Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Schematic diagram of the positions and orientations of syncytin-1, PEX1 and GATAD1 genes. Arrows show genes’ orientations. The patterned squares represent exons. Dark squares indicate the location of the CpG islands in GATAD1 gene. Light grey square represents the syncytin-1 5′ LTR region. The solid lines at the bottom show the positions of amplicons of real-time PCR. Note the opposite orientations of GATAD1 and syncytin-1 genes, which bring the 3 [prime] region of GATAD1 to a closer vicinity of syncytin-1 gene.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Information of PCR primers

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques:

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: PEX1 and GATAD1 mRNA levels in first-trimester (1N, n=8), third-trimester normal (3N, n=14) and third-trimester preeclamptic (3P, =7) placentas. Real-time PCR were performed as described in Materials and methods. Data were standardized by the results from β-actin internal control. The mRNA levels from third-trimester normal (3N) placentas were set as 1. The averages and standard errors of each group were presented. a PEX1 mRNA levels. No significant difference was found between 1N and 3P, or 3N and 3P groups. b GATAD1 mRNA levels. Significantly higher GATAD1 mRNA levels were observed in 3N than in 1N; A significant reduction of GATAD1 mRNA levels were found in 3P compared to 3N group. **p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Protein levels of GATAD1 in the first-trimester (1N1-1N6), third-trimester normal (3N1-3N6), and third-trimester preeclamptic (3P1-3P6) placentas. The sizes of GATAD1 and β-actin proteins are 29 kDa and 42 kDa, respectively. a Western blotting performed using GATAD1-specifc antibodies. b Results of densitometry analyses showing a similar trend of changes to that of mRNA levels: GATAD1 protein expression was higher in 3N than 1N, and lower in 3P than 3N. The GATAD1 expression data were standardized by the results from β-actin. The protein levels from third-trimester normal (3N) placentas were set as 1. The averages and standard errors from each group were presented. **p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Representative results of immunohistochemistry (40×10). The paraffin-embedded placental tissues were sliced into 4 μm sections. The sections were processed as describe under Materials and methods. As negative control (bottom right panel), a section of first-trimester normal placenta was processed with the same procedures except for the absence of primary antibodies. GATAD1 protein was stained brown color. The nuclei were stained blue with haematoxylin. GATAD1 protein localized mostly in the cytoplasm and membrane of syncytiotrophoblasts (ST), and to a less extent, the cytoplasm and membrane of cytotrophoblasts (CT). Higher level of GATAD1 expression was found in third-trimester (upper right) than first-trimester (upper left panel) placenta. Preeclamptic (bottom left) placentas expressed decreased levels of GATAD1 protein compared to normal placentas.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Immunohistochemistry, Negative Control, Staining, Expressing

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: GATAD1 gene methylation measured by COBRA. Following PCR amplification, DNA fragments representing the 5 [prime] and 3 [prime] regions of GATAD1 gene were digested with an excess of BstUI or TaqαI, respectively. Agarose gel electrophoresis was performed and DNA bands were visualized by ethidium bromide staining. a The absence of cleavage product (supposedly 147 bp and 107 bp) from 254 bp fragment indicated an largely unmethylated status of GATAD1 5 [prime] region in first-trimester (1N1 to 1N8), third-trimester normal (3N1 to 3N14) and Preeclamptic (3P1 to 3P7) placentas. b The 241 bp fragment representing the 3 [prime] region of GATAD1 was mostly cleaved, generating the 144 bp and 97 bp bands indicative of DNA methylation. c Densitometry analyses of the 3 [prime] methylation showing an increased methylation in 3N placentas compared to 1N, and decreased methylation levels in 3P placentas compared to 3N group. ** p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Methylation, Combined Bisulfite Restriction Analysis Assay, Amplification, Agarose Gel Electrophoresis, Staining, DNA Methylation Assay

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Bisulfite sequencing of the GATAD1 3 [prime] region. Bisulfite-converted DNA from 1N (n=5), 3N (n=5) and 3P (n=5) groups were PCR amplified, subcloned, and sequenced. a The typical sequencing result of the 3 [prime] region. Asterisks (*) mark CpG sites. The TaqαI recognition site used in COBRA is underlined. b GATAD1 3 [prime] bisulfate sequencing results. The solid and open circles represent the methylated and unmethylated cytosines, respectively, in CpGs dinucleotides contexts. The average methylation levels for each CpG site were presented in the bottom panels. c Quantitative comparison of the GATAD1 3 [prime] methylation among the three groups. 3N placentas displayed increased methylation levels compared to 1N, and 3P group exhibited decreased methylation levels compared to 3N. * p < 0.05; ** p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Methylation Sequencing, Amplification, Sequencing, Combined Bisulfite Restriction Analysis Assay, Methylation

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: The correlation between GATAD1 expression and GATAD1 3 [prime] methylation in human placentas (n=29). The Y-axis indicated GATAD1 mRNA levels and the X-axis represented GATAD1 3 [prime] methylation index. Spearman correlation analysis showed a highly significant positive correlation between GATAD1 mRNA levels and GATAD1 3 [prime] methylation levels among placental samples (r=0.62, p=0.0003).

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: Expressing, Methylation

Journal: Cellular signalling

Article Title: Decreased Expression and DNA Methylation Levels of GATAD1 in Preeclamptic Placentas

doi: 10.1016/j.cellsig.2014.01.013

Figure Lengend Snippet: Treatment with DNMT inhibitor led to a decreased GATAD1 3 [prime] DNA methylation and decreased GATAD1 expression. JAR cells were treated for 5 days with 0, 0.5, and 2.5 μM of 5-aza-deoxycytidine (ADC). a GATAD1 3 [prime] methylation was examined with COBRA. b Densitometry analyses indicated a dose-dependent decrease of GATAD1 3 [prime] DNA methylation following ADC treatment. c Results of real-time PCR showed a decrease of GATAD1 mRNA expression following ADC treatment. Data were standardized with the results from β-actin. Averages and standard errors were presented in the chart. **p < 0.01.

Article Snippet: Protein detection was carried out with primary antibodies, including rabbit anti-GATAD1 (1:500, Bioss, Inc., Woburn, MA, USA), mouse anti-β-actin (1:6000, SIGMA-ALDRICH, Saint Louis, MO, USA) and the matching secondary, peroxidase-labeled, antibodies (Anti-rabbit or anti–mouse; 1:6000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Techniques: DNA Methylation Assay, Expressing, Methylation, Combined Bisulfite Restriction Analysis Assay, Real-time Polymerase Chain Reaction

