Journal: Orphanet Journal of Rare Diseases

Article Title: Variants in a cis -regulatory element of TBX1 in conotruncal heart defect patients impair GATA6-mediated transactivation

doi: 10.1186/s13023-021-01981-4

Figure Lengend Snippet: GATA6 transcriptional regulation of TBX1 promoter. a Diagrammatic representation of the TBX1 cis -regulatory element showing putative potential GATA, NKX2-5, and SRF binding sites. b The influence of transcription factors on the p-138/+ 514 reporter gene in the C2C12 and NIH/3T3 cell lines. Luciferase analysis after cotransfection with luciferase reporter constructs containing the TBX1 cis -regulatory element TBX1 -138/+ 514 and the expression vector of GATA6, GATA4, NKX2-5, SRF or empty vector (pcDNA3.1). The results were normalized to Renilla and presented as the fold induction over the pcDNA3.1 vector. Data are shown as mean ± SEM, significance was calculated by one-way ANOVA with Dunnett’s multiple comparisons test, n = 3 independent experiments, ** p < 0.01 vs pcDNA3.1, *** p < 0.001 vs pcDNA3.1 in each cell line. c The influence of GATA6 on the TBX1 promoter reporter gene p-1179/+ 514 in the C2C12 and NIH/3T3 cell lines. Luciferase analysis after cotransfection with luciferase reporter p-1179/+ 514 and GATA6 expression vector or empty vector (pcDNA3.1). The results were normalized to Renilla and presented as the fold induction over the pcDNA3.1 vector. Data are shown as mean ± SEM, two-tailed unpaired t test was used for statistical calculation, n = 3 independent experiments, ** p < 0.01 vs pcDNA3.1, *** p < 0.001 vs pcDNA3.1 in each cell line. d and e The effects of the transcription factor GATA6 on the TBX1 promoter reporter gene in C2C12 and NIH/3T3 cell lines. Luciferase analysis after cotransfection of luciferase reporter and GATA6 expression construct or empty vector (pcDNA3.1). The results were normalized to Renilla and presented as the fold induction over basal reporter (pGL3-basic + pcDNA3.1). Data are shown as mean ± SEM, n = 3 independent experiments. Significance was calculated by two-way ANOVA with Sidak’s post hoc test, ### p < 0.001. Significance was calculated by two-way ANOVA with Tukey's multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For antibody lanes, assays were conducted in a similar manner with the following exceptions: addition of ∼5 μg of antibodies against GATA6 (sc-9055 X, Santa Cruz Biotechology Inc., USA) and incubation at room temperature for 20 min before the addition of the biotin-labeled DNA probe.

Techniques: Binding Assay, Luciferase, Cotransfection, Construct, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Orphanet Journal of Rare Diseases

Article Title: Variants in a cis -regulatory element of TBX1 in conotruncal heart defect patients impair GATA6-mediated transactivation

doi: 10.1186/s13023-021-01981-4

Figure Lengend Snippet: Histological staining analysis of TBX1 and GATA6 in human Carnegie Stage 13 (CS13) embryos. a Immunohistochemical staining for TBX1 and GATA6 in human CS13 embryos. Upper panel, TBX1 expression localized in pharyngeal arches (arrowheads); lower panel, GATA6 was expressed at pharyngeal arches (arrowheads) along with all atrial and ventricular chambers. Representative sagittal sections are shown (n = 3 embryos): dorsal is right; cranial is up. pa, pharyngeal arch; A, atrium; V, ventricle. Left panel, scale bars = 500 μm; middle panel, scale bars = 100 μm; right panel, scale bars = 50 μm. b Double immunofluorescence staining for TBX1 (red) and GATA6 (green) in human CS13 embryos. White arrows denote that both TBX1 and GATA6 are expressed in pharyngeal arches. Representative sagittal sections are shown (n = 3 embryos). pa, pharyngeal arch. Upper panel, scale bars = 100 μm; lower panel, scale bars = 50 μm

Article Snippet: For antibody lanes, assays were conducted in a similar manner with the following exceptions: addition of ∼5 μg of antibodies against GATA6 (sc-9055 X, Santa Cruz Biotechology Inc., USA) and incubation at room temperature for 20 min before the addition of the biotin-labeled DNA probe.

