Journal: Stem cell research

Article Title: Generation of an Alagille syndrome (ALGS) patient-derived induced pluripotent stem cell line (TRNDi032-A) carrying a heterozygous mutation (p.Cys682Leufs*7) in the JAG1 gene

doi: 10.1016/j.scr.2023.103231

Figure Lengend Snippet: Reagents details.

Article Snippet: Secondary Antibodies , anti-OCT3/4 Donkey Anti-Goat IgG (H + L) (Alexa Fluor 594) , 1:1000 , Jackson Immunoresearch, Cat#705-585-147 , RRID: AB_2340433.

Techniques: Immunocytochemistry, Flow Cytometry, Control, Mutagenesis

Journal:

Article Title: Orphan Nuclear Receptor GCNF Is Required for the Repression of Pluripotency Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation

doi: 10.1128/MCB.25.19.8507-8519.2005

Figure Lengend Snippet: Expression of Oct4 and GCNF and binding of GCNF to the Oct4 promoter in differentiated P19 and ES cells. (A) The protein expression levels of GCNF and Oct4 were analyzed by using the anti-GCNF and Oct4 antibodies. The amount of total protein was normalized by β-actin, which was used as a control. (B) The TRIF complex was detected in differentiated P19 and ES cells by EMSA analysis with an Oct4 DR0 probe. (C) The TRIF complex in differentiated P19 and ES cells can be supershifted by anti-GCNF antibodies. (D). Direct binding of GCNF to DR0 site located in the endogenous Oct4 gene proximal promoter in differentiated P19 and ES cells was demonstrated by ChIP assay. P19 and ES cells were differentiated by treatment with RA for the times indicated (hours).

Article Snippet: After fixation in 4% paraformaldehyde for 2 h and permeabilization with 0.2% Triton X-100 for 30 min and blocking with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA), embryoid bodies were incubated with rabbit anti-Nanog immunoglobulin G (Chemicon, Temecula, CA) and goat anti-Oct4 immunoglobulin G (Santa Cruz, CA) or normal rabbit and goat immunoglobulin G. After washing with phosphate-buffered saline, embryoid bodies were stained with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G and Texas Red-conjugated donkey anti-goat immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing, Binding Assay

Journal:

Article Title: Orphan Nuclear Receptor GCNF Is Required for the Repression of Pluripotency Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation

doi: 10.1128/MCB.25.19.8507-8519.2005

Figure Lengend Snippet: Isolation and characterization of GCNF−/− ES cells. (A) Genotyping of isolated ES cells from blastocyst outgrowths. Genomic DNA extracted from mouse tails was genotyped as controls (lanes 1 and 2). ES cells in lane 3, 4, and 5 were isolated from the same litter, corresponding to ES cell lines 2904d, 2904c, and 2412e, respectively. (B) Gender analysis of ES cell lines 2904d (lane 1), 2904c (lane 2), and 2412e (lane 3) and the PCR negative control (lane 4) was determined using the Y chromosome-specific primers and the myosin-specific primers as a control. (C) GCNF mRNA was analyzed by RT-PCR with DNA-binding domain-specific primers (10). Total RNAs were extracted from RA-differentiated cells and reverse-transcribed into cDNA (2904c, lane 1; 2412e, lane 2; and RT control, lane 3). (D) Western blot analysis of GCNF expression in GCNF−/− ES cells; 20 μg of total proteins extracted from RA-differentiated ES cells at different time points were analyzed. (E) EMSA analysis of the TRIF complex in the GCNF−/− ES cells using the Oct4 DR0 as a probe. Protein samples were the same as in panel D.

Article Snippet: After fixation in 4% paraformaldehyde for 2 h and permeabilization with 0.2% Triton X-100 for 30 min and blocking with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA), embryoid bodies were incubated with rabbit anti-Nanog immunoglobulin G (Chemicon, Temecula, CA) and goat anti-Oct4 immunoglobulin G (Santa Cruz, CA) or normal rabbit and goat immunoglobulin G. After washing with phosphate-buffered saline, embryoid bodies were stained with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G and Texas Red-conjugated donkey anti-goat immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Isolation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Western Blot, Expressing

Journal:

