Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: DNA methylation and isoform-specific expression within the promoter regions of FCGR3A and FCGR3B. A) Representation of the FCGR3 locus spanning approximately 100 kb of chromosome 1q23.3 displaying transcripts annotated in the RefSeq database (adapted from the UCSC Genome Browser). Below, promoter regions of FCGR3A and FCGR3B are enlarged displaying the various transcript variants annotated in the RefSeq database that differ in the 5′ region. MassARRAY amplicons (green bars) used are shown along with the positions of CpG dinucleotides (green markers). TSS, transcriptional start site. B) The MassARRAY EpiTYPER assay was used to interrogate the DNA methylation levels of CpGs across the promoter region of FCGR3A and FCGR3B in a gene-specific manner in sorted NK CD16a+ and CD16a- fractions. Methylation plots are aligned to enlargements of gene promoters (in B) according to the hg19 genome assembly. C, D) DNA methylation levels in NK cell lines and neutrophils, respectively. E) The expression ratio of FCGR3A to FCGR3B in CD16a+ NK cells and neutrophils as determined by the MassARRAY iPLEX assay. F) Quantitative RT-PCR analysis of 5′ transcript variants using primers specific for variant 1 or variants 3 and 2 indicating promoter usage in NK cells, neutrophils, and NK cell lines YT and NKL (both primer pairs do not distinguish FCGR3A vs. FCGR3B). D1-4 represents donor numbers 1–4. Expression values expressed relative to the average of four housekeeping (HK) genes.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: DNA Methylation Assay, Expressing, MassARRAY EpiTYPER assay, Methylation, Quantitative RT-PCR, Variant Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: Analysis of DNA methylation- and lineage-specific activity of FCGR3 promoter sequences. A) Illustration of FCGR3A and FCGR3B sequences cloned into luciferase constructs (black bars). B) Four separate promoter sequence fragments were cloned from either FCGR3A (left) or FCGR3B (right) and transfected in various cell lines. Luciferase assays showing sequence- and gene-specific activity (relative to empty-vector control). Pmed1-A and Pmed1-B were additionally methylated in vitro prior to transfection. Error bars represent SEM of n=3 individual experiments; ND, not done. C) Sequence alignment of Pmed1-A and Pmed1-B with numbered CpGs. All sequence variants are enlarged below; an 8 bp repeat occurring in the vicinity of CpG#6 is highlighted in blue, asterisks below the sequence indicate homology. D) Luciferase activity following site-directed mutagenesis of CpG#s 1&2 of Pmed1-A compared to unaltered FCGR3A and FCGR3B sequences in YT and K562 cells.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: DNA Methylation Assay, Activity Assay, Clone Assay, Luciferase, Construct, Sequencing, Transfection, Plasmid Preparation, Control, Methylation, In Vitro, Mutagenesis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: Identification of miR-218 as a potential regulator of FCGR3A. (A) Expression ratio of predicted miRNAs that were present in the miRNA expression array comparing CD16a+ and CD16a- NK cells freshly isolated from adult peripheral blood. A ratio <1 indicates low expression in CD16a+ NK cells while a ratio >1 indicates high expression in CD16a+ NK cells. (B) Predicted miRNA regulators of FCGR3A have putative binding sites in the FCGR3A 3′ UTR. (C) Validation of expression of predicted miRNA regulators of FCGR3A by qPCR (n=3). (D) Luciferase expression as a ratio of firefly/renilla for each expression vector (n=2). Data are presented as mean±SD, * indicates p<0.05.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: Expressing, Isolation, Binding Assay, Biomarker Discovery, Luciferase, Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development 1

doi: 10.4049/jimmunol.1701128

Figure Lengend Snippet: MiR-218 negatively regulates CD16a in primary human NK cells. Primary human NK cells were enriched by magnetic selection to >70% purity and infected with lentivirus containing either miR-218 or empty vector. 48hr after infection, NK cells were sorted as live/CD3-/CD56+/GFP+ lymphocytes. (A) Representative histogram plot (of one of six donors) of CD16a expression in live/CD3-/CD56+/GFP+ primary human NK cells infected with miR-218 or empty vector virus. (B) CD16a expression in primary human NK cells infected with miR-218 or empty vector virus (* indicates p=0.05). (C) Validation of miR-218 over-expression by real-time PCR (** indicates p<0.01). (D) FCGR3A mRNA expression assessed by real-time RT-PCR in sorted NK cells infected with either miR-218 or empty vector (*** indicates p<0.005). (B–D) Data are presented as mean±SD, n=6.

Article Snippet: Antibodies and flow cytometric analysis The following antibodies were used to stain human peripheral blood cells: CD3 (SK7, BD Biosciences), CD14 (TÜK4, Miltenyi), CD15 (VIMC6, Miltenyi), CD16 (VEP13, Miltenyi), CD16 (3G8, BD Biosciences), and CD56 (N901, Beckman Coulter).