Techniques: Staining, Immunohistochemical staining, Expressing, Double Immunofluorescence Staining

Journal: Orphanet Journal of Rare Diseases

Article Title: Variants in a cis -regulatory element of TBX1 in conotruncal heart defect patients impair GATA6-mediated transactivation

doi: 10.1186/s13023-021-01981-4

Figure Lengend Snippet: Electrophoretic gel mobility shift assays (EMSAs) revealed that GATA6 binds with the cis -regulatory element within the TBX1 promoter directly. EMSAs were performed using biotin-labeled oligonucleotide probes containing + 115/+ 302 bp of TBX1 cis -regulatory element and in vitro-translated TNT blank protein, TNT pcDNA3.1 protein and TNT GATA6 protein by reticulocyte lysates, respectively. A protein-DNA complex was formed (lane 3), which could be inhibited by the addition of 120-fold molar excess unlabeled consensus GATA6 competitor DNA (lane 4) or antibody targeting GATA6 (lane 5). The arrows indicate unbound biotin-labeled free probe, GATA6-DNA complex and GATA6 antibody-GATA6-DNA supershift

Article Snippet: For antibody lanes, assays were conducted in a similar manner with the following exceptions: addition of ∼5 μg of antibodies against GATA6 (sc-9055 X, Santa Cruz Biotechology Inc., USA) and incubation at room temperature for 20 min before the addition of the biotin-labeled DNA probe.

Techniques: Mobility Shift, Labeling, In Vitro

Journal: Orphanet Journal of Rare Diseases

Article Title: Variants in a cis -regulatory element of TBX1 in conotruncal heart defect patients impair GATA6-mediated transactivation

doi: 10.1186/s13023-021-01981-4

Figure Lengend Snippet: Mutation studies on the cis -regulatory element of the TBX1 promoter associated with CTD. a Chromatograms of sequence variants within the TBX1 cis -regulatory element. ( Top ) shows the heterozygous mutation of patients, ( Bottom ) shows healthy controls, and all the variants are marked with arrows. b Schematic representation of the identified TBX1 cis -regulatory element variants in CTD patients. The transcription start site is at the position of g.19756703 (+1) in the first non-coding exon (E1). The numbers represent the genomic DNA sequences of the TBX1 gene (NC_000022.11). c The effects of GATA6 on the TBX1 mutation luciferase reporter genes M-130, M-143, and M-200. The WT or mutant TBX1 promoter reporter gene were transfected alone into the C2C12 cell line or cotransfected with the GATA6 expression vectors. The luciferase activities were normalized to Renilla and presented as the fold increase relative to the activity of the reporter in the presence of an empty expression plasmid (pGL3-basic + pcDNA3.1). Patient variants are associated with a significant transcription activity reduction by GATA6. Data shown are mean ± SEM, n = 4 independent experiments. Significance was calculated by two-way ANOVA with Sidak’s post hoc test, ### p < 0.001. Significance was calculated by two-way ANOVA with Dunnett's multiple comparisons test, * p < 0.05 vs p -1179/+ 514, *** p < 0.001 vs p-1179/+ 514

Article Snippet: For antibody lanes, assays were conducted in a similar manner with the following exceptions: addition of ∼5 μg of antibodies against GATA6 (sc-9055 X, Santa Cruz Biotechology Inc., USA) and incubation at room temperature for 20 min before the addition of the biotin-labeled DNA probe.

