cited2 floxed mice  (Jackson Laboratory)

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