue from wild type (+/+) and Cited2 null (-/-) rats, n=4, log 2 -fold change in either direction>1.5, p<0.05. b . Heatmap showing select transcripts from RNA-seq analysis of wild type (+/+) and Cited2 null (-/-) rat 14.5 junctional zone tissue. c . Significantly upregulated and downregulated transcripts from RNA-seq analysis of differentiated wild type (+/+) and Cited2 null (-/-) rat trophoblast stem ( TS ) cells, n=3, log 2 -fold change in either direction>1.5, p<0.05. d . Heatmap showing select transcripts from RNA-seq analysis of differentiated rat TS cells from RNA-seq analysis of wild type (+/+) and Cited2 null (-/-) differentiated TS cells. e . Gene Ontology ( GO ) enriched terms for DEGs from wild type (+/+) versus Cited2 null (-/-) rat 14.5 junctional zone RNA-seq analysis. f . GO enriched terms for DEGs from wild type (+/+) versus Cited2 null (-/-) rat TS cell RNA-seq analysis.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Representative images of wild type (+/+) and Cited2 null (-/-) rat gd 13.5, 15.5 and 18.5 placentation sites immunostained for cytokeratin (green). Scale bars=1000 μm. b . Relative expression of transcripts associated with invasive trophoblast ( Prl7b1 ) from wild type (+/+) and Cited2 null (-/-) gd 13.5 decidual tissue and uterine placental interface tissue at gd 15.5 and 18.5. Graphs depict mean values ± SEM, n=11-24, unpaired t-test, *p<0.05. c . In situ hybridization showing Ceacam9 (red) and Prl7b1 (invasive trophoblast marker, green) transcript localization in gd 15.5 wild type (+/+) and Cited2 null (-/-) rat placentation sites, scale bar=1000 μm. d . Relative expression of Ceacam9 transcripts in gd 15.5 uterine placental interface tissue. Shown are mean values ± SEM, n=12-14, unpaired t-test, **p<0.01. Uterine placental interface ( UPI ), decidua ( DEC ), junctional zone ( JZ ), labyrinth zone ( LZ ).

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Expressing, In Situ Hybridization, Marker

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: Single cell-RNA sequencing was performed on wild type (+/+) and Cited2 null (-/-) gd 18.5 uterine-placental interface tissue samples. a . UMAP plot showing cell clustering in wild type (+/+) and Cited2 null (-/-) gd 18.5 uterine-placental interface tissue. UD, unidentified cluster. b . Number of analyzed cells in the invasive trophoblast cell cluster. Graph represents mean values ± SEM, n=3, unpaired t-test, *p<0.05. c . Bar plot showing select DEGs in the invasive cell cluster (upregulated shown in red; downregulated shown in blue). d . Gene Ontology enriched terms for DEGs from the invasive trophoblast cell cluster.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . Schematic of the experimental timeline for hypoxia exposure. b . Representative images of wild type (+/+) and Cited2 null (-/-) rat gd 13.5 placentation sites exposed to ambient or hypoxic (10.5% oxygen) conditions. Sections were immunostained for cytokeratin (green), perforin (red), and DAPI (blue). Scale bar=500 μm. Uterine placental interface ( UPI ), Decidua ( DEC ), Junctional Zone ( JZ ), labyrinth zone ( LZ ), and Spiral artery ( SpA ). c . Depth of invasion was quantified, and fold changes calculated for hypoxic relative to ambient conditions, n=5-9. d . Resorption rate assessed on gd 18.5 for individual genotypes from Cited2 +/- females bred to Cited2 +/- males and exposed to ambient of hypoxic conditions, n=20-33. e . Schematic of the experimental timeline for polyinosinic:polycytidylic acid ( polyI:C ) treatment, f . Relative expression of Isg15, Mx2, Ifi27I2b , and Oasl2 in junctional zone tissue from control (saline treated; CTRL ) and polyI:C treated ( IC ) wild type (+/+) and Cited2 null (-/-) animals.; n=8-17. Shown are mean values ± SEM, one-way analysis of variance, Tukey’s post-hoc test. *p<0.05, **p <0.01, ***p <0.001. ****p <0.0001.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: Expressing, Control, Saline

