cited2  (R&D Systems)

Journal: Cells

Article Title: Acute Depletion of Cited2 in Embryonic Stem Cells Disrupts Gene Networks Controlling Self-Renewal, Homeostasis, and Early Cell Fate Commitment

doi: 10.3390/cells15050450

Figure Lengend Snippet: Cited2 depletion alters gene expression and modulates the activin/nodal signaling pathway. ( A ) 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), 4HT for 48 h (Undiff. D0/4HT), and differentiated for 4 days upon treatment with ethanol (D4/Ethanol) or 4HT (D4/4HT) for the first 48 h. Each circle represents an individual sample. PC1 shows the main variability among the transcriptome differences and PC2 the second largest variability. ( B ) Volcano plot of differential gene expression in control C2 fl/fl [Cre] ESC treated with ethanol (vehicle) or 4HT for 48 h. Only the genes with a significant differential expression (adjusted p value < 0.05) and a positive or negative log2 fold-change >1 with a log2 were plotted. Each point represents the average value of one transcript in three biological independent experiments. Names of the top ten downregulated and upregulated genes are indicated. ( C ) Activity of the luciferase reporter (ARE)3-lux in C2 fl/fl [Cre]ESC treated with ethanol (Undiff. D0/Ethanol), 4HT for 48 h (Undiff. D0/4HT). Gene expression in Undiff. D0/Ethanol ESC was set to 1. Results are presented as mean ± SEM from three independent biological replicates, each measured in technical duplicate. ( D ) Luciferase activity of the (ARE)3-lux reporter in E14TG2A mouse ESC co-transfected with either pPyCAGIP-flagCITED2 (orange bars) or pPyCAGIP control vector (blue bars), with or without pCMV-flag-Smad2 or pCMV-flag-FoxH1. Cells were either left unstimulated or stimulated 24 h post-transfection with 30 ng/mL Activin A for 24 h. Reporter activity in cells transfected with (ARE)3-lux and control vectors, without Activin A stimulation, was set to 1. Data represent the mean ± SEM from three independent biological experiments, each measured in technical duplicate.

Article Snippet: Cited2 floxed mice [ ] were obtained from Dr. Sally Dunwoodie and rederived by Jackson Laboratory (Bar Harbor, ME, USA) using JR 664 C57BL/6 oocytes.

Techniques: Gene Expression, Control, Quantitative Proteomics, Activity Assay, Luciferase, Transfection, Plasmid Preparation

Journal: Cells

Article Title: Acute Depletion of Cited2 in Embryonic Stem Cells Disrupts Gene Networks Controlling Self-Renewal, Homeostasis, and Early Cell Fate Commitment

doi: 10.3390/cells15050450

Figure Lengend Snippet: Identification of biological processes affected in Cited2 -depleted cells. ( A ) Relative expression of the indicated genes determined by qRT-PCR in C2 fl/fl [Cre]ESC treated either with treated ethanol or 4HT for 48 h (Undiff. D0) and in cells differentiated for 4 days upon treatment with ethanol or 4HT for the first 48 h (Diff. D4). Gene expression in ethanol/vehicle conditions was set to 1. Bars represent mean ± SEM of three independent biological triplicates, each measured in technical duplicate. NS means “not significant”. ( B ) Relative expression of Bmi1 , Ennp2 , Nav2 and Zic5 genes by qRT-PCR Cited2 conditional-null (cKO) in neural progenitor-stage of embryos at E15.5. as previously described . Bars represent mean ± SEM of at least three independent biological triplicates, each measured in technical duplicate. NS means “not significant”. ( C ) Top gene ontology biological process terms for the genes downregulated (left) and upregulated (right) by Cited2 depletion at D0 of differentiation determined using Enrichr [ , ].

Article Snippet: Cited2 floxed mice [ ] were obtained from Dr. Sally Dunwoodie and rederived by Jackson Laboratory (Bar Harbor, ME, USA) using JR 664 C57BL/6 oocytes.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: Cells

Article Title: Acute Depletion of Cited2 in Embryonic Stem Cells Disrupts Gene Networks Controlling Self-Renewal, Homeostasis, and Early Cell Fate Commitment

doi: 10.3390/cells15050450

Figure Lengend Snippet: Cited2 and p300/CBP exhibit limited functional overlap in mouse ESC. ( A ) pNanog-L activity in E14TG2A ESC co-transfected with pPyCAGIP-flagCITED2 (black bar) or pPyCAGIP (white bar), with or without pcDNA3-p300 (blue bars) or pcDNA3-CBP (orange bars). Gene expression in cells transfected with pNanog-L and control vector was set to 1. Results are presented as the mean ± SEM of three biological experiments, each measured in technical duplicate. ( B ) Cited2 -depletion in ESC modestly increased global histone acetyltransferase activity. C2 fl/fl [Cre] ESC maintained in undifferentiating conditions, treated with ethanol (Undiff. D0/Ethanol) or 4HT (Undiff. D0/4HT) for 48 h. Cell extracts were subjected to immunoprecipitation using either an antibody specific to p300 (IP α-p300) or an anti-Flag antibody as a control (Control IP). Immunoprecipitated proteins were used to assess the histone acetyltransferase (HAT) activity, which were normalized to the total protein content. 40% of the total whole lysate used for IP (Input) was used for HAT assay. Results are shown as mean ± SEM from at least two biological replicates, each measured in technical duplicate.

Article Snippet: Cited2 floxed mice [ ] were obtained from Dr. Sally Dunwoodie and rederived by Jackson Laboratory (Bar Harbor, ME, USA) using JR 664 C57BL/6 oocytes.

