Journal: Journal of Virology

Article Title: Mechanical Barriers Restrict Invasion of Herpes Simplex Virus 1 into Human Oral Mucosa

doi: 10.1128/JVI.01295-17

Figure Lengend Snippet: Nectin-1 and HVEM are expressed in human oral epithelia and primary keratinocytes. (a) RNAs were isolated from epithelial sheets prepared from vestibular mucosa or from primary oral keratinocytes. RT-PCR demonstrates expression of nectin-1 and HVEM; as a control, GAPDH transcripts are shown. (b) Cells were dissociated from vestibular epithelial sheets by TrypLE Select treatment and stained for nectin-1. To investigate surface expression of HVEM, cells were dissociated from epithelial sheets by CDS treatment. Flow cytometric analyses indicate 92% and 40% of cells were positive for nectin-1 and HVEM, respectively. APC-A, allophycocyanin-conjugated antibody; FITC-A, fluorescein isothiocyanate-conjugated antibody. (c) Results from samples from 4 (nectin-1) or 3 (HVEM) different patients are shown as means ± standard deviation (SD), demonstrating that surface expression of HVEM is present on fewer cells than nectin-1.

Article Snippet: Cell suspensions prepared by TrypLE Select (Life Technologies) were incubated in PBS–5% FCS on ice for 45 min with mouse anti-nectin-1 (CK41; 1:100) ( 40 ), and nectin-1 was visualized with anti-mouse IgG-Cy5 (1:100 [Jackson ImmunoResearch Laboratories, Inc.]).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Staining, Standard Deviation

Journal: Immunology

Article Title: Identification of an HLA‐A*24:02‐restricted α ‐fetoprotein signal peptide‐derived antigen and its specific T‐cell receptor for T‐cell immunotherapy

doi: 10.1111/imm.13168

Figure Lengend Snippet: Functional analysis of KWV3.1‐transfected T cells. (a) Flow cytometric analysis of KWV3.1‐transfected T cells. The mRNA encoding KWV3.1 α‐ and β‐chains or buffer alone (control) were electroporated in T cells activated by CD3/CD28 beads. The electroporated T cells were stained with anti‐mouse T‐cell receptor‐β chain (mTRBC) antibody or AFP2–11/HLA‐A24 tetramer and analyzed on a flow cytometer. (b) Specific activation of KWV3.1‐transfected T cells against T2‐A24 cells pulsed with various concentrations of AFP2–11. Interferon‐γ (IFN‐γ) release was determined by elispot assay. The irrelevant peptide‐pulsed T2‐A24 and the unpulsed T2‐A24 were used as negative controls. (c) Specific activation of KWV3.1‐transfected T cells co‐cultured with tumor cell lines. KWV3.1‐transfected T cells were specifically activated by HepG2 (AFP+ HLA‐A24+) and SNU398‐AFP (AFP overexpressed, HLA‐A24+), but not by SNU398 (AFP− HLA‐A24+). IFN‐γ release was determined by elispot assay. A6 was used as a negative TCR control. (d) Cytotoxicity of KWV3.1‐transfected T cells against T2‐A24 loaded with varied concentrations of AFP2–11 peptide. Lactate dehydrogenase (LDH) assay was implemented at an effector : target ratio of 5 : 1. The A6‐transfected T cells incubated with T2‐A24 loaded with 10−6 m AFP2–11 were used as negative control. (e) The cytotoxic activity of KWV3.1‐transfected T cells against AFP‐expressing tumor cell lines at effector : target ratios of 1 : 1, 3 : 1, 5 : 1 and 10 : 1. A6 was used as a negative TCR control. Error bars indicate standard deviation of triplicate measurements

Article Snippet: Electroporation Before electroporation, the concentration of CD8 + T cells was adjusted to 5 × 10 5 cells/ml and stimulated by human anti‐CD3/CD28 dynabeads (Gibco) at a 2 : 1 ratio for 48 hr at 37° in complete RPMI‐1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin and 30 IU/ml IL‐2.