Journal: bioRxiv

Article Title: CITED2 IS A CONSERVED REGULATOR OF DEEP HEMOCHORIAL PLACENTATION

doi: 10.1101/2022.06.15.496287

Figure Lengend Snippet: a . In situ hybridization showing CITED2 transcript localization in first trimester human placenta. C ITED2 transcripts (black) were co-localized with E-cadherin ( CDH1 , red; marker of cytotrophoblast progenitor cells) transcripts. High magnification image of an EVT cell column and villus (yellow box) are shown in the right panel. The scale bar in the magnified image is 100 μm. b . Schematic showing human trophoblast stem ( TS ) cell differentiation from stem/progenitor state to EVT cells. c . Relative expression of CITED2 and EVT cell signature transcripts ( MMP2 and HLA-G ) in human TS cells in the stem state and following eight days of EVT cell differentiation, n=5. d . Relative expression of CITED2 transcript levels in EVT cells expressing control ( CTRL ) shRNA or CITED2 shRNA, n=3. Graphs represent mean values ± SEM, unpaired t-test, *p<0.05, ***p <0.001. ****p <0.0001. e . Significantly upregulated and downregulated transcripts from RNA-seq analysis of CTRL shRNA versus CITED2 shRNA samples, n=3, and log 2 -fold change ( FC ) in either direction>1.5, p<0.05. f . Heatmap showing select transcripts from RNA-seq analysis of CTRL shRNA versus CITED2 shRNA treated EVT cells. g . Gene Ontology enriched terms for DEGs from CTRL shRNA versus CITED2 shRNA treated EVT cells.

Article Snippet: Membranes were subsequently blocked with 5% BSA in Tris buffered saline with 0.1% Tween 20 ( TBST ) and probed using antibodies specific for CITED2 (1:500, AF5005, R&D Systems) and GAPDH (1:5,000, AM4300, Invitrogen).

Techniques: In Situ Hybridization, Marker, Cell Differentiation, Expressing, Control, shRNA, RNA Sequencing

Journal: Journal of Endocrinology

Article Title: CITED2 is expressed in human adrenocortical cells and regulated by basic fibroblast growth factor

doi: 10.1677/joe-06-0083

Figure Lengend Snippet: Figure 1 Representative sections of human embryonic adrenal glands at 8 weeks of gestation. Panel A: negative control without primary antibody (adZadrenal, 100!). Panel B: immunostaining with antibodies to CITED2 revealed that CITED2 is located in the definitive zone (arrows, 100!). Panel C: high magnification of a negative control demonstrates nuclear morphology in the definitive zone (1000!). Panel D: high magnification of a representative section of the definitive zone. Predominantly, small nuclei were stained with an antibody to CITED2 (arrows, 1000!). Panel E: immunohistochemistry with an antibody to 17-a-hydroxylase identified the fetal zone (zF) while the definitive zone (zD) did not stain (100!). Panel F: chromogranin A immunoreactive cells in the outside margin of the adrenal gland (arrows, 100!).

Article Snippet: Then cells were incubated with monoclonal anti-CITED2 antibodies (Novus Biologicals) in a dilution of 1:100 for 1 h at room temperature.

Techniques: Negative Control, Immunostaining, Staining, Immunohistochemistry

Journal: Journal of Endocrinology

Article Title: CITED2 is expressed in human adrenocortical cells and regulated by basic fibroblast growth factor

doi: 10.1677/joe-06-0083

Figure Lengend Snippet: Figure 2 Representative sections of stained normal adult adrenal glands and adrenocortical carcinomas. Panel A: negative control for normal adult adrenal gland without primary antibody (40!). Panel B: normal adult adrenal gland stained for CITED2. Arrows indicate immunoreactivity in the zonae glomerulosa and reticularis at the corticomedullary junction (40!). Panel C: adrenocortical carci- noma stained for CITED2 showed strong nuclear expression of CITED2 (200!). Panel D: gel of RT-PCR analysis of mRNA from normal adult human adrenal cortex (lane 2) and from NCI-H295R cells (lane 3) the 69 bp fragments corresponded to the predicted amplification product of CITED2. As control for PCR we used RNA skipping the reverse transcription reaction (lane 1).

Article Snippet: Then cells were incubated with monoclonal anti-CITED2 antibodies (Novus Biologicals) in a dilution of 1:100 for 1 h at room temperature.

Techniques: Staining, Negative Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription

Journal: Journal of Endocrinology

Article Title: CITED2 is expressed in human adrenocortical cells and regulated by basic fibroblast growth factor

doi: 10.1677/joe-06-0083

Figure Lengend Snippet: Figure 3 Regulation of CITED2-promotor activity and mRNA-level in NCI-H295R cells. Panel A: relative activity of the CITED2- promotor in NCI-H295R cells after exposure to bFGF in different concentrations either with or without PD98059 for 48 h. Panel B: relative levels of CITED2-mRNA in NCI-H295R cells after exposure to bFGF in different concentrations for 8 h. Panel C: effects of ACTH and forskolin (Forsk) on the CITED2-promotor activity after 48 h of stimulation (left panel) or on the CITED2-mRNA levels (right panel) after 8 h of stimulation in NCI-H295R cells. In all panels P-values indicate significant differences from the control (CNT).

