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: iScience

Article Title: METTL3 promotes peritoneal metastasis of colorectal cancer through regulating m6A modification of NRXN3 mRNA

doi: 10.1016/j.isci.2025.113165

Figure Lengend Snippet: METTL3 was upregulated in CRC peritoneal metastasis (A–C) IHC, RT-qPCR, and western blot analysis of METTL3 in CRC tissues and peritoneal metastasis tissues, Scale bar, 100 μm. (D and E) Me-ELISA and Me-dot blot analysis of the relative m6A levels in 24 CRC tissues and peritoneal metastasis tissues. (F) Western blot analysis of METTL3 protein levels in CT26-Luc cells after METTL3 was knocked down. (G) The images and quantification of the bioluminescence signals among mice upon intraperitoneal injection of CT26-Luc cells from Day 7 to Day 49 ( n = 3). (H) The survival curve of mice after intraperitoneal inoculation with METTL3 knockdown or negative control cells. (I) Images of parietal peritoneum and visceral peritoneum metastasis after the mice were sacrificed. Data are presented as SEM (A–E and G). Two-group comparisons were analyzed by Student’s t test (A–E and G), while survival differences were assessed using the log rank test (H). ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The EpiQuik m6A Methylation Quantification Kit (AmyJet Scientific, China) was utilized to quantitatively assess the total levels of m6A methylation in tissues or cells.

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Dot Blot, Injection, Knockdown, Negative Control

Journal: iScience

Article Title: METTL3 promotes peritoneal metastasis of colorectal cancer through regulating m6A modification of NRXN3 mRNA

doi: 10.1016/j.isci.2025.113165

Figure Lengend Snippet: METTL3-mediated m6A methylation sites existed in NRXN3 mRNA (A) MERIP-qPCR analysis of NRXN3 after silencing METTL3 . (B) RNA dot blot detection of the relative m6A levels in HCT116 and LoVo cells after silencing METTL3 . (C) The potential m6A site of NRXN3 mRNA via a m6A modification site predictor. (D) The structures of 3 high confidence m6A modification sites with “GGAC” motif in the NRXN3 CDS region. (E) Schematic diagram of m6A predicted sequence mutation. (F) Western blot detection of the protein level of METTL3 and FLAG after overexpression mutation. (G) RNA stability detection of NRXN3 mRNA after silencing METTL3 . Data are presented as mean ± SD (A, B, F, and G). Group differences were assessed using one-way ANOVA with Tukey’s test (A, B, and F) or two-way ANOVA with Tukey’s test (G). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. ns, not significant.

Article Snippet: The EpiQuik m6A Methylation Quantification Kit (AmyJet Scientific, China) was utilized to quantitatively assess the total levels of m6A methylation in tissues or cells.

Techniques: Methylation, Dot Blot, Modification, Sequencing, Mutagenesis, Western Blot, Over Expression

Journal: iScience

Article Title: METTL3 promotes peritoneal metastasis of colorectal cancer through regulating m6A modification of NRXN3 mRNA

doi: 10.1016/j.isci.2025.113165

Figure Lengend Snippet: METTL3 depended on YTHDC1 to regulate NRXN3 mRNA (A) Scatterplot of correlations between NRXN3 and m6A-readers in TCGA database. (B) Western blot detection of NRXN3α protein level after silencing m6A-readers. (C) Western blot detection of NRXN3 RNA pull-down results in HCT116 cell. (D) Stability detection of NRXN3 mRNA in HCT116 after silencing YTHDC1. (E) Western blot detection of protein levels of METTL3α, YTHDC1 and NRXN3 with indicated treatments. Data are presented as mean ± SD (B, D, and E). Pearson’s correlation to determine statistical significance of A, p value as shown. Differences were assessed using one-way ANOVA (B and E) or two-way ANOVA with Tukey’s test (D). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. ns, not significant.

Article Snippet: The EpiQuik m6A Methylation Quantification Kit (AmyJet Scientific, China) was utilized to quantitatively assess the total levels of m6A methylation in tissues or cells.

Techniques: Western Blot

Journal: Neuro-Oncology Practice

Article Title: Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival

doi: 10.1093/nop/npy028

Figure Lengend Snippet: Cox Regression Analysis of Overall Survival and Progression-Free Survival of 102 Primary Glioblastoma MGMT Unmethylated Patients With Substratification by MGMT Values

Article Snippet: Our patient cohort was derived retrospectively and consisted of newly diagnosed GBM patients seen at UCLA and KPLA between 2011 and 2016 with accessible LabCorp quantitative MGMT methylation values.

Techniques: Biomarker Discovery

Journal: Neuro-Oncology Practice

Article Title: Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival

doi: 10.1093/nop/npy028

Figure Lengend Snippet: Kaplan-Meier analysis is used to A, compare the MGMT (1-1.99) vs MGMT (<1) and MGMT (≥2) patients. The MGMT (1-1.99) group’s median OS (25.4 months) falls in between the MGMT (≥2) (38.8 months) and MGMT (<1) (17.3 months) median OS values (Log-rank P = .001). B, PFS showed the same trend, namely the MGMT (1-1.99) group generated a higher median OS of 11.8 months compared to the MGMT (<1) group but lower than the MGMT (≥ 2) group, yielding 11.8 months vs 7.92 months and 18.0 months (Log-rank P < .0001), respectively. MGMT indicates O-6-methylguanine-DNA methyltransferase; mo, months; OS, overall survival; PFS, progression-free survival.

Article Snippet: Our patient cohort was derived retrospectively and consisted of newly diagnosed GBM patients seen at UCLA and KPLA between 2011 and 2016 with accessible LabCorp quantitative MGMT methylation values.

Techniques: Generated

Journal: Neuro-Oncology Practice

Article Title: Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival

doi: 10.1093/nop/npy028

Figure Lengend Snippet: Cox Regression Analysis of Overall Survival and Progression-Free Survival of 165 Primary Glioblastoma Patients With Substratification by MGMT Values

Article Snippet: Our patient cohort was derived retrospectively and consisted of newly diagnosed GBM patients seen at UCLA and KPLA between 2011 and 2016 with accessible LabCorp quantitative MGMT methylation values.

Techniques: Biomarker Discovery