Techniques: Mutagenesis, Sequencing, Luciferase, Transfection, Expressing, Activity Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

doi: 10.1101/2023.05.16.540918

Figure Lengend Snippet: A. Schematic representation and immunostaining images of blastocysts and ICMs at stages E3.5 and E4.5. GATA6 and GATA4 are used as markers for PrE fate (green), and NANOG and SOX2 are used as markers for EPI fate (magenta) at stages E3.5 and E4.5, respectively. B. Quantification of total cell numbers in the ICM from blastocysts and isolated ICMs at stage E3.5, blastocysts and isolated ICMs at stage E4.5, and isolated ICMs cultured in vitro for 24 hours from stage E3.5 to E4.5. n=33, 30, 40, 21, 31 embryos for the different groups, respectively. Independent samples t-test between E3.5 blastocysts and E3.5 ICMs, p =0.106. One-way ANOVA between E4.5 Blastocysts, E4.5 ICMs, and E3.5 ICMs+24hr, p =0.145. C. Representative time-lapse imaging of ICMs isolated from E3.5 blastocysts expressing PrE-specific H2B-GFP ( Pdgfrα H2B-GFP , green) and ubiquitous H2B-mCherry ( R26-H2B mCherry , magenta), out of total 8 datasets from 3 independent experiments. Time is indicated in hh:mm, t=00:00 corresponds to start of live-imaging at stage E3.5+3hours, following completion of immunosurgery. D. Schematic representation of single-cell tracking of EPI and PrE cells from isolated ICMs from (C). Line plots indicating radial distances of all cells from one representative ICM until E4.0 stage. Colour of the line indicates cell fate – PrE, green and EPI, magenta. Shaded regions show spatial dispersion as mean ±SD of cell position along ICM radial axis. The geometric centroid of the ICM is considered as d=0.0 and ICM outer surface is considered as d=1.0 to normalise cell position across samples. Time-series plots for cell position were smoothed using a rolling average. E. Quantification of sorting score for isolated ICMs between stage E3.5 and E4.0. Data from n=8 ICMs. F. Line plots for radial cell position versus time from tracking of PrE and EPI cell movements in isolated ICMs. Time-series plots for cell position were smoothed using a rolling average. Cell tracking data pooled from n=160 PrE cells and n=133 EPI cells from 8 ICMs. G. Schematic diagram for analysis of PrE and EPI cell movements. Cell displacement is measured along the radial axis between consecutive timepoints and classified as inward or outward movement depending on the direction of displacement. H. Polar plots indicating preferential direction of cell movements among PrE and EPI. Cell position is plotted along radial axis, time is plotted along angular axis. Measurements are binned according to initial radial cell position and time. The mean displacement of each interval is plotted, colour indicates direction of movement. Scale bars 20μm. ns, non-significant

Article Snippet: Primary antibodies against GATA6 (R&D systems, AF1700), GATA4 (R&D systems, BAF2606), SOX2 (Cell Signaling, 23064), bi-phosphorylated myosin regulatory light chain (ppMRLC) (Cell Signaling, 3674), and Laminin (Novus Biologicals, NB300-14422) were diluted at 1:200.

Techniques: Immunostaining, Isolation, Cell Culture, In Vitro, Imaging, Expressing, Single Cell Tracking, Dispersion, Cell Tracking Assay

