Structured Review

Becton Dickinson cd45
BMSC characterization. A: control group. B: BMSCs were positive for CD29 and negative for <t>CD45.</t> C: BMSCs were positive for CD29 and negative for CD34.
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1) Product Images from "Effects of PB-EPCs on Homing Ability of Rabbit BMSCs via Endogenous SDF-1 and MCP-1"

Article Title: Effects of PB-EPCs on Homing Ability of Rabbit BMSCs via Endogenous SDF-1 and MCP-1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0145044

BMSC characterization. A: control group. B: BMSCs were positive for CD29 and negative for CD45. C: BMSCs were positive for CD29 and negative for CD34.
Figure Legend Snippet: BMSC characterization. A: control group. B: BMSCs were positive for CD29 and negative for CD45. C: BMSCs were positive for CD29 and negative for CD34.

Techniques Used:

2) Product Images from "Monitoring Caspase-3 Activation with a Multimodality Imaging Sensor in Living Subjects"

Article Title: Monitoring Caspase-3 Activation with a Multimodality Imaging Sensor in Living Subjects

Journal:

doi: 10.1158/1078-0432.CCR-07-5244

Serial bioluminescence imaging of caspase-3 activation by staurosporine and UCN-01 in living mice
Figure Legend Snippet: Serial bioluminescence imaging of caspase-3 activation by staurosporine and UCN-01 in living mice

Techniques Used: Imaging, Activation Assay, Mouse Assay

Serial bioluminescence imaging of caspase-3 activation by staurosporine and UCN-01 in living mice
Figure Legend Snippet: Serial bioluminescence imaging of caspase-3 activation by staurosporine and UCN-01 in living mice

Techniques Used: Imaging, Activation Assay, Mouse Assay

Induction in mRFP1, FL, and TK activities through cleavage of the MTF fusion protein by activated caspase-3, as measured by FACS, enzyme assays, Western blot analysis, and fluorescence microscopy in response to different doses of staurosporine. A, FACS
Figure Legend Snippet: Induction in mRFP1, FL, and TK activities through cleavage of the MTF fusion protein by activated caspase-3, as measured by FACS, enzyme assays, Western blot analysis, and fluorescence microscopy in response to different doses of staurosporine. A, FACS

Techniques Used: FACS, Western Blot, Fluorescence, Microscopy

Indirect measure of caspase-3 activation and cell viability from B16F10-mtf-hrl cells by fl and hrl gene expression at different doses of staurosporine treatments. A, imaging of B16F10-mtf-hrl cells for fl and hrl gene expression from control and staurosporine-treated
Figure Legend Snippet: Indirect measure of caspase-3 activation and cell viability from B16F10-mtf-hrl cells by fl and hrl gene expression at different doses of staurosporine treatments. A, imaging of B16F10-mtf-hrl cells for fl and hrl gene expression from control and staurosporine-treated

Techniques Used: Activation Assay, Expressing, Imaging

Multimodality imaging of FL and TK activation from caspase-3 – cleaved MTF fusion protein from the B16F10mtf-hrl tumors treated with staurosporine by bioluminescence and microPET in living mice. A, bioluminescence and microPET imaging of a living
Figure Legend Snippet: Multimodality imaging of FL and TK activation from caspase-3 – cleaved MTF fusion protein from the B16F10mtf-hrl tumors treated with staurosporine by bioluminescence and microPET in living mice. A, bioluminescence and microPET imaging of a living

Techniques Used: Imaging, Activation Assay, Mouse Assay

Graphical representation of the ratio metric analyses of serial bioluminescence imaging of the B16F10mtf-hrl tumors treated with staurosporine and UCN-01. A, ratiometric analysis of bioluminescence imaging of B16F10mtf-hrl tumors treated with staurosporine.
Figure Legend Snippet: Graphical representation of the ratio metric analyses of serial bioluminescence imaging of the B16F10mtf-hrl tumors treated with staurosporine and UCN-01. A, ratiometric analysis of bioluminescence imaging of B16F10mtf-hrl tumors treated with staurosporine.