Techniques: Functional Assay, Transfection, Staining, Flow Cytometry, Activation Assay, Enzyme-linked Immunospot, Cell Culture, Lactate Dehydrogenase Assay, Incubation, Negative Control, Activity Assay, Expressing, Standard Deviation

Journal: World Journal of Gastroenterology

Article Title: Expression of exogenous rat collagenase in vitro and in a rat model of liver fibrosis

doi: 10.3748/wjg.v8.i5.901

Figure Lengend Snippet: RT-PCR assay of the mRNA expression of recombinant collagenase in NIH 3T3 cells. The PCR-amplified 652 bp fragment of recombinant collagenase cDNA derived from their respective mRNA was found in NIH 3T3 cells which were transfected with pCMV-RC-F (lane 1), while no signal was detected in cells which transfected with control plasmid pTargeTM (lane 2). Lane M is the molecular weight marker (DNA/Hind III, BamH I).

Article Snippet: Collagenase mRNA expression in rat liver Total RNA was isolated from the liver tissues using Trizol (Gibco BRL) according to the manufacturer's directions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Recombinant, Amplification, Derivative Assay, Transfection, Control, Plasmid Preparation, Molecular Weight, Marker

Journal: World Journal of Gastroenterology

Article Title: Expression of exogenous rat collagenase in vitro and in a rat model of liver fibrosis

doi: 10.3748/wjg.v8.i5.901

Figure Lengend Snippet: RT-PCR assay for the expression of recombinant interstitial collagenase in rat livers. The 652 bp PCR amplified fragment of recombinant collagenase cDNA which was derived from their respective mRNA could be detected in liver samples from pCMV-RIC-F plasmid transduced rat groups C1 (lane 1, 2) and C2 (lane 3, 4) but not in the normal control group A (lane 5, 6) and disease control group B (lane 7). 335 bp fragment was PCR amplified β-actin cDNA. Lane M was 100 bp-1200 bp DNA marker ladders.

Article Snippet: Collagenase mRNA expression in rat liver Total RNA was isolated from the liver tissues using Trizol (Gibco BRL) according to the manufacturer's directions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Recombinant, Amplification, Derivative Assay, Plasmid Preparation, Control, Marker

Journal: World Journal of Gastroenterology

Article Title: Expression of exogenous rat collagenase in vitro and in a rat model of liver fibrosis

doi: 10.3748/wjg.v8.i5.901

Figure Lengend Snippet: RT-PCR assay for the endogenous expression of collagenase in rat livers. The 753 bp PCR-amplified fragment of endogenous collagenase cDNA which was derived from their respective mRNA could be detected in all liver samples from the normal control groups A (lane 1) and disease control group B (lane 4), as well as pCMV-RIC-F plasmid transduced rat groups C1 (lane 3) and C2 (lane 4).335 bp fragment was PCR amplified β-actin cDNA. Lane M was 100 bp-1200 bp DNA marker ladders.

Article Snippet: Collagenase mRNA expression in rat liver Total RNA was isolated from the liver tissues using Trizol (Gibco BRL) according to the manufacturer's directions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Derivative Assay, Control, Plasmid Preparation, Marker