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 1. Genomic organization of the human ELF5 locus and transcript isoform expression in placenta and trophoblast cell lines. (A) Diagram of the exon–intron structure of the human ELF5 locus and annotated splice var- iants. Position of primers used is indicated. Filled boxes represent open- reading frames and open boxes represent untranslated regions. (B) RT–PCR analysis with isoform-specific and common primers reveals that ELF5-2b is the expressed splice variant in placenta and the trophoblast-like cell line TCL-1, but that it is absent from the first trimester mesenchymal-like cell line TCL-2. (C) RT–PCR with primers spanning exons 3 and 4 demonstrates that the annotated ELF5-2bDex3/4 variant is not present in placenta and chor- iocarcinoma and trophoblast-like cell lines JEG-3 and TCL-1.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Variant Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 2. Expression of trophoblast stem cell genes and epigenetic regulation of ELF5 in placenta throughout gestation. (A) RT–PCR analysis of ELF5, CDX2 and EOMES (i.e. genes important for trophoblast stem cell self-renewal and proliferation in the mouse) on human placental villous samples ranging from 7 weeks of gestation to term. Four independent term placental samples were investigated. The choriocarcinoma cell line JEG-3 was included as control. Colour-inverted photographs of ethidium bromide stained gels are shown. All three genes are expressed in placenta, but CDX2 is not detected from the second trimester onwards even when the PCRs are over-cycled. (B) Quantitative RT–PCR (qPCR) analysis of ELF5, CDX2 and EOMES on the same samples used in (A). ELF5 is down- regulated in second and third trimesters, whereas no overall regulation with gestational age was observed for EOMES. (C) Comparison of expression levels between first trimester and term. ELF5 expression is significantly reduced at term when compared with first trimester, CDX2 is absent from term placentas. (D) Bisulphite sequencing analysis of the ELF5 promoter region. Filled circles indicate methylated cytosine residues. ELF5 is extremely hypomethylated in the first trimester and acquires higher DNA methylation levels in second and third trimester, correlating with transcriptional down-regulation at these stages. (E) DNA methylation analysis of an extended region between 2400 bp and +400 bp around the transcriptional start site of ELF5. Hypomethylation correlates with ELF5 expression in JEG-3 cells and, conversely, ELF5 is hypermethylated and not expressed in TCL-2 cells. The methylation pattern in TCL-1 cells reveals a critical stretch of five CpG residues (grey box) at the immediate transcriptional start site that needs to be unmethylated for ELF5 to be expressed.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Quantitative RT-PCR, Comparison, Bisulfite Sequencing, Methylation, DNA Methylation Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 3. Immunofluorescence localization of ELF5 to cytotrophoblasts in the human placenta. (A) Overview of 11 week placental villous cross-section shows ELF5 localization to nuclei of villous cytotrophoblasts, but absence from nuclei of the overlying syncytiotrophoblast layer. Cytotrophoblasts are a proliferative cell population that continuously divide to replenish the overlying syncytium. (B) Co-localization with cytokeratin 7 (CK7) confirms the trophoblast identity of ELF5-positive cells. (C) Confocal image of a double staining of ELF5 and the villous cytotrophoblast marker SPINT1 (also known as HAI-1) shows that every ELF5-positive nucleus resides within the cytotrophoblast layer. Top row 6 week, bottom row 11 week placenta. (D) Confocal image analysis of an 11 week villous section stained for ELF5 and the extravillous cytotrophoblast (EVT) marker integrin alpha-5 (ITGA5). ELF5 is detected only in nuclei at the proliferative base, but not further distal along the EVT column where cells adopt an invasive phenotype and lose proliferative potential. (E) ELF5 is also absent from post- mitotic interstitial and endovascular EVTs within the decidual bed.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Double Staining, Marker, Staining

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 4. CDX2 identifies a subset of ELF5-positive cytotrophoblasts as a TS-like compartment that is regulated by FGFR2. (A) ELF5 co-localizes with FGFR2 in villous cytotrophoblasts as identified by confocal image analysis of double immunofluorescence stainings of 11 week placental sections. Since FGF signalling has been implicated in TS cell proliferation in mice and humans and can activate ELF5 expression in other tissues, FGF/FGFR2 may induce ELF5 expression within a putative TS cell niche in the human placenta. (B) Double staining of a 6 week placental section for ELF5 and CDX2. Larger groups of CDX2-positive cells are detected only in early gestation up to 8.5–9 weeks. CDX2 is mostly co-expressed with ELF5 (arrowheads). (C) Dual labelling of 6 week placental section for CDX2 and the proliferation marker Ki67. CDX2-expressing cytotrophoblasts preferentially stain positive for Ki67, indicating their high proliferation rate. CDX2 and Ki67 are restricted to the proximal end of cytotrophoblast cell columns (highlighted by the boxed area). The white arrows indicate the direction of progressive extravillous trophoblast (EVT) differentiation and migration.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Double Staining, Marker, Staining, Migration

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 5. Inter-regulatory network of trophoblast transcription factors CDX2, EOMES and ELF5. (A) Chromatin immunoprecipitation assays show that CDX2 binds to the ELF5 promoter region in JEG-3 and TCL-1 cells where ELF5 is hypomethylated and expressed, but not in TCL-2 cells where ELF5 is hypermethylated and repressed. (B) In turn, ELF5 binds to the CDX2 and EOMES promoter regions in JEG-3 and TCL-1 cells where it is expressed, but not in TCL-2 cells from which it is absent, thereby establishing a transcrip- tional feedback loop between all three transcription factors. Binding to the EOMES promoter region was more consistent and is indicative of a more effi- cient, stronger interaction than with the CDX2 upstream region, consistent with results observed in mouse trophoblast (9).