Techniques Used: Imaging

3) Product Images from "A Single-Amino-Acid Substitution in Herpes Simplex Virus 1 Envelope Glycoprotein B at a Site Required for Binding to the Paired Immunoglobulin-Like Type 2 Receptor ? (PILR?) Abrogates PILR?-Dependent Viral Entry and Reduces Pathogenesis ▿"

Article Title: A Single-Amino-Acid Substitution in Herpes Simplex Virus 1 Envelope Glycoprotein B at a Site Required for Binding to the Paired Immunoglobulin-Like Type 2 Receptor ? (PILR?) Abrogates PILR?-Dependent Viral Entry and Reduces Pathogenesis ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01166-10

Effect of heparin on recombinant virus adsorption. Subconfluent Vero cells were infected with YK333 (wild-type/EGFP), YK704 (gB-T53A/EGFP), or YK705 (gB-TA-repair/EGFP) (A) or with YK333 (wild-type/EGFP), YK708 (gB-T53/480A/EGFP), or YK709 (gB-TATA-repair/EGFP) (B) at an MOI of 0.1 at 4°C. After 2 h of adsorption at 4°C, cells were washed three times at 4°C with medium 199 supplemented with 1% FCS and 0, 1, 10, or 100 μg heparin/ml. Medium 199 supplemented with 1% FCS, at 37°C, was then added, and the cells were placed into a 37°C incubator. At 10 h postinfection, EGFP-positive cells were determined by using a FACSCalibur instrument. These results are the averages and standard errors of data from three independent experiments, calculated relative to the sample with no heparin.
Figure Legend Snippet: Effect of heparin on recombinant virus adsorption. Subconfluent Vero cells were infected with YK333 (wild-type/EGFP), YK704 (gB-T53A/EGFP), or YK705 (gB-TA-repair/EGFP) (A) or with YK333 (wild-type/EGFP), YK708 (gB-T53/480A/EGFP), or YK709 (gB-TATA-repair/EGFP) (B) at an MOI of 0.1 at 4°C. After 2 h of adsorption at 4°C, cells were washed three times at 4°C with medium 199 supplemented with 1% FCS and 0, 1, 10, or 100 μg heparin/ml. Medium 199 supplemented with 1% FCS, at 37°C, was then added, and the cells were placed into a 37°C incubator. At 10 h postinfection, EGFP-positive cells were determined by using a FACSCalibur instrument. These results are the averages and standard errors of data from three independent experiments, calculated relative to the sample with no heparin.

Techniques Used: Recombinant, Adsorption, Infection

4) Product Images from "Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation"

Article Title: Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation

Journal: Nature Communications

doi: 10.1038/ncomms11730

LncRNA NRON represses HIV-1 replication. ( a ) The expression levels of lncRNAs in activated primary CD4 +  T lymphocytes were detected with real-time qRT–PCR. The expression of  T-bet  was detected as positive control ( n =3). ( b ) Real-time qRT–PCR detection of the lncRNAs expression level differences between the resting and activated primary CD4 +  T lymphocytes from a same donor ( n =3). ( c ) The activated primary CD4 +  T lymphocytes were transfected with siRNAs against indicated lncRNAs or nonspecific control and were infected with HIV-1 NL4-3  viruses. HIV-1 productions in the cultures were detected by p24 ELISA at 7 days post infection ( n =3). ( d ) Northern blotting detection of NRON expression in the resting (R) or activated (A) primary CD4 +  T lymphocytes from the same donors. Numbers indicated the fold change related to control. ( e ) The activated primary CD4 +  T lymphocytes were transfected with siRNAs against NRON or nonspecific control and were infected with HIV-1 NL4-3  viruses. HIV-1 productions in the cultures were detected with p24 ELISA at indicated time points post infection ( n =3). The results in  a – c , e  show mean±s.d. (error bars). * P
Figure Legend Snippet: LncRNA NRON represses HIV-1 replication. ( a ) The expression levels of lncRNAs in activated primary CD4 + T lymphocytes were detected with real-time qRT–PCR. The expression of T-bet was detected as positive control ( n =3). ( b ) Real-time qRT–PCR detection of the lncRNAs expression level differences between the resting and activated primary CD4 + T lymphocytes from a same donor ( n =3). ( c ) The activated primary CD4 + T lymphocytes were transfected with siRNAs against indicated lncRNAs or nonspecific control and were infected with HIV-1 NL4-3 viruses. HIV-1 productions in the cultures were detected by p24 ELISA at 7 days post infection ( n =3). ( d ) Northern blotting detection of NRON expression in the resting (R) or activated (A) primary CD4 + T lymphocytes from the same donors. Numbers indicated the fold change related to control. ( e ) The activated primary CD4 + T lymphocytes were transfected with siRNAs against NRON or nonspecific control and were infected with HIV-1 NL4-3 viruses. HIV-1 productions in the cultures were detected with p24 ELISA at indicated time points post infection ( n =3). The results in a – c , e show mean±s.d. (error bars). * P

Techniques Used: Expressing, Quantitative RT-PCR, Positive Control, Transfection, Infection, Enzyme-linked Immunosorbent Assay, Northern Blot

Depletion of NRON reactivates HIV-1 viruses in latently infected CD4 +  T lymphocytes. ( a ) Primary resting CD4 +  T lymphocytes were nucleofected with HIV-1 promoter reporter system plasmids, pcDNA3.1-Tat-HA and siRNAs against NRON or nonspecific control. The promoter activity was determined with dual-luciferase reporter assay at 48 h after transfection ( n =3). ( b ) Tat and control GFP were detected by western blotting on NRON knockdown in nucleofected primary resting CD4 +  T lymphocytes. Numbers indicated the fold change related to the control. ( c ) The latently infected cells were transfected with NRON siRNAs or nonspecific control, or were transfected with siRNAs in combination with the treatment of SAHA, and detected by FACS at 48–72 h post transfection. The GFP+ ratio indicated the reactivation level ( d ;  n =3). ( e ) Resting CD4 +  T lymphocytes isolated from HIV-1-infected individuals on suppressive cART were transfected with siRNAs in combination with the treatment of SAHA. After 48 h, HIV-1 virion-associated RNAs in the supernatants were isolated and detected with real-time qRT–PCR ( n =3). ( f ) The intracellular HIV-1 RNA and NRON RNA expression levels were detected in resting CD4 +  T lymphocytes isolated from HIV-1-infected individuals on suppressive cART ( n =20), and the correlation between the HIV-1 RNA and NRON RNA levels was shown. The simple linear regression analysis was performed and linear regression line was shown. Data in  a , d , e  show mean±s.d. (error bars). Results in  b  represent three independent experiments. * P
Figure Legend Snippet: Depletion of NRON reactivates HIV-1 viruses in latently infected CD4 + T lymphocytes. ( a ) Primary resting CD4 + T lymphocytes were nucleofected with HIV-1 promoter reporter system plasmids, pcDNA3.1-Tat-HA and siRNAs against NRON or nonspecific control. The promoter activity was determined with dual-luciferase reporter assay at 48 h after transfection ( n =3). ( b ) Tat and control GFP were detected by western blotting on NRON knockdown in nucleofected primary resting CD4 + T lymphocytes. Numbers indicated the fold change related to the control. ( c ) The latently infected cells were transfected with NRON siRNAs or nonspecific control, or were transfected with siRNAs in combination with the treatment of SAHA, and detected by FACS at 48–72 h post transfection. The GFP+ ratio indicated the reactivation level ( d ; n =3). ( e ) Resting CD4 + T lymphocytes isolated from HIV-1-infected individuals on suppressive cART were transfected with siRNAs in combination with the treatment of SAHA. After 48 h, HIV-1 virion-associated RNAs in the supernatants were isolated and detected with real-time qRT–PCR ( n =3). ( f ) The intracellular HIV-1 RNA and NRON RNA expression levels were detected in resting CD4 + T lymphocytes isolated from HIV-1-infected individuals on suppressive cART ( n =20), and the correlation between the HIV-1 RNA and NRON RNA levels was shown. The simple linear regression analysis was performed and linear regression line was shown. Data in a , d , e show mean±s.d. (error bars). Results in b represent three independent experiments. * P

Techniques Used: Infection, Activity Assay, Luciferase, Reporter Assay, Transfection, Western Blot, FACS, Isolation, Quantitative RT-PCR, RNA Expression

5) Product Images from "Polyfunctional anti-human epidermal growth factor receptor 3 (anti-HER3) antibodies induced by HER3 vaccines have multiple mechanisms of antitumor activity against therapy resistant and triple negative breast cancers"

Article Title: Polyfunctional anti-human epidermal growth factor receptor 3 (anti-HER3) antibodies induced by HER3 vaccines have multiple mechanisms of antitumor activity against therapy resistant and triple negative breast cancers

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-018-1023-x

In vivo effects of human epidermal growth factor receptor 3 (HER3) vaccine-induced antibody (HER3-VIA) in lapatinib-refractory rBT474 SCID tumor xenografts. a Flow cytometry analysis of HER2/HER3 expression (filled histograms) by BT474 and rBT474 cells. Mean fluorescence intensities are shown in each histogram. b The lapatinib-refractory cell line rBT474 was implanted into SCID mice and HER3-VIA was administered and tumor size was measured every 2-3 days : * p
Figure Legend Snippet: In vivo effects of human epidermal growth factor receptor 3 (HER3) vaccine-induced antibody (HER3-VIA) in lapatinib-refractory rBT474 SCID tumor xenografts. a Flow cytometry analysis of HER2/HER3 expression (filled histograms) by BT474 and rBT474 cells. Mean fluorescence intensities are shown in each histogram. b The lapatinib-refractory cell line rBT474 was implanted into SCID mice and HER3-VIA was administered and tumor size was measured every 2-3 days : * p

Techniques Used: In Vivo, Flow Cytometry, Cytometry, Expressing, Fluorescence, Mouse Assay