Journal: bioRxiv

Article Title: Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

doi: 10.1101/2023.05.16.540918

Figure Lengend Snippet: A. Immunofluorescence image of a 3x blastocyst at stage E3.75 showing laminin distribution around PrE cells. White dotted line, ICM-cavity interface. White arrowhead marks GATA6-expressing nucleus of a PrE cell enriched for laminin expression. B. Immunofluorescence image of a 3x blastocyst at stage E3.75 showing PKCλ+ζ distribution in PrE cells. White arrowhead marks leading edge of a PrE cell with PKCλ+ζ localisation. C. Immunofluorescence image of an E3.75 ICM showing PKCλ+ζ localisation in PrE and EPI cells. White dotted lines mark cell boundaries. Yellow line indicates the line segments from cell inner edge (towards ICM centroid) to cell outer edge (towards ICM-fluid interface) along which fluorescence intensity is measured. D. Line plots for normalised fluorescence intensity of PKCλ+ζ in individual inside cells from E3.75 isolated ICMs. Colour of the line indicates GATA6-expression level of the cell. n=260 cells from 32 ICMs. Each of the thin lines corresponds to measurement from one cell. Bold line and shaded region indicate mean±SD of aPKC intensity for GATA6-high and GATA6-low cells. E. Schematic description of polarisation index. Polarisation index is calculated as the ratio between mean aPKC intensity at 1/4 th distance from outer edge and mean aPKC intensity at 1/4 th distance from inner edge. Boxplots for comparison of the polarisation index in PrE (GATA6 high) versus EPI cells (GATA6 low). GATA6 expression level is categorised as high or low by thresholding the bimodal distribution of GATA6 fluorescence intensity. Colour of the line indicates GATA6-expression level of the cell. n=136 GATA6-high and 124 GATA6-low cells from 32 ICMs. One-way ANOVA, p =6.03e -20 . F. Scatterplot of polarisation index of cells versus radial distance of the cell from the ICM centroid. Colour of the datapoint indicates GATA6-expression level of the cell. Black dotted line, linear regression with Pearson’s R=0.079, p =0.205. n=260 cells from 32 ICMs. G. Immunofluorescence images of control and Gö6983-treated E4.0 isolated ICMs and quantification of sorting score. n=16,24 ICMs for control and Gö6983-treated ICMs respectively. Independent samples t-test, p = 8.01e -04 H. Immunofluorescence images of representative WT, Prkci +/+ ;Prkcz -/- , and Prkci +/- ;Prkcz -/- E4.5 blastocysts and quantification of number of ectopic PrE cells in E4.5 blastocysts from each group. n= 25, 17, 12 blastocysts for WT, Prkci +/+ ;Prkcz -/- , and Prkci +/- ;Prkcz -/- respectively. Mann-Whitney U test, p = 2.43e -04 , 6.36e -04 Scale bars 20μm. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: Primary antibodies against GATA6 (R&D systems, AF1700), GATA4 (R&D systems, BAF2606), SOX2 (Cell Signaling, 23064), bi-phosphorylated myosin regulatory light chain (ppMRLC) (Cell Signaling, 3674), and Laminin (Novus Biologicals, NB300-14422) were diluted at 1:200.

Techniques: Immunofluorescence, Expressing, Fluorescence, Isolation, Comparison, Control, MANN-WHITNEY

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Quantitative real-time PCR primer sequences

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: GATA6 is expressed in human hepatocytes during early gestation but not in normal perinatal or adult hepatocytes. A–C: immunohistochemical staining of GATA6 protein in normal human liver. Brown indicates positive staining in nuclei. In fetal liver (FL) at gestational week (GW) 13 (FL GW13), GATA6 protein is expressed in all hepatocytes (arrowhead) and other cell types of the liver (A). At GW37, GATA6 protein expression is diminished in hepatocytes (arrowhead) and restricted to cholangiocytes lining the bile ducts (arrow) (B). The expression pattern of GATA6 protein in adult liver (AL) is similar to that of late FL with positive cholangiocytes (arrow) and negative hepatocytes (arrowhead) (C). D–G: in situ hybridization of GATA6 mRNA in FL GW37 (D and E) and AL (F and G). Green indicates positive signal in cytoplasm. Cholangiocytes show positive signal (arrow); hepatocytes show weak or negative signal (arrowhead). Scale bar = 200 µm (A–C) and 20 µm (D–G).