Article Snippet: Then cells were incubated with monoclonal anti-CITED2 antibodies (Novus Biologicals) in a dilution of 1:100 for 1 h at room temperature.

Techniques: Activity Assay, Control

Journal: Journal of Endocrinology

Article Title: CITED2 is expressed in human adrenocortical cells and regulated by basic fibroblast growth factor

doi: 10.1677/joe-06-0083

Figure Lengend Snippet: Figure 4 Immunofluorescence technique demonstrates CITED2 protein expression in NCI-H295R cells. Panel A: unstimulated control. Blue fluorescence dye DAPI marks nuclei. CITED2 protein is represented by a green fluorescence signal in the nuclei. Panel B: exposure of NCI-H295R cells for 24 h to bFGF (10 ng/ml) increased CITED2 nuclear expression in comparison with the control. Panel C: exposure of NCI-H295R cells to bFGF (10 ng/ml) in combination with PD98059 (20 mM) for 24 h attenuated CITED2 nuclear fluorescence signal in comparison with the stimulation with bFGF (10 ng/ml) alone. Panel D: exposure of NCI-H295R cells to ACTH (100 nM) for 24 h did not exert an effect on CITED2-protein expression in comparison with the control.

Article Snippet: Then cells were incubated with monoclonal anti-CITED2 antibodies (Novus Biologicals) in a dilution of 1:100 for 1 h at room temperature.

Techniques: Expressing, Control, Comparison

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: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: The expression levels of stem cell markers importin13, c-kit, CD146, and telomerase are decreased in endometrial polyps

doi: 10.12659/MSM.881901

Figure Lengend Snippet: The expression levels of IPO13 and caspase3 in the endometrial tissues were detected by immunohistochemistry (400×). ( A ) Control endometrium at the proliferation phase. ( B ) Control endometrium at the secretion phase. ( C ) EP at the proliferation phase. ( D ) EP at the secretion phase. ( E ) Control endometrium at the proliferation phase. ( F ) Control endometrium at the secretion phase. ( G ) EP at the proliferation phase. ( H ) EP at the secretion phase. * p<0.05 ( vs. control endometrium at the proliferation phase); ** p<0.05 ( vs. control endometrium at the secretion phase); error bars, SEM.

Article Snippet: Anti-IPO13 goat polyclonal antibody (1:100, NB100-1369, Novus Biologicals), anti-telomerase rabbit polyclonal antibody (1: 100, ZA-0239, Beijing Golden Bridge Biotechnology Co., Ltd.), anti-CD146 mouse monoclonal antibody (1:50, ZM-0299, Beijing Golden Bridge Biotechnology Co., Ltd.), anti-caspase3 rabbit polyclonal antibody (1:100, BS1518, Bioworld Technology, Inc.), anti-bax rabbit polyclonal antibody (1:100, BS1725, Bioworld Technology, Inc.) and anti-bcl-2 rabbit monoclonal antibody (1:100, bs-0522R, Bioworld Technology, Inc.) were incubated with the sections overnight at 4°C.

Techniques: Expressing, Immunohistochemistry, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: The expression levels of stem cell markers importin13, c-kit, CD146, and telomerase are decreased in endometrial polyps

doi: 10.12659/MSM.881901

Figure Lengend Snippet: The mRNA expression of IPO13, c-kit, bcl-2 and bax was detected by real-time PCR. (A) Control endometrium at the proliferation phase. (IB) Control endometrium at the secretion phase. (IC) EP at the proliferation phase. (ID) EP at the secretion phase. (IIA) Control endometrium at the proliferation phase. (IIB) Control endometrium at the secretion phase. (IIC) EP at the proliferation phase. (IID) EP at the secretion phase. (IIIA) Control endometrium at the proliferation phase. (IIIB) Control endometrium at the secretion phase. (IIIC) EP at the proliferation phase. (IIID) EP at the secretion phase. (IVA) Control endometrium at the proliferation phase. (IVB) Control endometrium at the secretion phase. (IVC) EP at the proliferation phase. (IVD) EP at the secretion phase. ** p<0.05 ( vs. control endometrium at the secretion phase); error bars, SEM.

