Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Scientific Reports

Article Title: Evaluation of the upper airway microbiome and immune response with nasal epithelial lining fluid absorption and nasal washes

doi: 10.1038/s41598-020-77289-3

Figure Lengend Snippet: Common microbial ecology indices of the upper airway microbiome in children with paired samples based on 16S ribosomal RNA sequencing and according to sample type. ( A ) Grouped bar graphs of selected indices showing no differences in community richness between nasal filters and nasal washes at the amplicon sequence variant unit (ASV) level. ( B ) Box-and-whisker plots of selected indices showing no differences in community α-diversity between nasal filters than in nasal washes at the ASV level. ( C ) Principal coordinates analysis (PCoA) plot of Bray–Curtis dissimilarities showing no distinct clustering by sample type at the ASV level. The lines connect samples with their group centroids. ( A , B ) were generated with the R package ggplot2 version 3.0.0 ( https://cran.r-project.org/web/packages/ggplot2/index.html) ; ( C ) was generated in vegan version 2.5-2 ( https://cran.r-project.org/web/packages/vegan/index.html ) and minor aesthetic edits were performed with Inkscape version 1.0.

Article Snippet: First, eukaryotic DNA was depleted using the NEBNext Microbiome Enrichment Kit (New England Biolabs).

Techniques: RNA Sequencing Assay, Amplification, Sequencing, Variant Assay, Whisker Assay, Generated

Journal: Scientific Reports

Article Title: Evaluation of the upper airway microbiome and immune response with nasal epithelial lining fluid absorption and nasal washes

doi: 10.1038/s41598-020-77289-3

Figure Lengend Snippet: Common microbial ecology indices of the upper airway microbiome in adults with paired samples based on 16S ribosomal RNA sequencing and according to sample type. ( A ) Grouped bar graphs of selected indices showing higher community richness in nasal filters than in nasal washes at the amplicon sequence variant (ASV) level. ( B ) Box-and-whisker plots of selected indices showing higher community α-diversity in nasal filters than in nasal washes at the ASV level. ( C ) Principal coordinates analysis (PCoA) plot of Bray–Curtis dissimilarities showing distinct clustering by sample type at the ASV level. The lines connect samples with their group centroids. ( A , B ) were generated with the R package ggplot2 version 3.0.0 ( https://cran.r-project.org/web/packages/ggplot2/index.html ) ; ( C ) was generated in vegan version 2.5–2 ( https://cran.r-project.org/web/packages/vegan/index.html ) and minor aesthetic edits were performed with Inkscape.

Article Snippet: First, eukaryotic DNA was depleted using the NEBNext Microbiome Enrichment Kit (New England Biolabs).

Techniques: RNA Sequencing Assay, Amplification, Sequencing, Variant Assay, Whisker Assay, Generated

Journal:

Article Title: Egr-1 Regulates Expression of the Glial Scar Component Phosphacan in Astrocytes after Experimental Stroke

doi: 10.2353/ajpath.2008.070648

Figure Lengend Snippet: Egr-1 binds to the phosphacan promoter in vivo. A: HeLa cells were transiently transfected with Egr-1 full-length human cDNA and subjected to ChIP with anti-Egr-1 antibody or rabbit IgG isotype control. Specific primers were used to amplify the area containing the putative Egr-1 site (lanes 1), or a distal, 4.5-kb upstream fragment (lanes 2). Genomic DNA input shows that the primers amplify bands of the expected size. Immunoprecipitation of sheared chromatin (ChIP) with anti-Egr-1 (α-Egr-1) produces a band of stronger intensity as compared with the distal site product and to control isotype IgG. B: HeLa cells were stimulated for 2 hours with PMA and ionomycin to induce endogenous Egr-1 and subjected to ChIP with anti-Egr-1 antibody or rabbit IgG isotype control. Precipitated DNA fragments were amplified with the same primers as in A surrounding the Egr-1 site (lanes 1) or the distal area (lanes 2). Genomic DNA input shows that primers amplify bands of the expected size. Immunoprecipitation of sheared chromatin (ChIP) with anti-Egr-1 (α-Egr-1) produces a band of stronger intensity as compared with the distal site product and to control isotype IgG. C: Immunoprecipitated DNA from the analysis in B was amplified and quantified by real-time PCR using primers surrounding the Egr-1 binding site or the distal area. Fold differences in the amounts of PCR products obtained with anti-Egr-1 antibody relative to isotype controls were calculated as described in Materials and Methods. Average values with SEM from five independent experiments are depicted. There is ∼6- to 10-fold higher amplification of the Egr-1 site fragment compared with the control fragment from the distal area.

Article Snippet: The three fragments were amplified using human genomic DNA as template (prepared with the Wizard genomic DNA purification kit, Promega) and inserted into the pGL3 promoter vector (Promega).

Techniques: In Vivo, Transfection, Control, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay