Journal: Epigenetics

Article Title: Genome-wide oxidative bisulfite sequencing identifies sex-specific methylation differences in the human placenta

doi: 10.1080/15592294.2018.1429857

Figure Lengend Snippet: Hierarchical clustering of tissues based on the relative expression level of CSMD1 exons. Each column represents a tissue and each row represents an Ensembl CSMD1 exon. The relative expression is measured in the log-scale of FPKM (see Methods) and it is colored from red (high) to dark blue (low). The columns (tissues) are hierarchically clustered by the similarity of the expression pattern and the rows (exons) are ordered by the position of exons within the CSMD1 transcript: transcript-start (top) to transcript-end (bottom). Except the placenta (this study), the RNA-Seq datasets of other tissues are from the following three participating members of NIH Roadmap Epigenomics Mapping Consortium: Schultz et al. (GEO accession: GSE16256), UCSF-UBC (GEO accession: GSE16368), and Broad Institute (GEO accession: GSE17312). Exons that contain canonical and placenta-specific transcription start site (TSS) are shown in blue and red, respectively.

Article Snippet: Other Roadmap [ ] whole-genome bisulfite sequencing datasets generated at the Broad Institute and UCSF-UBC were downloaded from the GEO website ( https://www.ncbi.nlm.nih.gov/geo/roadmap/epigenomics ), as follows: fetal muscle (fMU, GSM1172596); fetal thymus (fMU, GSM1172595); hippocampus (HC, GSM1112838 and GSM916050); brain germinal matrix (GM, GSM941747); breast luminal epithelial (LE, GSM1127125), and testis spermatozoa (SP, GSM1127117).

Techniques: Expressing, RNA Sequencing

Journal: Journal of biomedical science

Article Title: SLCO4A1-AS1 promotes colorectal tumourigenesis by regulating Cdk2/c-Myc signalling.

doi: 10.1186/s12929-022-00789-z

Figure Lengend Snippet: Fig. 2 Promoter hypomethylation promotes SLCO4A1-AS1 expression in CRC. a Schematic map illustrating a predicted CpG island and its DNA methylation probes in the promoter of SLCO4A1-AS1. TSS, transcription start site. b The β-value of methylation of SLCO4A1-AS1 was lower in tumour samples than in normal tissues according to the CRC dataset of TCGA. The β-value of methylation of SLCO4A1-AS1 was linearly related to SLCO4A1-AS1 expression in CRC tissues from TCGA (c) and cell lines from CCLE (d). e Relative expression of SLCO4A1-AS1 in CRC cell lines was measured using qRT-PCR (the left panel). The methylation levels of SLCO4A1-AS1 in CRC cell lines and leukocyte cells were determined by bisulfite sequencing PCR. A total of 5 individual clones were randomly picked for sequencing (the right panel). f The mean methylation levels of these CpG sites were negatively associated with the expression levels of SLCO4A1-AS1 in CRC cells. g SLCO4A1-AS1 expression in CRC cells treated with DNA methyltransferase inhibitor (5-aza-dC). h DNA methylation analyses of SLCO4A1-AS1 in paired CRC tissues and noncancerous tissues using methylation-specific PCR assay. N, adjacent noncancerous tissue; T, tumour tissue; M, DNA marker

Article Snippet: Genomic DNA was extracted from cancer cells or human leukocytes using a Genomic DNA Mini Preparation Kit (Beyotime, China) and then bisulfite-modified using an EpiJET Bisulfite Conversion Kit (Thermo Fisher, USA).

Techniques: Expressing, DNA Methylation Assay, Methylation, Quantitative RT-PCR, Methylation Sequencing, Clone Assay, Sequencing, Marker

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Promoter DNA demethylation of Keap1 gene in diabetic cardiomyopathy

doi:

Figure Lengend Snippet: The bisulphate DNA sequencing analysis of Keap1 promoter DNA in remote, peri-infarct zones in myocardium of non-diabetic (NDM) and type-2 diabetic (DM-T2). A. Represent the methylation status of CpG dinucleotides in Keap1 promoter region (between -430 and -110); B. Represent the percentage of demethylation calculated from the bisulphate DNA sequencing.

Article Snippet: The genomic DNA isolated from biopsy samples were subjected to bisulfite conversion by EZ DNA Methylation-Direct™ kit (Zymo Research Corporation, Orange, CA).

Techniques: DNA Sequencing, 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: 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