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Immunohistochemical staining, Staining, Expressing, In Situ Hybridization

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Hepatocyte expression of GATA6 is elevated in biliary atresia (BA) and decreased after portoenterostomy (PE). A: boxplot showing the relative GATA6 mRNA expression, as determined by qPCR, in different liver sample groups. Dots represent individual samples, the box represents the interquartile range, and the whiskers represent the 1st and 4th quartile. The line inside the box is the median, and the dashed line represents the mean. *P < 0.05, **P < 0.01 compared with BA group. The P values of other paired comparisons are indicated in Table 2. B–E: in situ hybridization of GATA6 in BA samples demonstrates strongly positive hepatocytes (arrowhead) (B and C), whereas in situ hybridization in BA-post-PE samples shows weak or negligible signal in hepatocytes (arrowhead) (D and E). F–H: GATA6 immunohistochemistry from normal adult liver (AL) with negative hepatocytes and positive cholangiocytes (F), BA with strong immunoreactivity in hepatocyte nuclei (G), and BA-post-PE with less immunoreactivity in hepatocytes compared with BA (H). I: Western blotting of GATA6 protein in AL, BA, BA-post-PE, and disease control (DC) samples. NBI, normalized band intensity. J: paired sample analysis of GATA6 protein expression from patients before BA and after PE (BA-post-PE). Green (in situ hybridization) and brown (immunohistochemistry) indicate positive staining. Scale bars = 150 µm (B and C), 20 µm (C and E), and 200 µm (F and H).

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Western Blot, Staining

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: P values from comparisons of mRNA levels of different liver sample types (in Figs. 2A and 7A, C, and E) using Wilcoxon method

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: GATA6 immunoreactivity in liver sample groups

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Double in situ hybridization in biliary atresia (BA) liver. A and B: double in situ hybridization was performed on 2 different samples of BA liver. GATA6 expression is high in both ductular reaction (DR) area and liver parenchyma (hepatocytes); these 2 histological compartments are denoted in the figures by dashed lines. C and D: CK7 (C) is strongly expressed in DR area, whereas its expression is weaker in hepatocytes. CFTR (D) expression is limited to DR area. E and F: merged images of DAPI, GATA6, and CK7 (E) or DAPI, GATA6, and CFTR (F). Bile duct epithelium (arrow) is positive for both GATA6 and CK7/CFTR, but hepatocytes (arrowhead) express only GATA6. G and I: higher-magnification images from E. H and J: higher-magnification images from F. Green indicates positive signal for GATA6, and red indicates positive signal for CK7 or CFTR. Blue indicates DAPI staining in the nuclei of all cell types. Scale bars = 50 µm (A–F) and 10 µm (G–J).

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: In Situ Hybridization, Expressing, Staining

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: GATA6 is overexpressed in hepatocytes in 2 mouse models of biliary obstruction. A: GATA6 protein is expressed in bile duct epithelium (arrow) but not in hepatocytes (arrowhead) of normal murine liver (4 wk after sham surgery). B: in mice subjected to bile duct ligation (BDL), hepatocytes (arrowhead) in the periportal area strongly express GATA6. C and D: at postnatal day 15 (P15), before the onset of ductular reaction (DR), there remains weak expression of GATA6 in normal hepatocytes (arrowhead), and there is no difference in GATA6 expression between control (Ctrl) and Alb-Cre;Rbpjflox/flox;Hnf6flox/flox double knockout (DKO) mice. E and F: at P30 Ctrl hepatocytes are GATA6 negative, whereas the Alb-Cre;Rbpjflox/flox;Hnf6flox/flox liver with severe DR shows strong GATA6 immunoreactivity in hepatocytes. G and H: at P120 in Alb-Cre;Rbpjflox/flox;Hnf6flox/flox liver, the DR has diminished, and liver histology, as well as GATA6 expression, is similar to Ctrl liver with immunoreactivity only in bile duct epithelial cells (arrow). n = 3 in each group. Scale bars = 50 µm.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Ligation, Expressing, Double Knockout