Techniques: Selection, Infection, Plasmid Preparation, Expressing, Virus, Biomarker Discovery, Over Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Functional Significance and Therapeutic Potential of miRNA-20b-5p in Esophageal Squamous Cell Carcinoma

doi: 10.1016/j.omtn.2020.05.015

Figure Lengend Snippet: DNA Methylation Status of miR-20b-5p by MassARRAY (A) Methylation profile of CpG sites for the miR-20b-5p gene. The color of the circles is related to the percentage of methylation at each CpG site. Boxes indicate the different methylation patterns between 14 ESCC samples and the corresponding tissues. (B) Hierarchical cluster analysis of CpG site methylation profiles of the miR-20b-5p promoter region in esophageal squamous cell carcinoma (n = 14) and control esophageal tumors (n = 14). The color gradient between green and red indicates methylation of each miR-20b-5p unit in each sample ranging from 0% to 60%. Gray represents technically inadequate or missing data. (C) Evaluation of CpG methylation within the miR-20b-5p promoter. The distribution of the seven analyzed CpG units within miR-20b-5p is shown. Data are presented as the mean value ± SD from triplicate experiments. @@ p < 0.01.

Article Snippet: The methylation status was quantified for evaluation with the MassARRAY platform (Agena Bioscience, USA).

Techniques: DNA Methylation Assay, Methylation, Control, CpG Methylation Assay

Journal: Cell Death & Disease

Article Title: MBD2 facilitates tumor metastasis by mitigating DDB2 expression

doi: 10.1038/s41419-023-05804-1

Figure Lengend Snippet: A The B16F10 melanoma tumor volume in each group was determined at the indicated time points. B Images of tumor nodules at the endpoint of the experiment. C The weight of the tumor nodules from each group at the endpoint of the experiment. D Western blot analysis of MBD2 in each group. E , F RT-PCR analysis of Cdh1 ( E ) and Cdh2 ( F ) in the indicated groups. G Representative images of E-cadherin and N-cadherin coimmunostaining in the tumor nodule sections. H Images of pulmonary metastatic nodules from the indicated groups. I Representative H&E staining of pulmonary metastatic nodules from the indicated groups. J MBD2 binds to methylated CpG DNA within the DDB2 promoter and thus represses DDB2 expression, resulting in tumor metastasis. Intravenous administration of liposomes carrying MBD2 siRNA suppressed tumor growth in an animal model bearing tumor metastasis. L-Scr siRNA: liposome-based Scrambled siRNA; L- MBD2 siRNA: liposome-based MBD2 siRNA. The data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Global DNA methylation was evaluated by a MethylFlashTM Methylated DNA Quantification Kit (Epigentek, NY, USA) according to the instructions and bisulfite DNA sequencing was carried by Sequenom MassARRAY platform (BGI.write, Beijing, China) as previously reported [ ].

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Methylation, Expressing, Liposomes, Animal Model

Journal: Cancer Research

Article Title: Xenoestrogen-Induced Epigenetic Repression of microRNA-9-3 in Breast Epithelial Cells

doi: 10.1158/0008-5472.can-08-4914

Figure Lengend Snippet: Figure 1. Preexposure of MDECs to diethylstilbestrol and immunofluorescence analysis of nuclear ER-a. A, treatment scheme. Breast progenitor cells were propagated as nonadherent spherical colonies, called mammospheres, and treated with 70 nmol/L diethylstilbestrol or DMSO solvent control for 3 wk. To induce differentiation, cells were seeded on a collagen substratum in the absence of diethylstilbestrol for 3 wk. Expression profiling (microRNA microarray), immunofluorescence image (IFA), and epigenetic [chromatin immunoprecipitation-PCR (ChIP-PCR) and MassARRAY] analyses were then done on the progeny epithelial cells. B, increased internalization of ER-a in diethylstilbestrol-preexposed MDECs. After the preexposure to diethylstilbestrol (DES) or DMSO, MDECs were subjected to immunofluorescence analysis. The percentage of subcellular localization of ER-a–positive cells, independently scored by two researchers, is shown in the bar chart. Columns, mean of five independent sets of MDEC samples; bars, SE. P < 0.001 (Student’s t test). C, nuclear trafficking of ER-a in MDECs on diethylstilbestrol treatment. DMSO-preexposed MDECs were exposed to 70 nmol/L diethylstilbestrol in the indicated time points. Translocation of ER-a protein (green) from the cytoplasm to the nucleus was observed, suggesting functional estrogen signaling. Nuclei were stained with 4¶,6-diamidino-2-phenylindole (DAPI; blue). The turquoise signals (merged) highlight the localization of ER-a in the nucleus.

Article Snippet: The MassARRAY platform was used to perform quantitative methylation analysis (Sequenom).

Techniques: Immunofluorescence, Solvent, Control, Expressing, Microarray, Chromatin Immunoprecipitation, Translocation Assay, Functional Assay, Staining