Journal: Cell Death & Disease

Article Title: Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

doi: 10.1038/cddis.2016.346

Figure Lengend Snippet: EHF regulates the expression of HER receptors and the activities of their downstream signaling pathways in gastric cancer cells. ( a ) qRT-PCR assay was used to evaluate mRNA expression of EHF and HER receptors in primary gastric cancers ( n =30). Linear regression analysis was performed to assess the correlations between them. 18S rRNA was used as a normalized control. ( b ) qRT-PCR assay was performed to investigate the effect of EHF knockdown on the expression of HER receptors. Expression levels of these genes were normalized with 18S rRNA levels. Data were presented as mean±S.E. ( c ) The effect of EHF depletion on the expression of HER2-4 was determined in the indicated cells by western blot analysis. GAPDH was used as loading control. ( d ) Cells transfected with si-EHF-979 or si-NC were lysed and lysates were subjected to western blot analysis. The antibodies against phospho-Erk (p-Erk), total Erk (t-Erk), phospho-Akt (p-Akt) and total Akt (t-Akt) were used to determine the effect of EHF knockdown on the activities of the MAPK/Erk and PI3K/Akt cascades. GAPDH was used as a loading control

Article Snippet: The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

doi: 10.1038/cddis.2016.346

Figure Lengend Snippet: EHF is identified as a new HER2 transcription factor and the modulator of HER3 and HER4 in gastric cancer. ( a ) BGC823 cells were transiently transfected with pGL3-Basic or luciferase reporter constructs containing various lengths of the promoter region of HER2 gene, as indicated (F1: −607/+11; F2: −175/+11; F3: −607/−175) (left panels). Cotransfection with empty vector was used as a control. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays (right panels). ( b ) The luciferase reporter gene assay was performed to evaluate the effect of EHF knockdown on promoter activity of HER2 in BGC823 cells. The ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( c ) HEK293T cells were cotransfected pGL3-HER2-Luc-F1 and various amounts of pcDNA3.1(-)A-EHF or empty vector, respectively. Promoter activities of HER2 were measured by luciferase reporter gene assays. All the ratio of the Luc/Renilla activity is shown as means±S.E. of three independent assays. ( d ) Putative promoter regions of HER2 (−607/+11), HER3 (−997/+440) and HER4 (−697/+306) were inserted into the pGL3-Basic to construct the luciferase reporter plasmid pGL3-HER2-Luc, pGL3-HER3-Luc and pGL3-HER4-Luc (upper panels). P1-P7 represent the regions analyzed by ChIP assays for HER2 , HER3 and HER4 , respectively. BGC823 cells were transiently transfected with pcDNA3.1/myc-His(-)A-EHF or empty vector, and were subjected to ChIP-qRT-PCR assays using anti-Myc tag antibody. Flod enrichment was shown as means±S.E. of three independent assays (lower panels). ( e ) EMSA assay was performed to confirm the interaction between EHF and HER2 promoter. Shown are specific DNA-binding of in vitro translated EHF protein to an oligonucleotide sequence (SH2) containing ETS responsive element (GAGGAA) from the HER2 promoter. Unlabeled mutated probes contain specific mutations in the GGAA ETS core or flanking nucleotides of core sequence, as indicated by MT1 and MT2. Unlabeled wild-type (WT) and mutated (MT1 or MT2) competitor probes were added at 100-fold molar excess. Statistically significant differences were indicated: * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc).

Techniques: Transfection, Luciferase, Construct, Cotransfection, Plasmid Preparation, Activity Assay, Reporter Gene Assay, Quantitative RT-PCR, Binding Assay, In Vitro, Sequencing

Journal: Cell Death & Disease

Article Title: Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer

doi: 10.1038/cddis.2016.346

Figure Lengend Snippet: HER2 depletion attenuates proliferation-promoting effect of EHF in gastric cancer cells. Inhibitory effect of HER2 depletion on cell proliferation in GES-1 and MGC803 cells overexpressing EHF were evaluated by MTT assay. The data were presented as mean±S.E. Statistically significant differences were indicated: * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc).

Techniques: MTT Assay

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

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: The CD40-CD154 co-stimulation pathway mediates innate immune injury in adriamycin nephrosis.

doi: 10.1093/ndt/gfp569

Figure Lengend Snippet: Fig. 4. CD40 expression in mouse kidney. Immunohistochemical staining of frozen kidney sections demonstrated marked upregulation of CD40 expression in ADR+IgG mice (B) compared with ADR+MR1 (C) and normal mice (A). Quantitation was performed by a blinded observer by counting the number of stained cells per high power field in at least five sections per mouse (D). To identify which cells express CD40, kidney sections were stained for F4/80 (primary ab: rat anti-mouse F4/80, secondary ab: rhodamine conjugated rabbit anti-rat IgG) and CD40 (primary ab: goat anti-mouse CD40, secondary ab: FITC-conjugated donkey anti-goat IgG). Whole kidney CD40 mRNA expression correlated with interstitial volume (E) but not with glomerulosclerosis or tubular cell height (data not shown). Some sections as illustrated in F showed lack of co-localization of staining, whilst there was co-localization of staining (yellow colour) (G) in other parts of the kidney. Therefore, CD40 is expressed on both F4/80 positive and negative cells in vivo. F4/80 staining alone (H) is shown for comparison. CD40 was not expressed in glomeruli (I). White arrows—glomeruli, blue arrows—double stained cells (yellow) positive for CD40 and F4/80.