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Schematic showing the domain organization of the reference HER2-specific CAR constructs and modifications made to introduce programmed membrane protein (proMP) transmembrane domains (TMDs). Bold, boxed sequence indicates the human CD28 TMD in the reference CD28TM and no cys CARs and designed proMP sequences in the monomeric (proCAR-1), dimeric (proCAR-2), and trimeric (proCAR-3) receptors. ( b ) BW5147 murine thymoma cells stably expressing proCARs and a destabilized GFP NF-κB reporter were surface labeled with anti-Myc antibody and analyzed by flow cytometry to assess surface expression levels. ( c ) Live cells from ( b ) were coated with polyclonal anti-IgG to bind CARs through the scFv domain and immunoprecipitated using protein G beads. Products were separated by nonreducing SDS-PAGE and immunoblotted using anti-Myc antibody to visualize surface-expressed CAR proteins. Molecular weight of the unglycosylated CAR polypeptide is 55 kDa. ( d, e ) Cells from ( b ) were co-cultured with HER2+ SKBR3 human breast adenocarcinoma cells for the indicated times and analyzed by flow cytometry for upregulation of activation marker CD69 ( d ) and GFP expression from the NF-κB reporter ( e ). All activation levels are normalized to the 8 hr time point in cells expressing the CD28TM CAR (% CD28TM Max). Bars represent the mean ± SD, and dots show the individual data points for three independent experiments. ( f ) Maximum target killing percentage at 20:1 effector to target ratio from 4 hr 51 Cr release assay. Bars show mean ± SEM with each data point representing an individual experiment (n = 3). p-Values determined from paired t -tests. ( g ) Cytokine production by primary mouse HER2 proCAR T cells following 24 hr co-culture with MC57-HER2 target tumor cells. Bars show mean concentration ± SEM with each data point representing an individual experiment (n = 5). Significance was determined from one-way ANOVA with multiple comparisons. Cytokine production on antigen-negative parental MC57 cells shown separately in .

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Construct, Introduce, Sequencing, Stable Transfection, Expressing, Labeling, Flow Cytometry, Immunoprecipitation, SDS Page, Molecular Weight, Cell Culture, Activation Assay, Marker, Release Assay, Co-Culture Assay, Concentration Assay

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Model of the CD28TM interface generated by mutagenesis of the CD3ζ TMD (PDB: 2HAC). Polar residues of the CD28 dimerization motif (orange) with predicted hydrogen bonds depicted (dotted lines). ( b ) Surface expression and ( c ) SDS-PAGE and immunoblot analysis of HER2 CARs possessing WT CD28TM or CD28TM mutations depicted in ( a ) expressed in the BW5147 cell line. ( d ) Quantitation of target cell killing measured by chromium release assay and cytokine production by primary mouse CD8 + CAR T cells in response to the MC57-HER2 target cell line (n = 4). Experiments performed as in . p-Values determined by paired t -tests. ( e ) Representative immunofluorescent confocal images of CAR-CD28 co-clustering in primary mouse CAR T cells. CAR clustering was induced with anti-Myc primary followed by crosslinking with fluorescent secondary antibody (magenta). Cells were then labeled for CD28 (cyan). Images are Z-projections over 12 m, scale bar represents 3 m. ( f ) Quantitation of CAR-CD28 co-clustering, each dot representing the percentage of CAR clusters in one cell that co-localized with a CD28 cluster. Lines show mean CAR-CD28 co-clustering percentage/per cell ± SEM, n ≥ 30 cells. p-Values determined by unpaired t -tests.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Generated, Mutagenesis, Expressing, SDS Page, Western Blot, Quantitation Assay, Release Assay, Labeling

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Flow cytometry gating strategy to determine the transduction efficiency of primary murine CAR T cells. Lymphocytes selected via morphology, live cells selected as zombie aqua negative, T cells selected as CD3 + CD8 + and mCherry + cells defined as CAR T cells. c-Myc co-expression with mCherry indicates surface CAR expression. ( b ) Example 2D plots showing extracellular c-Myc labeling (y-axis) vs. intracellular mCherry (x-axis) of CD3 + CD8 + T cells on day 5 post-transduction with CD28TM CARs and ProCARs 1–3, demonstrating the percentage of cells expressing the CARs. Empty mCherry vector included as c-Myc-negative control.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Flow Cytometry, Transduction, Expressing, Labeling, Plasmid Preparation, Negative Control

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: Example 2D plots showing extracellular c-Myc labeling (y-axis) vs. intracellular mCherry (x-axis) of CD3 + CD8 + T cells on day 5 post-transduction with CD28TM chimeric antigen receptors (CARs) and proCAR-4, demonstrating the percentage of cells expressing the CARs. Empty mCherry vector included as c-Myc-negative control.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Labeling, Transduction, Expressing, Plasmid Preparation, Negative Control

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Flow Cytometry, Fluorescence, Microscopy, Recombinant, Sequencing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Modification, Software