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: In patients with biliary atresia (BA), GATA6 protein expression in hepatocytes correlates to bile duct expansion (BDE), age at portoenterostomy (PE), and the liver injury marker alanine aminotransferase. Hepatocyte GATA6 protein expression was divided into 2 groups (<70% positive nuclei = low/intermediate vs. >70% positive nuclei = high) and correlated to BDE rate (P = 0.0094) (A), age at PE (B), and plasma (P)-alanine aminotransferase (C). A: contingency tabling coupled with χ2 test was employed to test the statistical significance. B and C: dots represent individual samples, the box represents the interquartile range, and the whiskers represent the 1st and 4th quartile. The line inside the box is the median, and the dashed line represents the mean. *P < 0.05.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing, Marker

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Enforced expression of GATA6 causes alterations in expression of genes regulating cholangiocyte and hepatocyte differentiation. The human hepatocellular cell line HepG2 and primary human hepatocytes were transiently transfected with pCDNA3-GATA6 or pCDNA3 plasmid alone. After 48 h, RNA was harvested and subjected to qRT-PCR analysis. A: in HepG2 cells, 3 genes related to cholangiocyte differentiation (HNF1β, HNF6, and JAG1) were significantly upregulated in cells overexpressing GATA6 compared with control (Ctrl) cells. B: in primary human hepatocytes, the relative expression of 4 genes related to cholangiocyte differentiation (HNF1β, HNF6, JAG1, and DKK1) and 1 gene related to hepatocyte differentiation (HNF4α) were upregulated in cells with GATA6 overexpression compared with control cells. For both cell types, 5 independent experiments were performed in triplicate. Each bar depicts the logarithm of the ratio of mRNA expression GATA6 overexpression vs. control cells. *P < 0.05, **P < 0.01.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Expression of HNF1β, HNF6, and JAG1 is elevated in biliary atresia (BA) liver, decreases after successful portoenterostomy (PE), and correlates to GATA6. A, C, and E: boxplots showing relative mRNA expression of HNF1β (A), HNF6 (C), and JAG1 (E), as measured by qPCR, in BA vs. other liver specimens. Dots represent individual samples, the box represents the interquartile range, and the whiskers represent the 1st and 4th quartile. The line inside the box is the median, and the dashed line represents the mean. *P < 0.05, **P < 0.01. The P values of other paired comparisons are indicated in Table 2. B, D, and F: linear regression analyses of HNF1β (B), HNF6 (D), and JAG1 (F) mRNA expression vs. GATA6 mRNA expression in BA-post-PE samples. FL, fetal liver; AL, adult liver; DC, disease control.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing

Journal: bioRxiv

Article Title: The origin of mouse extraembryonic endoderm stem cell lines

doi: 10.1101/2021.12.27.474300

Figure Lengend Snippet: ( A ) Cryosections of two E6.5 (R26-tauGFP41 x Sox17-Cre) F1 deciduae. Thickness is 12 µ m. Intrinsic green fluorescence of GFP, counterstaining with DAPI (blue). ( B ) E6.5 embryo without ectoplacental cone and same embryo after disaggregation. Top: bright-field image; bottom, intrinsic green fluorescence of GFP. ( C ) There were two types of colonies on day 4: flat and GFP- and XEN-like and GFP+. ( D ) Post-XEN cell line X-E6.5-AC1456-4 on day 18, derived from a pool of XEN-like colonies picked on day 5. Most cells in the culture are GFP+. ( E ) GFP+ XEN-like cells (X-E6.5-AC1456-4) surrounding GFP-flat colonies on days 10 and 22. On day 35 there were no more GFP-flat colonies. ( F ) Immunofluorescence was performed on outgrowths from GFP-E6.5 embryos on day 7. ( G ) Flat colonies displayed GATA6 and SOX7 expression, strong OCT4 expression, and weak or no NANOG expression on day 18. ( H ) Most cells from flat colonies had the typical appearance of XEN-like cells, with strong expression of GATA6 and SOX7 and no OCT4 or NANOG expression on day 36.

Article Snippet: Primary antibodies from R&D Systems were against GATA6 (#AF1700), SOX7 (#AF2766), and SOX17 (#AF1924).