Article Snippet: Soluble CD154 levels were measured in culture supernatants using the soluble CD40 ligand ELISA kit (R&D systems, MN, USA) following the manufacturer's instructions.

Techniques: Expressing, Immunohistochemical staining, Staining, Quantitation Assay, In Vivo, Comparison

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: The CD40-CD154 co-stimulation pathway mediates innate immune injury in adriamycin nephrosis.

doi: 10.1093/ndt/gfp569

Figure Lengend Snippet: Fig. 5. CD40 expression in vitro. Single cell suspensions of primary renal tubular epithelial cells (RTEC), macrophages (J774 cell line) and mesangial cells (CRL-1927 cell line) were stained for PE-CD40 or isotype control and analysed by flow cytometry. RTEC constitutively expressed high levels of CD40 (A), whereas macrophages (B) and mesangial cells (C) expressed only low amounts of CD40. Interferon-γ stimulation upregulated CD40 on macrophages (strongly) and mesangial cells (weakly) but not RTEC. Data show percentage of cells staining for PE-CD40.

Article Snippet: Soluble CD154 levels were measured in culture supernatants using the soluble CD40 ligand ELISA kit (R&D systems, MN, USA) following the manufacturer's instructions.

Techniques: Expressing, In Vitro, Staining, Control, Flow Cytometry

Journal: PloS one

Article Title: Activated human T cells secrete exosomes that participate in IL-2 mediated immune response signaling.

doi: 10.1371/journal.pone.0049723

Figure Lengend Snippet: Figure 3. Cytokine production from autologous exosome stimulated CD3+ T cells at day zero (0 h) and day five (120 h). Relative quantification of spot intensities was performed using Quantity One software (Bio-Rad). Each bar represents an average of the intensity from two protein spots. White bars represent 0 h and grey bars represent 120 h (day 5). The exosomes appeared to contain significant amounts of CCL5 (RANTES) immediately after the addition of exosomes (at 0 h) since the supernatants showed relatively large amounts of RANTES. These levels were decreased at day five. doi:10.1371/journal.pone.0049723.g003

Article Snippet: Analysis of cytokines in the supernatant was carried out using Proteome ProfilerTMArray, Human Cytokine Array Panel A (cat#ARY005, R&D Systems Europe) according to manufacturer’s instructions.

Techniques: Quantitative Proteomics, Software

Journal: PloS one

Article Title: Activated human T cells secrete exosomes that participate in IL-2 mediated immune response signaling.

doi: 10.1371/journal.pone.0049723

Figure Lengend Snippet: Figure 5. Cytokine production from IL-2 stimulated CD3+ T cells at day zero (0 h) and day five (120 h). Relative quantification of spot intensities was performed using Quantity One software (Bio- Rad). Each bar represents an average of the intensity from two protein spots. White bars represent 0 h and grey bars represent 120 h (day 5). Cytokines IL-5, MIF, and GM-CSF (CSF2) were present at a high level in the supernatant after five days. doi:10.1371/journal.pone.0049723.g005

Article Snippet: Analysis of cytokines in the supernatant was carried out using Proteome ProfilerTMArray, Human Cytokine Array Panel A (cat#ARY005, R&D Systems Europe) according to manufacturer’s instructions.

Techniques: Quantitative Proteomics, Software

Journal: PloS one

Article Title: Activated human T cells secrete exosomes that participate in IL-2 mediated immune response signaling.

doi: 10.1371/journal.pone.0049723

Figure Lengend Snippet: Figure 6. Cytokine production from exosome+IL-2 stimulated CD3+ T cells at day zero (0 h) and day five (120 h). Relative quantification of spot intensities was performed using Quantity One software (Bio-Rad). Each bar represents an average of the intensity from two protein spots. White bars represent 0 h and grey bars represent 120 h (day 5). The cytokines IL-5, IL-13 and GM-CSF as well as the chemokines CCL3 and CCL4 were present at higher levels at day five. doi:10.1371/journal.pone.0049723.g006

Article Snippet: Analysis of cytokines in the supernatant was carried out using Proteome ProfilerTMArray, Human Cytokine Array Panel A (cat#ARY005, R&D Systems Europe) according to manufacturer’s instructions.

Techniques: Quantitative Proteomics, Software