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Chromatin Immunoprecipitation, Binding Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 6. Trophoblast transcription factor expression and epigenetic regulation of ELF5 in human ES cells and derived trophoblast cell lines. (A) Initial bisul- phite sequencing analysis of two pooled hES cell lines and derived trophoblast cells indicates a high degree of DNA methylation at the ELF5 promoter despite the limited trophoblast differentiation potential. (B) RT–PCR and (C) qPCR analysis for trophoblast transcription factors ELF5, CDX2 and EOMES on six differ- ent hES cells lines (Shef1, Shef4–7, H7), including one subclone with an abnormal karyotype (Shef5a), two derived cytotrophoblast cell lines (TrophH7 and TrophShef4), the JEG-3, TCL-1 and TCL-2 cell lines, an 8+4 week placenta for relative comparison of expression levels and a colorectal cancer cell line (DKO4) as positive control for CDX2 expression (27). Colour-inverted photographs of ethidium bromide stained gels are shown. ELF5 is detectable in some hES cell lines, albeit at very low levels. Higher expression levels of CDX2 and EOMES may relate to their function within the embryonic lineage and is not directly indicative of trophoblast differentiation potential. Strikingly, in contrast to their expression in placenta, all three genes are absent from the hES-derived tropho- blast cell lines. (D) Normalization of qPCR data to Shef6, one of the most highly ELF5 expressing hES cell lines, in comparison with JEG-3, TCL-1 and TCL-2 cell lines as well as a first trimester placenta sample demonstrates the comparatively negligible amount of ELF5 expression in hES cells that is approximately 300-fold less than in normal trophoblast in vivo. (E) Bisulphite sequencing analysis of the ELF5 promoter in three different hES cell lines and two derived trophoblast cell lines shows relatively little epigenetic variability between different hES cell lines. Hypermethylation correlates with extremely low ELF5 expression levels. (F) Elf5 is also highly methylated in three independent mouse epiblast stem cell lines and (G) in two human-induced pluripotent stem cell lines derived from kereatinocytes and fibroblasts.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Derivative Assay, Sequencing, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Comparison, Positive Control, Staining, In Vivo, Bisulfite Sequencing, Methylation

Journal: Frontiers in Genetics

Article Title: Bisulfite-Converted DNA Quantity Evaluation: A Multiplex Quantitative Real-Time PCR System for Evaluation of Bisulfite Conversion

doi: 10.3389/fgene.2021.618955

Figure Lengend Snippet: Concept of a multiplex quantitative real-time PCR evaluation system for bisulfite conversion (BisQuE) and an example. Genomic DNA (gDNA) and bisulfite-converted DNA (BS-DNA) undergoes the developed BisQuE method, including cytosine-free PCR primers and probes for two different-sized targets. Also, standard curves and the short-T to C transforming equation (-*->, highlighted in pink color) were obtained with standard DNA and C-T indicators, respectively. With the results of each gDNA and BS-DNA, the three key features (conversion efficiency, degradation level, and recovery) were calculated.

Article Snippet: Six BS conversion kits (EZ DNA Methylation-Lightning Kit, Premium Bisulfite kit, MethylEdge ® Bisulfite Conversion System, EpiJET Bisulfite Conversion Kit, EpiTect Fast DNA Bisulfite Kit, and NEBNext ® Enzymatic Methyl-seq Conversion Module) were tested in 20 samples using 50 ng of genomic DNA as an input with the BisQuE.