Techniques: Fluorescence, Derivative Assay, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: The origin of mouse extraembryonic endoderm stem cell lines

doi: 10.1101/2021.12.27.474300

Figure Lengend Snippet: ( A ) outgrowths from disaggregated E7.5 embryo. ( B ) Post-XEN cells contributed to ExEn in E7.5 stage embryos. ( C ) E7.5 embryo (bottom) and Reichert’s membrane (RM) (top). The bright-field image was merged with the fluorescence image. ( D ) RM. ( E ) Embryo without RM. ( F ) Outgrowth of RM (PE) on days 3 and 10 and post-XEN cell line X-E7.5-PE-Z1544-1 on day 38. All cells were GFP-. Top: bright-field image; bottom, intrinsic green fluorescence of GFP. ( G ) VE (GFP-) and epiblast (GFP+); outgrowth of VE on day 3; a small number of XEN cells on day 10 were GFP+. Top: bright-field image; bottom, intrinsic green fluorescence of GFP. ( H, I ) Post-XEN cell lines X-E7.5-PE-Z1544-5 ( H ) and X-E7.5-VE-Z01966-1 ( I ). Intrinsic green fluorescence of GFP and immunofluorescence for GATA4, GATA6, SOX7, SOX17, DAB2, OCT4, NANOG, and CDX2. DAPI was used as nuclear strain.

Article Snippet: Primary antibodies from R&D Systems were against GATA6 (#AF1700), SOX7 (#AF2766), and SOX17 (#AF1924).

Techniques: Membrane, Fluorescence, Immunofluorescence

Journal: bioRxiv

Article Title: The origin of mouse extraembryonic endoderm stem cell lines

doi: 10.1101/2021.12.27.474300

Figure Lengend Snippet: (A) Isolated single cells from an ICMs. (B) A single PrE formed outgrowth that was GFP- and with the phenotype of XEN-like on day 3, with a few GFP+ cells on day 5, day 8 and day 30. (C) A single epiblast formed outgrowth that was strongly GFP+ expression with an ES-like phenotype on day 3, day 5, day 8, and day 30 and converted to the XEN cell line on day 50. (D) Performed outgrowths could be derived from single epiblast cells from GFP+ ICMs for immunofluorescence on day 7. Performed outgrowths with few or no GFP cells and with an XEN-like phenotype could be derived from single PrE cells for immunofluorescence on day 7. (E) Immunofluorescence staining of ES-AC1558-2-8 from a single epiblast. (F) Immunofluorescence staining of cXEN-AC1558-2-8 converted from ES-AC1558-2-8 on day 61. DAB2, GATA4, GATA6, SOX17 and SOX7 were expressed in a single epiblast cell-derived XEN cell line. OCT4, NANGO and CDX2 were no expression.

Article Snippet: Primary antibodies from R&D Systems were against GATA6 (#AF1700), SOX7 (#AF2766), and SOX17 (#AF1924).

Techniques: Isolation, Expressing, Derivative Assay, Immunofluorescence, Staining

Journal: Developmental cell

Article Title: Single-cell analysis of bidirectional reprogramming between early embryonic states identify mechanisms of differential lineage plasticities in mice