In vivo effects of human epidermal growth factor receptor 3 (HER3) vaccine-induced antibody (HER3-VIA) in triple negative MDA-MB-468 SCID tumor xenografts. a Flow cytometry analysis of HER family expression by MDA-MB-468 cells. Cells were stained with phycoerythrin (PE)-conjugated anti-epidermal growth factor receptor (anti-EGFR), anti-HER2, or anti-HER3 monoclonal antibodies (mAb) (upper three histograms), or were incubated with HER3-VIA or green fluorescence protein (GFP)-VIA (1:100 dilution), followed by PE-conjugated anti-mouse IgG (lower two histograms). b Effect of HER3-VIA on signaling pathway was analyzed by western blotting. MDA-MB-468 cells were incubated with HER3-VIA (1:100 dilution) in vitro at 37 °C for the indicated time period. c Passive transfer of HER3-VIA retarded the growth of established MDA-MB-4684 in SCID mice: * p
Figure Legend Snippet: In vivo effects of human epidermal growth factor receptor 3 (HER3) vaccine-induced antibody (HER3-VIA) in triple negative MDA-MB-468 SCID tumor xenografts. a Flow cytometry analysis of HER family expression by MDA-MB-468 cells. Cells were stained with phycoerythrin (PE)-conjugated anti-epidermal growth factor receptor (anti-EGFR), anti-HER2, or anti-HER3 monoclonal antibodies (mAb) (upper three histograms), or were incubated with HER3-VIA or green fluorescence protein (GFP)-VIA (1:100 dilution), followed by PE-conjugated anti-mouse IgG (lower two histograms). b Effect of HER3-VIA on signaling pathway was analyzed by western blotting. MDA-MB-468 cells were incubated with HER3-VIA (1:100 dilution) in vitro at 37 °C for the indicated time period. c Passive transfer of HER3-VIA retarded the growth of established MDA-MB-4684 in SCID mice: * p

Techniques Used: In Vivo, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Expressing, Staining, Incubation, Fluorescence, Western Blot, In Vitro, Mouse Assay

Vaccine-induced antibody (VIA) in serum from mice immunized with adenovirus encoding full length human 3 epidermal growth factor receptor (HER3-VIA) induces receptor internalization and downregulation of human epidermal growth factor receptor 3 (HER3), but not epidermal growth factor receptor (EGFR) nor HER2. SKBR3, BT474-M1 ( a ) and MDA-MB-468 ( b ) were incubated with 1:100 HER3-VIA or green fluorescence protein (GFP)-VIA at 37 °C for 1 or 3 h, respectively. After washing and permeabilization, RedTM-conjugated anti-mouse IgG (H + L) was used to visualize internalization of the VIA-bound proteins with a fluorescence microscope. SKBR3 cells ( c ) or MDA-MB-468 cells ( d ) were incubated with 1:100 HER3-VIA or GFP-VIA at 37 °C for 3 h. Cells were fixed, permeabilized and stained with anti-HER2 antibody (trastuzumab, 40 μg/mL) ( c ) or anti-EGFR antibody (cetuximab, 40 μg/mL) ( d ), followed by Cy2-conjugated anti-human IgG antibody
Figure Legend Snippet: Vaccine-induced antibody (VIA) in serum from mice immunized with adenovirus encoding full length human 3 epidermal growth factor receptor (HER3-VIA) induces receptor internalization and downregulation of human epidermal growth factor receptor 3 (HER3), but not epidermal growth factor receptor (EGFR) nor HER2. SKBR3, BT474-M1 ( a ) and MDA-MB-468 ( b ) were incubated with 1:100 HER3-VIA or green fluorescence protein (GFP)-VIA at 37 °C for 1 or 3 h, respectively. After washing and permeabilization, RedTM-conjugated anti-mouse IgG (H + L) was used to visualize internalization of the VIA-bound proteins with a fluorescence microscope. SKBR3 cells ( c ) or MDA-MB-468 cells ( d ) were incubated with 1:100 HER3-VIA or GFP-VIA at 37 °C for 3 h. Cells were fixed, permeabilized and stained with anti-HER2 antibody (trastuzumab, 40 μg/mL) ( c ) or anti-EGFR antibody (cetuximab, 40 μg/mL) ( d ), followed by Cy2-conjugated anti-human IgG antibody

Techniques Used: Mouse Assay, Multiple Displacement Amplification, Incubation, Fluorescence, Microscopy, Staining

6) Product Images from "A double helical motif in OCIAD2 is essential for its localization, interactions and STAT3 activation"

Article Title: A double helical motif in OCIAD2 is essential for its localization, interactions and STAT3 activation

Journal: Scientific Reports

doi: 10.1038/s41598-018-25667-3

OCIAD2 localizes to endosomes and mitochondria. ( A – G ) Confocal images and co-localization plots of OCIAD2_FL-EGFP transfected HEK293 cells showing localization of OCIAD2 with various markers detected by immunofluorescence staining (red). ( A ) endogenous OCIAD2; ( B ) cytochrome oxidase subunit IV (CoxIV); ( C ) early endosome, Rab5; ( D ) late endosome, Rab7; ( E ) recycling endosome, Rab11; ( F ) lysosome, Lysotracker; ( G ) Golgi, GM130. Insets (A, B and C) show magnified view of the boxed region. Scale bars = 5 µm in C, D, E and 10 µm in A, B, F and G.
Figure Legend Snippet: OCIAD2 localizes to endosomes and mitochondria. ( A – G ) Confocal images and co-localization plots of OCIAD2_FL-EGFP transfected HEK293 cells showing localization of OCIAD2 with various markers detected by immunofluorescence staining (red). ( A ) endogenous OCIAD2; ( B ) cytochrome oxidase subunit IV (CoxIV); ( C ) early endosome, Rab5; ( D ) late endosome, Rab7; ( E ) recycling endosome, Rab11; ( F ) lysosome, Lysotracker; ( G ) Golgi, GM130. Insets (A, B and C) show magnified view of the boxed region. Scale bars = 5 µm in C, D, E and 10 µm in A, B, F and G.

Techniques Used: Transfection, Immunofluorescence, Staining

Phylogenetic analysis of the OCIAD proteins. ( A ) Schematic representation of the mouse OCIAD1/Asrij and OCIAD2 protein organization. ( B ) Multiple sequence alignment of OCIAD1/Asrij and OCIAD2 protein sequences (using MUSCLE) from mouse and human showing conserved regions. Dark shading shows amino acids identical in all sequences and light shading indicates similar amino acids. Numbers at the end of each sequence indicate amino acid positions. ( C ) Multiple sequence alignment of the predicted mouse, rat and human OCIAD2 open reading frames (using MUSCLE). ( D . Three gene duplication events (diamonds) were identified in the tree. Open diamonds mark duplication nodes with low bootstrap values and blue diamond marks duplication node with highest bootstrap value (47). Given that there is only one copy of OCIA-domain containing proteins in invertebrates, these have been referred to as OCIAD. The extra copy of OCIAD1 protein present in Danio rerio and Clupea harengus has been listed as OCIAD1-like protein in the tree. ( E ) Sequence conservation analysis of vertebrate OCIAD1 (red line) and OCIAD2 (blue line) with respect to a conserved protein Histone H3 (black line) across different taxa. The dotted lines represent best-fit whereas solid lines represent the original values.
Figure Legend Snippet: Phylogenetic analysis of the OCIAD proteins. ( A ) Schematic representation of the mouse OCIAD1/Asrij and OCIAD2 protein organization. ( B ) Multiple sequence alignment of OCIAD1/Asrij and OCIAD2 protein sequences (using MUSCLE) from mouse and human showing conserved regions. Dark shading shows amino acids identical in all sequences and light shading indicates similar amino acids. Numbers at the end of each sequence indicate amino acid positions. ( C ) Multiple sequence alignment of the predicted mouse, rat and human OCIAD2 open reading frames (using MUSCLE). ( D . Three gene duplication events (diamonds) were identified in the tree. Open diamonds mark duplication nodes with low bootstrap values and blue diamond marks duplication node with highest bootstrap value (47). Given that there is only one copy of OCIA-domain containing proteins in invertebrates, these have been referred to as OCIAD. The extra copy of OCIAD1 protein present in Danio rerio and Clupea harengus has been listed as OCIAD1-like protein in the tree. ( E ) Sequence conservation analysis of vertebrate OCIAD1 (red line) and OCIAD2 (blue line) with respect to a conserved protein Histone H3 (black line) across different taxa. The dotted lines represent best-fit whereas solid lines represent the original values.