Techniques: Multiplex Assay, Real-time Polymerase Chain Reaction

Journal: Frontiers in Genetics

Article Title: Bisulfite-Converted DNA Quantity Evaluation: A Multiplex Quantitative Real-Time PCR System for Evaluation of Bisulfite Conversion

doi: 10.3389/fgene.2021.618955

Figure Lengend Snippet: Six BS conversion kits and an overview of the results.

Article Snippet: Six BS conversion kits (EZ DNA Methylation-Lightning Kit, Premium Bisulfite kit, MethylEdge ® Bisulfite Conversion System, EpiJET Bisulfite Conversion Kit, EpiTect Fast DNA Bisulfite Kit, and NEBNext ® Enzymatic Methyl-seq Conversion Module) were tested in 20 samples using 50 ng of genomic DNA as an input with the BisQuE.

Techniques: Methylation

Journal: PLoS ONE

Article Title: FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans

doi: 10.1371/journal.pone.0001612

Figure Lengend Snippet: (A) The sorted CD4 + CD25 lo , CD4 + CD25 hi and CD19 + cells were analyzed by flow cytometry. (B) FOXP3 expression in the sorted populations as determined by intracellular flow cytometry. Plots showing results from one representative donor out of four analyzed, except for plot showing FOXP3 expression in CD19 + cells where one single donor was analyzed (C) Sorted CD4 + CD25 + cells suppress the proliferation of CD4 + CD25 − cells. CD4 + CD25 − cells were activated with CD3 and CD28 antibodies together with CD4 − feeder cells and increasing numbers of CD4 + CD25 + cells in triplicate samples. Proliferation was measured as incorporation of [ 3 H]Thymidine for 18 hours, here illustrated as counts per minute (cpm) on the y-axis. The CD4 + CD25 + to CD25 − cell ratio is displayed on the x-axis. Squares indicate coculture of CD25 − and CD25 + cells. Triangles indicate control samples with only CD4 + CD25 − cells. (D) FOXP3 mRNA expression of sorted cell populations. FOXP3 mRNA was measured by real-time PCR in FACS sorted CD4 + CD25 hi , CD4 + CD25 lo and CD19 cells. Data was normalized to the expression in CD4 + CD25 lo cells using the 2 −ΔΔCt method and RPII as housekeeping gene. Data represent mean of triplicate samples from one single donor.

Article Snippet: CD4 + CD25 lo cells isolated from male donors were activated for 48 hours with anti-CD3/CD28 Dynabeads (Invitrogen) (proportion cell∶beads 1∶1) in AIM-V containing 10% HUS, 180 U rIL-2 and 1% Penicillin Streptamycin (PeSt), after which the stimulus was removed and cultures followed for an additional 14 days of culture in AIM-V containing 10% HUS, 180 U rIL-2 and 1% PeSt.

Techniques: Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans

doi: 10.1371/journal.pone.0001612

Figure Lengend Snippet: Plots showing results from one representative donor out of four analyzed. (A) FACS analysis of CD4 + CD25 lo cells stimulated at day 0 with CD3/CD28 Dynabeads (proportion cell∶beads 1∶1) in presence of 180 U IL-2. Stimuli removed after 48 h and cells followed for an additional 2 weeks with regards to their expression of CD25 (left) and FOXP3 (middle). Left column demonstrating the relationship between CD25 (y-axis) and FOXP3 (x-axis) expression. (B) Expression of CD25 (grey filled bars) and FOXP3 (open bars) in stimulated cells as described above, displayed as mean fluorescent intensity (MFI).

Article Snippet: CD4 + CD25 lo cells isolated from male donors were activated for 48 hours with anti-CD3/CD28 Dynabeads (Invitrogen) (proportion cell∶beads 1∶1) in AIM-V containing 10% HUS, 180 U rIL-2 and 1% Penicillin Streptamycin (PeSt), after which the stimulus was removed and cultures followed for an additional 14 days of culture in AIM-V containing 10% HUS, 180 U rIL-2 and 1% PeSt.