Article Snippet: Anti-IPO13 goat polyclonal antibody (1:100, NB100-1369, Novus Biologicals), anti-telomerase rabbit polyclonal antibody (1: 100, ZA-0239, Beijing Golden Bridge Biotechnology Co., Ltd.), anti-CD146 mouse monoclonal antibody (1:50, ZM-0299, Beijing Golden Bridge Biotechnology Co., Ltd.), anti-caspase3 rabbit polyclonal antibody (1:100, BS1518, Bioworld Technology, Inc.), anti-bax rabbit polyclonal antibody (1:100, BS1725, Bioworld Technology, Inc.) and anti-bcl-2 rabbit monoclonal antibody (1:100, bs-0522R, Bioworld Technology, Inc.) were incubated with the sections overnight at 4°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: The expression levels of stem cell markers importin13, c-kit, CD146, and telomerase are decreased in endometrial polyps

doi: 10.12659/MSM.881901

Figure Lengend Snippet: The relative expression levels of IPO13 and bcl-2 proteins in normal endometrial and EP tissues.

Article Snippet: Anti-IPO13 goat polyclonal antibody (1:100, NB100-1369, Novus Biologicals), anti-telomerase rabbit polyclonal antibody (1: 100, ZA-0239, Beijing Golden Bridge Biotechnology Co., Ltd.), anti-CD146 mouse monoclonal antibody (1:50, ZM-0299, Beijing Golden Bridge Biotechnology Co., Ltd.), anti-caspase3 rabbit polyclonal antibody (1:100, BS1518, Bioworld Technology, Inc.), anti-bax rabbit polyclonal antibody (1:100, BS1725, Bioworld Technology, Inc.) and anti-bcl-2 rabbit monoclonal antibody (1:100, bs-0522R, Bioworld Technology, Inc.) were incubated with the sections overnight at 4°C.

Techniques: Expressing, Control

Journal: Arthritis and rheumatism

Article Title: Cited2 modulates hypoxia-inducible factor-dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc.

doi: 10.1002/art.24073

Figure Lengend Snippet: Figure 6. Regulation of vascular endothelial growth factor (VEGF) expression by HIF and cited2. A, Real-time reverse transcription– polymerase chain reaction analysis of the relative expression levels of VEGF in normoxic and hypoxic nucleus pulposus cells. B, Nucleus pulposus cells were cotransfected with VEGF promoter plasmid along with 100 ng of either constitutively active (CA) HIF-2 plasmid (pCA-HIF-2) or pCA-HIF-1 (401603) plasmid or with empty backbone vector pcDNA3 together with 100 ng pARNT (HIF-1) plasmid. C, Nucleus pulposus cells were cotransfected with VEGF promoter plasmid along with increasing doses (100–300 ng) of dominant-negative HIF-1 plasmid (DN-HIF-1). Triangles indicate addition of increasing dose of plasmid. D, Nucleus pulposus cells were cotransfected with VEGF luciferase reporter along with either cited2 expression plasmid or empty backbone plasmid pCMV6 under nor- moxic conditions. E, Nucleus pulposus cells were cotransfected with cited2 reporter construct along with either cited2 small interfering RNA (siRNA) (Si-Cited2) or control siRNA duplexes (Co) and cultured under normoxic or hypoxic conditions for 24 hours. F, Relative expression of VEGF mRNA after partial silencing of cited2 under hypoxic conditions. Values are the mean and SD from 3 independent experiments performed in triplicate. P 0.05. See Figure 2 for other definitions.

Article Snippet: Cited2 expression plasmid (#SC116345) and plasmid pCMV6 were purchased from Origene (Rockville, MD).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Plasmid Preparation, Dominant Negative Mutation, Luciferase, Construct, Small Interfering RNA, Control, Cell Culture

Journal: PLoS ONE

Article Title: (-)-Phenserine Attenuates Soman-Induced Neuropathology

doi: 10.1371/journal.pone.0099818

Figure Lengend Snippet: Information on primers and probes used in the study.

Article Snippet: Cited2 , Rat , Rn00586705_m1 , NM_053698.2 , 1–2 , 223 , 71 , Probe spans exons.

Techniques:

Journal: PLoS ONE

Article Title: (-)-Phenserine Attenuates Soman-Induced Neuropathology

doi: 10.1371/journal.pone.0099818

Figure Lengend Snippet: Administration of (−)-phenserine thirty minutes prior to soman differentially modulates gene expression in the piriform cortex three hours after soman.

Article Snippet: Cited2 , Rat , Rn00586705_m1 , NM_053698.2 , 1–2 , 223 , 71 , Probe spans exons.

Techniques: Gene Expression