Techniques Used: Sequencing

Knockdown of OCIAD2 retards migration of HEK293 cells. ( A ) Western Blotting confirming ociad2 knockdown (KD) in OCIAD2_ shRNA (1, 2 and 3) transfected HEK293 cells as compared to non-silencing (NS) shRNA. Graph shows relative OCIAD2 levels in NS and KD. n = 4 ( B ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in NS and KD. Data are representative of three independent experiments and graph shows relative STAT3/pSTAT3 ratio upon ociad2 . n = 4 ( C ) Flow cytometry analysis of Ki-67 + population in ociad2 KD HEK293 cells. Graph shows results obtained from four independent experiments (mean ± SEM). ( D ) Reduced migration of ociad2 KD cells observed in wound healing assays. Monolayers of control and ociad2 knockdown HEK293 cells were wounded and the degree of recovery was measured at 0, 12 and 24 hours post-wounding. Representative phase and fluorescence images, 4× magnification. Measurement and estimation of wound recovery was based on the initial wound size. Graph shows quantification of results obtained from three independent experiments (mean ± SEM) with at least 5 fields analyzed per sample per experiment. ( E ) Representative images (4× magnification) showing reduced migration of ociad2 KD HEK293 cells after 48 hours in a trans-well migration assays. Graphs represent quantification of cell migration (absorbance at 570 nm) obtained from two independent trans-well experiments performed in two technical replicates (mean ± SEM). * p
Figure Legend Snippet: Knockdown of OCIAD2 retards migration of HEK293 cells. ( A ) Western Blotting confirming ociad2 knockdown (KD) in OCIAD2_ shRNA (1, 2 and 3) transfected HEK293 cells as compared to non-silencing (NS) shRNA. Graph shows relative OCIAD2 levels in NS and KD. n = 4 ( B ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in NS and KD. Data are representative of three independent experiments and graph shows relative STAT3/pSTAT3 ratio upon ociad2 . n = 4 ( C ) Flow cytometry analysis of Ki-67 + population in ociad2 KD HEK293 cells. Graph shows results obtained from four independent experiments (mean ± SEM). ( D ) Reduced migration of ociad2 KD cells observed in wound healing assays. Monolayers of control and ociad2 knockdown HEK293 cells were wounded and the degree of recovery was measured at 0, 12 and 24 hours post-wounding. Representative phase and fluorescence images, 4× magnification. Measurement and estimation of wound recovery was based on the initial wound size. Graph shows quantification of results obtained from three independent experiments (mean ± SEM) with at least 5 fields analyzed per sample per experiment. ( E ) Representative images (4× magnification) showing reduced migration of ociad2 KD HEK293 cells after 48 hours in a trans-well migration assays. Graphs represent quantification of cell migration (absorbance at 570 nm) obtained from two independent trans-well experiments performed in two technical replicates (mean ± SEM). * p

Techniques Used: Migration, Western Blot, shRNA, Transfection, Flow Cytometry, Cytometry, Fluorescence

Intron-exon structure of the ociad1/2 genes and comparison of their transcript levels in human tissues and cell lines. ( A ) Intron-exon structures of the genes coding for OCIAD1 and OCIAD2 in human, mouse and zebra fish. Intron-exon structure for the longest transcript was obtained from the Ensemble database (Accession numbers: ENST00000381473.7, ENSMUST00000031038.10, ENSDART00000103365.4, ENST00000508632.5, ENSMUST00000087195.8 and ENSDART00000164503.1). Colored boxes correspond to exons, grey-color indicates untranslated regions. Black solid lines represent introns. The size of introns and exons in nucleotides is mentioned. Introns are not drawn to scale. Exons coding for the double helical domain of OCIAD proteins have been indicated. ( B ) Comparison of transcript levels of ociad1 and ociad2 ).
Figure Legend Snippet: Intron-exon structure of the ociad1/2 genes and comparison of their transcript levels in human tissues and cell lines. ( A ) Intron-exon structures of the genes coding for OCIAD1 and OCIAD2 in human, mouse and zebra fish. Intron-exon structure for the longest transcript was obtained from the Ensemble database (Accession numbers: ENST00000381473.7, ENSMUST00000031038.10, ENSDART00000103365.4, ENST00000508632.5, ENSMUST00000087195.8 and ENSDART00000164503.1). Colored boxes correspond to exons, grey-color indicates untranslated regions. Black solid lines represent introns. The size of introns and exons in nucleotides is mentioned. Introns are not drawn to scale. Exons coding for the double helical domain of OCIAD proteins have been indicated. ( B ) Comparison of transcript levels of ociad1 and ociad2 ).

Techniques Used: Fluorescence In Situ Hybridization

The double helical motif of OCIAD2 is necessary for interaction with OCIAD1. ( A ) Schematic representation of full-length (FL), N-terminal (N), hydrophobic region (Hph) and C-terminal (C) fragments of OCIAD2 generated for this study. Numbers indicate amino acid positions. Hx: predicted alpha helix. ( B – H ) Confocal images of HEK293 cells transfected with OCIAD2 reporter constructs, showing localization of ectopic OCIAD2-reporter with endogenous OCIAD2 ( B – D ) or endogenous OCIAD1 ( E – H ) as indicated, detected by fluorescence immunostaining with respective antibodies. Insets show magnified view of the boxed region. Co-localization plots are to the right of each panel. Scale bar: ( B – D ): 10 µm; ( E – H ): 5 µm. ( I – O ) Cell lysates from untransfected HEK293 cells (I) or those expressing various OCIAD2 (J, K, L, O) or OCIAD1/Asrij ( M , N .
Figure Legend Snippet: The double helical motif of OCIAD2 is necessary for interaction with OCIAD1. ( A ) Schematic representation of full-length (FL), N-terminal (N), hydrophobic region (Hph) and C-terminal (C) fragments of OCIAD2 generated for this study. Numbers indicate amino acid positions. Hx: predicted alpha helix. ( B – H ) Confocal images of HEK293 cells transfected with OCIAD2 reporter constructs, showing localization of ectopic OCIAD2-reporter with endogenous OCIAD2 ( B – D ) or endogenous OCIAD1 ( E – H ) as indicated, detected by fluorescence immunostaining with respective antibodies. Insets show magnified view of the boxed region. Co-localization plots are to the right of each panel. Scale bar: ( B – D ): 10 µm; ( E – H ): 5 µm. ( I – O ) Cell lysates from untransfected HEK293 cells (I) or those expressing various OCIAD2 (J, K, L, O) or OCIAD1/Asrij ( M , N .

