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: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A and B) OSS and Wnt/β-catenin signaling do not enhance FOXC2 and GATA2 expression in human umbilical vein endothelial cells (HUVECs). Primary human lymphatic endothelial cells (HLECs) or HUVECs were cultured under OSS for 48 hr (A) or in the presence of recombinant Wnt3a (rWnt3a) for 24 hr (B). Subsequently, RNA was extracted and analyzed by real-time qPCR for AXIN2, FOXC2 , and GATA2 . The expression levels were normalized to that of GAPDH. (C and D) PROX1 provides competence to HUVECs to respond to OSS and Wnt/β-catenin signaling and enhance the expression of FOXC2 and GATA2. HUVECs were infected with lentiviral particles expressing GFP or human PROX1 for 48 hr. Subsequently, the cells were exposed to (C) OSS for 48 hr or to (D) the Wnt agonist BIO for 12 hr. (C) PROX1-expressing HUVECs were cultured under static or OSS conditions with or without the Wnt antagonist iCRT3. Western blot was performed to quantify FOXC2 expression. (D) PROX1-expressing HUVECs were cultured with BIO or DMSO for 24 hr, and the expression of FOXC2 and GATA2 was quantified by real-time qPCR. (E and F) PROX1 is necessary for OSS- and Wnt/β-catenin signaling-mediated expression of FOXC2 and GATA2 in HLECs. HLECs were infected with lentiviral particles expressing shRNAs that target GFP (sh-Con) or PROX1 (sh-P1#1 and sh-P1#2) for 48 hr. Subsequently, HLECs were additionally cultured under OSS for 48 hr (E) or with rWnt3a for 24 hr (F). Real-time qPCR was performed to quantify the expression of FOXC2 and GATA2 . Statistics: n = 3 for all experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars in graphs represent ± SEM.

Article Snippet: Briefly, 1.0 X 10 7 HLECs (Lonza) at 90% confluency were treated with DMSO or Bio (0.5 μM) for 3 hours.

Techniques: Expressing, Cell Culture, Recombinant, Infection, Western Blot

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet: (A) PROX1 enhances the expression of Wnt/β-catenin signaling target genes in HLECs. HLECs were infected with lentiviral particles expressing shRNAs that target GFP (sh-GFP) or PROX1 (shPROX1#1 and sh-PROX1#2) for 72 hr. Subsequently, RNA was extracted, and real-time qPCR was performed for the expression of Wnt/β-catenin target genes. (B–D) PROX1 synergizes with β-catenin to enhance Wnt/β-catenin signaling. (B) 293T cells were co-transfected with TOPFlashor FOPFlash luciferase reporters with PROX1- and or β-catenin-expressing vectors. The TCF/LEF binding sites of TOPFLASH are inactivated to generate FOPFlash as a negative control for Wnt/β-catenin signaling. (C and D) 293T cells were co-transfected with TOPFlash- and PROX1-expressing vectors together with AXIN-expressing (C) or ΔN-TCF7L2expressing (D) plasmids. DN-TCF7L2 cannot interact with β-catenin and functions as a dominant-negative mutant. A Renilla luciferase-expressing plasmid was used as an internal control, and luciferase activities were measured 36 hr after transfection. (E–G) PROX1 associates with β-catenin. (E) 293T cells were transfected with Myc-tagged β-catenin and FLAG-tagged PROX1 plasmids. 48 hr later, the cell lysate was subjected to a co-immunoprecipitation assay using anti-FLAG antibody or anti-Myc antibody. The precipitates were probed by western blot using the indicated antibodies. (F) HLEC lysate was immunoprecipitated using anti-PROX1 antibody and probed by western blot using anti-PROX1 or anti-β-catenin antibodies. (G) HLECs were treated with BIO for the indicated number of hours. The lysates were analyzed as in (F). (H) HLECs were treated with DMSO or BIO (0.5 μM) for 3 hr, and ChIP was performed using anti-PROX1 antibody. qPCR was performed using primers flanking the TCF/LEF-binding site in the 3.5 kb location. As a negative control, primers flanking a TCF/LEF-binding site that is located at a more upstream location (5.5 kb) were used. Statistics: n = 3 for all experiments. **p < 0.01, ***p < 0.001. Error bars in graphs represent ± SEM.

Article Snippet: Briefly, 1.0 X 10 7 HLECs (Lonza) at 90% confluency were treated with DMSO or Bio (0.5 μM) for 3 hours.

Techniques: Expressing, Infection, Transfection, Luciferase, Binding Assay, Negative Control, Dominant Negative Mutation, Plasmid Preparation, Control, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

Journal: Cell reports

Article Title: Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling

doi: 10.1016/j.celrep.2018.09.049

Figure Lengend Snippet:

Article Snippet: Briefly, 1.0 X 10 7 HLECs (Lonza) at 90% confluency were treated with DMSO or Bio (0.5 μM) for 3 hours.

Techniques: Virus, Recombinant, Electron Microscopy, Transfection, SYBR Green Assay, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Software