Techniques: Expressing

Journal: PLoS ONE

Article Title: FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans

doi: 10.1371/journal.pone.0001612

Figure Lengend Snippet: The methylation level of activated cells derived from the CD25 lo population was monitored with the COBRA based analysis described in . (A) Cultures from three separate donors were setup and stimulated as in , after which the methylation status in the CD25 hi and CD25 lo fraction was monitored for 16 days. Cultures of T regulatory cells from two donors were kept in parallel and likewise analyzed with respect to methylation at day 0 as well as indicated time points. Shown are mean values±SEM. (B) FOXP3 expression in the CD25 hi and CD25 lo cells isolated at day 0 was determined by intracellular flow cytometry (left graph). During activation, FOXP3 expression was also determined in the sorted populations derived from stimulated CD25 lo cells (right graph). Data represent mean values from three separate donors±SEM. (C) CD25 hi and CD25 lo cells from three donors were isolated on day 5 post-activation. To examine the suppressive capacity of these activated CD25 lo derived cells, they were put into co-culture with autologous CFSE-stained non-activated CD4 + T cells in the presence of anti-CD3 and anti-CD28 antibodies, and autologous CD4 − cells as feeder cells. A control sample was included for each donor where only responder and feeder cells were included in the culture. Suppressive activity was evaluated on day 3 of co-culture as the mean fluorescence intensity ratio of CFSE + responder cells in each sample relative the control sample.

Article Snippet: CD4 + CD25 lo cells isolated from male donors were activated for 48 hours with anti-CD3/CD28 Dynabeads (Invitrogen) (proportion cell∶beads 1∶1) in AIM-V containing 10% HUS, 180 U rIL-2 and 1% Penicillin Streptamycin (PeSt), after which the stimulus was removed and cultures followed for an additional 14 days of culture in AIM-V containing 10% HUS, 180 U rIL-2 and 1% PeSt.

Techniques: Methylation, Derivative Assay, Combined Bisulfite Restriction Analysis Assay, Expressing, Isolation, Flow Cytometry, Activation Assay, Co-Culture Assay, Staining, Activity Assay, Fluorescence

Journal: PLoS ONE

Article Title: FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans

doi: 10.1371/journal.pone.0001612

Figure Lengend Snippet: CD25 and FOXP3 expression was monitored in CD4 + CD25 lo cells during activation with (A) CD3/CD28 dynabeads as described in or repeated stimulation with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies every 7 days. CD4 + CD25 hi Tregs were also monitored as they were stimulated continuously with CD3/CD28 dynabeads. (B) CD4 + CD25 lo cells stimulated with high (10 µg/mL), medium (5 µg/mL) or low (0.5 µg/mL) anti-CD3 antibody together with 1 µg/mL anti-CD28 antibody. (C) CD4 + CD25 lo cells stimulated with anti-CD3 (5 µg/mL) and anti-CD28 (1 µg/mL) antibodies together with TGF-β (5 ng/mL), TGF-β (5 ng/mL) and IL-10 (10 ng/mL) or IL-10 only (10 ng/mL). Stimuli were removed on day 2 after stimulation and FACS analysis performed on days 0, 2, 5, 7, 10, 14 and 21. Data represent mean values from three separate donors±SEM, except for expanded Tregs where data represent mean values from two separate donors. Methylation status of the −77 reporter position was monitored during conditions described above (D–F). Evaluation of methylation status was performed on DNA from one donor at days 0, 7, 14 and 21 for each separate population. Methylation of expanded Tregs at day 7, 12 and 16 represent mean values from two separate donors.

Article Snippet: CD4 + CD25 lo cells isolated from male donors were activated for 48 hours with anti-CD3/CD28 Dynabeads (Invitrogen) (proportion cell∶beads 1∶1) in AIM-V containing 10% HUS, 180 U rIL-2 and 1% Penicillin Streptamycin (PeSt), after which the stimulus was removed and cultures followed for an additional 14 days of culture in AIM-V containing 10% HUS, 180 U rIL-2 and 1% PeSt.

Techniques: Expressing, Activation Assay, Methylation