Techniques Used: Generated, Transfection, Construct, Fluorescence, Immunostaining, Expressing

Comparison of the genomic organization for ociad1 and ociad2 across different species. Schematics depict genomic region encompassing ociad1 (red) and ociad2 (blue) and flanking genes fryl , cwh43 or dcun1d4 as indicated. Chromosome numbers and position of genes on sense or antisense strand are indicated. Direction of arrows indicates direction of transcription. Phyla are grouped into colored boxes.
Figure Legend Snippet: Comparison of the genomic organization for ociad1 and ociad2 across different species. Schematics depict genomic region encompassing ociad1 (red) and ociad2 (blue) and flanking genes fryl , cwh43 or dcun1d4 as indicated. Chromosome numbers and position of genes on sense or antisense strand are indicated. Direction of arrows indicates direction of transcription. Phyla are grouped into colored boxes.

Techniques Used:

The double helical motif of OCIAD2 interacts with STAT3 and promotes its activation. ( A ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in HEK293 cells transfected with various OCIAD2 constructs as indicated. UT: untransfected, V: empty vector transfected; FL: OCIAD2_FL-EGFP; N: OCIAD2_N-EGFP; Hph: OCIAD2_Hph-dsRed and C: OCIAD2_C-dsRed. Graph represents pSTAT3/STAT3 ratio obtained upon overexpression of various OCIAD2 constructs relative to control (shown as a dotted line). n = 4 ( B ) Validation of transfection of FLAG-STAT3 construct into HEK293 cells by Western Blotting. ( C ) FLAG-STAT3 transfected HEK293 cell lysate subjected to immunoprecipitation with anti-FLAG antibody and assessed for interaction with OCIAD1 and OCIAD2. ( D – G ) HEK293 cell lysates expressing various OCIAD2 constructs (FL, N, Hph and C) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) to probe for interaction with STAT3. Protein size markers (in kDa) are indicated to the left. Data for STAT3/pSTAT3 Western Blotting are representative of three independent experiments and * p .
Figure Legend Snippet: The double helical motif of OCIAD2 interacts with STAT3 and promotes its activation. ( A ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in HEK293 cells transfected with various OCIAD2 constructs as indicated. UT: untransfected, V: empty vector transfected; FL: OCIAD2_FL-EGFP; N: OCIAD2_N-EGFP; Hph: OCIAD2_Hph-dsRed and C: OCIAD2_C-dsRed. Graph represents pSTAT3/STAT3 ratio obtained upon overexpression of various OCIAD2 constructs relative to control (shown as a dotted line). n = 4 ( B ) Validation of transfection of FLAG-STAT3 construct into HEK293 cells by Western Blotting. ( C ) FLAG-STAT3 transfected HEK293 cell lysate subjected to immunoprecipitation with anti-FLAG antibody and assessed for interaction with OCIAD1 and OCIAD2. ( D – G ) HEK293 cell lysates expressing various OCIAD2 constructs (FL, N, Hph and C) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) to probe for interaction with STAT3. Protein size markers (in kDa) are indicated to the left. Data for STAT3/pSTAT3 Western Blotting are representative of three independent experiments and * p .