Techniques: Functional Assay, Activity Assay, Transfection, Gene Expression, Control, Plasmid Preparation, Immunoprecipitation, HAT Assay

Journal: Cells

Article Title: Acute Depletion of Cited2 in Embryonic Stem Cells Disrupts Gene Networks Controlling Self-Renewal, Homeostasis, and Early Cell Fate Commitment

doi: 10.3390/cells15050450

Figure Lengend Snippet: Cited2 depletion in ESC increases the risk for DNA damage. ( A ) Relative expression of p53-target and other upregulated genes and DNA repair related genes determined by qRT-PCR in C2 fl/fl [Cre]ESC treated either with treated ethanol or 4HT for 48 h. Gene expression in ethanol/vehicle conditions was set to 1. Means were compared with the control condition using a t -test considering the p value threshold of 0.05 for significance. Bars represent mean ± SEM of three independent biological triplicates, each measured in technical duplicate. NS means “not significant”. ( B ) Detection of γH2AX nuclei in C2 fl/fl [cre] cells cultured in undifferentiating conditions treated with 1 µM 4HT (4HT) for 16 h, 30 min with 100 µM hydrogen peroxide (H 2 O 2 ) or ethanol as a control, using an anti-γH2AX antibody (green). Nuclei were stained with DAPI (blue). Nuclei and the γH2AX speckles were determined (Segment.) using the segmentation pipeline in Cell Profiler. Scale bars represent 10 µm. ( C ) Images acquired as described in B, were used to count the number of γH2AX speckles per nucleus. ( D ) The mean of the intensity of the γH2AX speckles per nucleus was also determined. Means were compared with the control condition using a t -test considering the p value threshold of 0.05 for significance. The results are from three independent biological experiments, and 321 to 411 nuclei were analyzed per condition. ( E ) γH2AX protein levels determined by western blotting in protein extracts from cells treated as in B. Bottom panel: Total protein transferred on the PVDF membrane stained with No-Stain whole protein labelling (Thermo Fisher, A44717) used to control for loading. ( F ) Proliferation of C2 fl/fl [Cre]ESC treated either with treated ethanol and DMSO vehicle (Ethanol/DMSO), 4HT and DMSO (4HT/DMSO) or 4HT plus increasing concentrations of pifithrin-α (4HT/pifithrin-α). Left panel: representative images of the colonies at 48 h after treatment. Scales bars, 250 µm. Right panel: Cell counts at 48 h (mean ± SEM; n = 9 wells per group, from three independent biological triplicates, each measured in technical triplicate). Data were analyzed by one-way ANOVA followed by Dunnett’s test versus 4HT/DMSO (F (4,40) ) = 3.194, p = 0.0229). NS means “not significant”.

Article Snippet: Cited2 floxed mice [ ] were obtained from Dr. Sally Dunwoodie and rederived by Jackson Laboratory (Bar Harbor, ME, USA) using JR 664 C57BL/6 oocytes.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Control, Cell Culture, Staining, Western Blot, Membrane

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 3 | Overexpression of CITED2 at the animal level is able to alleviate PH. (A) The results of gene enrichment analysis in normoxia and hypoxia showed that CITED2 was downregulated in hypoxia, showing a negative correlation. (B–D) Expression of CITED2 mRNA and protein in the pulmonary arteries of model mice. (E) Schematic diagram of lentivirus model mouse construction. (F) The decreased cardiopulmonary function of model mice was effectively reversed by overexpression of CITED2. (G) Right ventricular systolic blood pressure and right ventricular specific gravity were improved in the CITED2 overexpression group. (H, I) H&E and Masson staining of tissue sections of mouse pulmonary arterioles. (K) The ex- pression of PCNA was downregulated in the pulmonary arteries of mice in the lentivirus-treated group. (J) Tissue immunofluorescence experiments showed that the expression of Ki67 in small pulmonary vessels decreased significantly after CITED2 overexpression. A: artery. (L, M) The expression of cycle-related proteins in the pulmonary arteries of mice in the lentivirus-treated group was downregulated. LV: lentivirus. (Bar = mean ± S.E.M, **p < 0.01; ***p < 0.001).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Expressing, Staining, Immunofluorescence

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 4 | Overexpression of CITED2 can affect the proliferation of smooth muscle cells and hinder cell cycle progression. (A) Western blot re- sults of efficiency detection after overexpression of CITED2. (B) The results of the CCK8 experiment indicated that overexpression of CITED2 could affect the growth and viability of mPASMCs. (C) Overexpression of CITED2 affected the expression of PCNA in cells. (D) After overexpression of CITED2, the proportion of cells in the G2/M and S phase was reduced, which affected cell division. (E, F) The results of the EdU cell fluorescence assay and Ki67 cell fluorescence assay indicated that overexpression of CITED2 could reduce cell proliferation. Red and green represent remark- ably proliferating cells. (G, H) Overexpression of CITED2 at the cellular level can affect the expression levels of multiple proteins in the cell cycle. (Bar = mean ± S.E.M, **p < 0.01; ***p < 0.001).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Western Blot, Expressing, Fluorescence

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 6 | FOXJ3 cooperates with SEs to regulate the transcription of CITED2. (A) Schematic representation of overexpression plasmid con- struction and transfection. (B–D) In the dual-luciferase experiment, plasmids containing three SEs motifs were co-transfected with FOXJ3 plasmids into mPASMCs. When both were present, the luciferase activity was the greatest. (Bar = mean ± S.E.M, *p < 0.05).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Plasmid Preparation, Transfection, Luciferase, Activity Assay

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 9 | Differential effects of CITED2 on PASMC proliferation under normoxia and hypoxia. Normally, the H3K27ac modification of the SEs and promoter regions of CITED2 is significant. Downstream proteins influenced by CITED2, such as cyclin proteins, CDKs and PCNA, decreased. SMC proliferation is also diminished. However, the phenomenon is reversed under hypoxia.

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Modification