doi: 10.1016/j.devcel.2024.11.022

Figure Lengend Snippet: (A) Time course flow cytometry-based analysis of pluripotency-associated markers SSEA-1 and Oct4 -GFP, and XEN-associated marker PDGFRα during XEN reprogramming. Representative contour plots show expression of SSEA-1 (AlexaFluor647-conjugated), Oct4 -GFP and PDGFRα (PE-Cy7-conjugated) at days 2, 8 and 16 of reprogramming. Population percentage indicated within each gate. (B) (Top) Tracking the reprogramming efficiencies of four major subpopulations sorted at day 14 of reprogramming. At day 28, populations arising from each sorted subpopulation were determined using flow cytometry. (Bottom, left) Stacked bar charts depicting the mean proportion of the population represented by each subpopulation at day 14, and at day 28 (bottom, right) . S-O-P+ subpopulation constitutes the remainder of the population (not shown) to amount to 100% and represents non-reprogramming XEN cells. N = 6 (2 independent experiments with 3 replicates each). (C) Time course tracking of ES-to-iXEN conversion using flow cytometry analysis of SSEA-1, PDGFRα and Gata6 -Venus reporter. Representative contour plots show expression of PDGFRα (PE-Cy7-conjugated), Gata6 -Venus and SSEA-1 (BV421-conjugated) at indicated timepoints. Population percentage is indicated within each gate. (D) Summarized schematic of XEN-to-iPS and ES-to-iXEN lineage conversions. (Left) Hypothesized reprogramming route taken by XEN cells. Sizes and number of arrows reflect the likelihood of cells progressing from one state to the next (smaller/single arrows = few cells progress; larger/more arrows = many cells progress). Reprogramming XEN cells initiate expression of Oct4 -GFP, then downregulate PDGFRα. This downregulation step represents a bottleneck during reprogramming since few Oct4 -GFP+ cells progress to this state. Following PDGFRα downregulation, a large proportion of cells upregulate SSEA-1. (Right) Hypothesized lineage conversion of ES to iXEN cells. Initial upregulation of both, PDGFRα and Gata6 -Venus, is followed by downregulation of SSEA-1. No obvious bottlenecks are detected during ES-to-iXEN conversion.

Article Snippet: Samples were incubated in PCR strip tubes with 1:50 dilutions of primary antibodies against Gata6 (R&D Systems), Gata4 (Santa Cruz Biotech), or anti-IgG (EpiCypher) in Antibody Buffer (Wash Buffer with 2mM EDTA and 0.01% Digitonin (Millipore Sigma)) overnight with gentle rocking at 4°C.

Techniques: Flow Cytometry, Marker, Expressing

Journal: Developmental cell

Article Title: Single-cell analysis of bidirectional reprogramming between early embryonic states identify mechanisms of differential lineage plasticities in mice

doi: 10.1016/j.devcel.2024.11.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Samples were incubated in PCR strip tubes with 1:50 dilutions of primary antibodies against Gata6 (R&D Systems), Gata4 (Santa Cruz Biotech), or anti-IgG (EpiCypher) in Antibody Buffer (Wash Buffer with 2mM EDTA and 0.01% Digitonin (Millipore Sigma)) overnight with gentle rocking at 4°C.

Techniques: Negative Control, Virus, Recombinant, SYBR Green Assay, Electron Microscopy, Immunoprecipitation, Reverse Transcription, RNA HS Assay, Plasmid Preparation, Software

Journal: PeerJ

Article Title: Proteomics (SWATH-MS) informed by transcriptomics approach of tropical herb Persicaria minor leaves upon methyl jasmonate elicitation

doi: 10.7717/peerj.5525

Figure Lengend Snippet: Differentially expressed proteins in Persicaria minor leaf proteome upon methyl jasmonate (MeJA) treatment. Mean peak areas (three biological replicates) for control (Mean C) and treated (Mean T) samples were generated from SWATH-MS proteomics analysis. Differentially expressed proteins were selected based on three statistical analyses detailed in Methods ( p < 0.05) as well as having normalized fold change (FC.norm) of control over treated samples greater than 1.5 (significantly up-regulated proteins) or lesser than 0.67 (significantly down- regulated proteins). Protein ID, protein name, UniProt accession number and organism were retrieved from the protein annotation of the P. minor transcriptome ( Rahnamaie-Tajadod et al., 2017 ). Please refer to Tables S4 and S5 for detailed descriptions of the raw data and analyses performed.

Article Snippet: cds.c37568_g1_i1—m.8344 , 102,097.61 , 37,178.92 , 0.53 , Transcription factor GATA-6 , P43693 , Gallus gallus.

Techniques: Control, Generated, Data-independent acquisition, Glycoproteomics, Binding Assay, Ubiquitin Proteomics, Variant Assay, Membrane