Article Title: Orphan Nuclear Receptor GCNF Is Required for the Repression of Pluripotency Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation

doi: 10.1128/MCB.25.19.8507-8519.2005

Figure Lengend Snippet: ES cell phenotype of GCNF−/− ES cells. (A) Morphology of GCNF−/− ES cells cultured under the control of LIF as monolayer (panels a to f) and embryoid bodies (EB, panels g to h). (B) Immunofluorescent staining of embryoid bodies. Embryoid bodies were incubated with rabbit anti-Nanog and goat anti-Oct4 antibodies and visualized by fluorescein isothiocyanate-labeled donkey anti-rabbit and Texas Red-labeled donkey anti-goat secondary antibodies. The images were taken under bright field (panels a to c), fluorescein isothiocyanate filter (panels d to f), and Texas Red filter (panels g to i) and merged images from fluorescein isothiocyanate and Texas Red filters (panels j to l) using a Zeiss laser scanning confocal microscope (LSM510). (C) RT-PCR analysis of pluripotent marker gene expression in GCNF−/− ES cells cultured as a monolayer for 2 days in the presence of LIF. Wild-type ES cells were cultured and analyzed as controls.

Article Snippet: After fixation in 4% paraformaldehyde for 2 h and permeabilization with 0.2% Triton X-100 for 30 min and blocking with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA), embryoid bodies were incubated with rabbit anti-Nanog immunoglobulin G (Chemicon, Temecula, CA) and goat anti-Oct4 immunoglobulin G (Santa Cruz, CA) or normal rabbit and goat immunoglobulin G. After washing with phosphate-buffered saline, embryoid bodies were stained with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G and Texas Red-conjugated donkey anti-goat immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Cell Culture, Staining, Incubation, Labeling, Microscopy, Reverse Transcription Polymerase Chain Reaction, Marker, Expressing

Journal:

Article Title: Orphan Nuclear Receptor GCNF Is Required for the Repression of Pluripotency Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation

doi: 10.1128/MCB.25.19.8507-8519.2005

Figure Lengend Snippet: Loss of repression of Oct4 gene expression in differentiated GCNF−/− ES cells. (A) Oct4 expression was detected by Northern blot analysis in RA-differentiated ES cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was detected to control for loading amounts and integrity of the RNAs. (B) Oct4 protein was detected by Western blot analysis in RA-differentiated ES cells and β-actin was used as a loading and integrity control. (C) Direct binding of GCNF to the DR0 element located in the proximal promoter of the endogenous Oct4 gene in RA-differentiated wild-type and GCNF−/− ES cells was analyzed by ChIP assay. Preimmunized serum immunoglobulin G was used as a negative control.

Article Snippet: After fixation in 4% paraformaldehyde for 2 h and permeabilization with 0.2% Triton X-100 for 30 min and blocking with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA), embryoid bodies were incubated with rabbit anti-Nanog immunoglobulin G (Chemicon, Temecula, CA) and goat anti-Oct4 immunoglobulin G (Santa Cruz, CA) or normal rabbit and goat immunoglobulin G. After washing with phosphate-buffered saline, embryoid bodies were stained with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G and Texas Red-conjugated donkey anti-goat immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing, Northern Blot, Western Blot, Binding Assay, Negative Control

Journal:

Article Title: Orphan Nuclear Receptor GCNF Is Required for the Repression of Pluripotency Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation

doi: 10.1128/MCB.25.19.8507-8519.2005

Figure Lengend Snippet: List of known GCNF response elements

Article Snippet: After fixation in 4% paraformaldehyde for 2 h and permeabilization with 0.2% Triton X-100 for 30 min and blocking with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA), embryoid bodies were incubated with rabbit anti-Nanog immunoglobulin G (Chemicon, Temecula, CA) and goat anti-Oct4 immunoglobulin G (Santa Cruz, CA) or normal rabbit and goat immunoglobulin G. After washing with phosphate-buffered saline, embryoid bodies were stained with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G and Texas Red-conjugated donkey anti-goat immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Sequencing, Binding Assay

Journal:

