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: Heliyon

Article Title: Epigenetic regulation of FOXI2 promotes clear cell renal cell carcinoma progression

doi: 10.1016/j.heliyon.2024.e29218

Figure Lengend Snippet: Hypermethylation in CpG sites of FOXI2 in six paired ccRCC tissues (A, B) methylation state in FOXI2 promoter determined by Sequenom MassARRAY analysis in six paired ccRCC and adjacent normal tissues. Red arrows show profile of several methylation of CpG sites in the FOIX2 promoter region, with significant differences between tumor and normal tissues, p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Quantitative methylation analysis of the FOXI2 promoter region was conducted using the Sequenom MassARRAY platform (oebiotech, Shanghai, China), following the manufacturer's protocol.

Techniques: Methylation

Journal: International Journal of Molecular Sciences

Article Title: DNA Demethylation Induces Tree Peony Flowering with a Low Deformity Rate Compared to Gibberellin by Inducing PsFT Expression under Forcing Culture Conditions

doi: 10.3390/ijms23126632

Figure Lengend Snippet: Effect of 5-azaC and GA treatments on DNA methylation levels in the promoter regions of flowering-pathway-related genes and photosynthetic analysis. ( A ) Related expression of DNA methylation regulation genes PsDRM , PsCMT, and PsMET by RT-qPCR (Duncan’s test at p < 0.05, error bars indicate the ±SD and different letters for each day indicate significant differences, n = 3). ( B ) DNA methylation level in the promoter of flowering-pathway-related genes determined by McrBC-PCR; + and − indicate with and without GTP, respectively, in the digestion solution ( n = 3, representative results shown here). ( C ) DNA methylation in the promoter of PsFT based on MassARRAY (Duncan’s test at p < 0.05, error bars indicate the ±SD and n = 3). ( D ) CG context prediction 3 kb before the ATG site in the PsFT promoter and the cis -element prediction in the tested region. The demethylation level in the above CGs 1–2 and 3–5 are also shown here. ( E ) Morphological levels used for photosynthetic determination. ( F ) Analysis of net photosynthetic rate (Pn), stomatic conductance (Gs), intercellular CO 2 concentration (Ci), and transpiration rate (Tr) in the third leaf of each plant at 28 d. (Duncan’s test at p < 0.05 after analysis of variance; data are shown as the mean ±SD, and the different letters indicates significant differences among different treatments, n = 10).

Article Snippet: Quantitative methylation analysis of bisulfite-treated genomic DNA was conducted through Agena’s MassARRAY EpiTYPER system (Agena Bioscience, San Diego, CA, USA).

Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR, Concentration Assay