Article Title: Orphan Nuclear Receptor GCNF Is Required for the Repression of Pluripotency Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation

doi: 10.1128/MCB.25.19.8507-8519.2005

Figure Lengend Snippet: Direct repression of Nanog expression by GCNF binding to DR0 elements in the Nanog gene. (A) Localization of three DR0 elements in the promoter and 3′ untranslated region of the mouse Nanog gene and description of the Nanog luciferase reporter constructs. Arrows indicate the DR0 elements and their relative orientations. (B) Analysis of binding of GCNF to the Nanog DR0 sequence by EMSA. GCNF-overexpressing COS1 extracts (lanes 1 and 2) or RA-differentiated P19 cell extracts (lanes 3 and 4) or GCNF+/+ ES cell extracts (lanes 5 and 6) or GCNF−/− ES cell extracts (lanes 7 and 8) were incubated with the Nanog DR0 probe in the absence or presence of anti-GCNF antibodies. (C) GCNF dose-dependently repressed the Nanog reporter gene expression in transiently transfected CHO-K1 cells; 200 ng/well of three Nanog-Luc reporters, Nanog5P, Nanog3P, and Nanog5/3P, and Oct4-Luc as a positive control were cotransfected with different amounts of HA-GCNF expression vector (0, 20, 50, and 100 ng/well). (D) Direct binding of GCNF to the endogenous Nanog gene DR0 elements was demonstrated by ChIP assay with GCNF antibodies. GCNF+/+ and GCNF−/− ES cells were differentiated by treatment with RA for the times indicated (hours).

Article Snippet: After fixation in 4% paraformaldehyde for 2 h and permeabilization with 0.2% Triton X-100 for 30 min and blocking with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA), embryoid bodies were incubated with rabbit anti-Nanog immunoglobulin G (Chemicon, Temecula, CA) and goat anti-Oct4 immunoglobulin G (Santa Cruz, CA) or normal rabbit and goat immunoglobulin G. After washing with phosphate-buffered saline, embryoid bodies were stained with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G and Texas Red-conjugated donkey anti-goat immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing, Binding Assay, Luciferase, Construct, Sequencing, Incubation, Transfection, Positive Control, Plasmid Preparation

Journal:

Article Title: Orphan Nuclear Receptor GCNF Is Required for the Repression of Pluripotency Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation

doi: 10.1128/MCB.25.19.8507-8519.2005

Figure Lengend Snippet: Model for regulation of pluripotency gene expression by GCNF in ES cells and embryos. Expression of Oct4 (blue) and Nanog (green) maintains self-renewal of ES cells. Oct4 also activates expression of other pluripotency genes, such as Sox2 and FGF4. The target genes of Nanog are currently unknown (?). Upon RA treatment, induction of GCNF (red) and p53 (magenta) represses the expression of Oct4, Nanog, and other unknown genes facilitating the differentiation of ES cells. GCNF also represses the expression of Oct4 and Nanog in the somatic cells of developing embryos, such as neuronal cells, during gastrulation and thus indirectly restricts their expression to primordial germ cells. LRH-1 (aquamarine) activates the expression of Oct4 in undifferentiated ES cells and the epiblast of embryos.

Article Snippet: After fixation in 4% paraformaldehyde for 2 h and permeabilization with 0.2% Triton X-100 for 30 min and blocking with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA), embryoid bodies were incubated with rabbit anti-Nanog immunoglobulin G (Chemicon, Temecula, CA) and goat anti-Oct4 immunoglobulin G (Santa Cruz, CA) or normal rabbit and goat immunoglobulin G. After washing with phosphate-buffered saline, embryoid bodies were stained with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G and Texas Red-conjugated donkey anti-goat immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA).

Techniques: Expressing

Journal: Biotechnology progress

Article Title: Induced pluripotent stem cells can utilize lactate as a metabolic substrate to support proliferation.

doi: 10.1002/btpr.3090

Figure Lengend Snippet: Figure 8. Comparison of pluripotency gene and protein expression for K3 iPSCs after one and five passages under different initial glucose and lactate concentrations. A) Normalized gene expression after 1-passage for Oct4, Sox2, and Nanog. B) Normalized protein expression after 1-passage for Oct4. C) Normalized gene expression after 5-passages for Oct4, Sox2, and Nanog. D) Normalized protein expression after 5-passages for Oct4. Control media – blue; Low Glucose media – light blue; High Lactate media – red; and Low Glucose + High Lactate – light red. The asterisk (*) indicates that the normalized gene or protein expressions were significantly different from the control (p ≤ 0.05). Error bars represent standard error.

Article Snippet: The secondary antibody incubations were conducted for 1-h at room temperature at a 1:1000 dilution in TBST using a goat anti-rabbit IgG HRP- linked antibody for β-actin and a goat anti-mouse IgG HRP-linked antibody for Oct4 (Bio-Rad Laboratories, Inc., Hercules, CA).

Techniques: Comparison, Expressing, Gene Expression, Control