Techniques Used: Activation Assay, Western Blot, Transfection, Construct, Plasmid Preparation, Over Expression, Immunoprecipitation, Expressing

7) Product Images from "Direct Binding of the Ligand PSG17 to CD9 Requires a CD9 Site Essential for Sperm-Egg Fusion"

Article Title: Direct Binding of the Ligand PSG17 to CD9 Requires a CD9 Site Essential for Sperm-Egg Fusion

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E03-04-0244

Binding of PSG17N-Myc-His to CD9-transfected 293T cells. HEK 293T cells were transfected with empty plasmid (EP), pCD9-eGFP (wild-type CD9:WT-CD9), pCD9-F174A-eGFP (single mutant CD9: SM-CD9), or pCD9-SFQ to AAA (173-175)-eGFP (triple mutant CD9: TM-CD9). Forty-eight hours posttransfection, the cells were incubated with 10 μg/ml PSG17N-Myc-His and bound PSG17N-Myc-His was detected after treatment with HRP-conjugated anti-Myc mAb and TMB/peroxidase substrate. The data are expressed as mean absorbance ± SE. Each data point represents four identical wells and the experiment was repeated two independent times with similar results.
Figure Legend Snippet: Binding of PSG17N-Myc-His to CD9-transfected 293T cells. HEK 293T cells were transfected with empty plasmid (EP), pCD9-eGFP (wild-type CD9:WT-CD9), pCD9-F174A-eGFP (single mutant CD9: SM-CD9), or pCD9-SFQ to AAA (173-175)-eGFP (triple mutant CD9: TM-CD9). Forty-eight hours posttransfection, the cells were incubated with 10 μg/ml PSG17N-Myc-His and bound PSG17N-Myc-His was detected after treatment with HRP-conjugated anti-Myc mAb and TMB/peroxidase substrate. The data are expressed as mean absorbance ± SE. Each data point represents four identical wells and the experiment was repeated two independent times with similar results.

Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Incubation

FACS analysis of PSG17N-Myc-His binding to HEK 293T wild-type and mutated CD9-transfected cells. HEK 293T cells were transfected with pCD9-eGFP (wild-type CD9: WT-CD9) (A) or pCD9-F174A-eGFP (single mutant CD9: SM-CD9) (B), after which they were sequentially incubated with 10 μg/ml PSG17N-Myc-His, anti-Myc mAb, and PE-labeled rat anti-mouse IgG1.
Figure Legend Snippet: FACS analysis of PSG17N-Myc-His binding to HEK 293T wild-type and mutated CD9-transfected cells. HEK 293T cells were transfected with pCD9-eGFP (wild-type CD9: WT-CD9) (A) or pCD9-F174A-eGFP (single mutant CD9: SM-CD9) (B), after which they were sequentially incubated with 10 μg/ml PSG17N-Myc-His, anti-Myc mAb, and PE-labeled rat anti-mouse IgG1.

Techniques Used: FACS, Binding Assay, Transfection, Mutagenesis, Incubation, Labeling

Binding of PSG17N-Myc-His to the oocyte. Zona-free eggs from wild-type (A-D) or CD9 knockout animals (E and F) were incubated in 50 μg/ml XylE (a His-tagged control protein) (A and B) or PSG17N-Myc-His (C-F) for 30 min, and stained by indirect immunofluorescence by using an anti-Myc antibody, followed by Oregon Green-conjugated secondary antibody.
Figure Legend Snippet: Binding of PSG17N-Myc-His to the oocyte. Zona-free eggs from wild-type (A-D) or CD9 knockout animals (E and F) were incubated in 50 μg/ml XylE (a His-tagged control protein) (A and B) or PSG17N-Myc-His (C-F) for 30 min, and stained by indirect immunofluorescence by using an anti-Myc antibody, followed by Oregon Green-conjugated secondary antibody.

Techniques Used: Binding Assay, Knock-Out, Incubation, Staining, Immunofluorescence

PSG17N binds to the extracellular loop 2 of murine CD9. Cobalt chelate beads were incubated with GFP-His (lane 1), CEACAM1a[1-4]-His (lane 2) or PSG17N-Myc-His (lanes 3 and 4) used as baits. The beads were then incubated with GST-CD9EC2 (lanes 1-3) or the same construct carrying the triple mutation SFQ (173-175) to AAA (GST-CD9EC2-TM) (lane 4). After several washes, the proteins were eluted with a buffer containing 290 mM imidazole, separated on a 4-20% NuPAGE gel, and detected by Western blot by using an anti-CD9 antibody.
Figure Legend Snippet: PSG17N binds to the extracellular loop 2 of murine CD9. Cobalt chelate beads were incubated with GFP-His (lane 1), CEACAM1a[1-4]-His (lane 2) or PSG17N-Myc-His (lanes 3 and 4) used as baits. The beads were then incubated with GST-CD9EC2 (lanes 1-3) or the same construct carrying the triple mutation SFQ (173-175) to AAA (GST-CD9EC2-TM) (lane 4). After several washes, the proteins were eluted with a buffer containing 290 mM imidazole, separated on a 4-20% NuPAGE gel, and detected by Western blot by using an anti-CD9 antibody.

Techniques Used: Incubation, Construct, Mutagenesis, Western Blot

8) Product Images from "Identifying Small Molecules which Inhibit Autophagy: a Phenotypic Screen Using Image-Based High-Content Cell Analysis"

Article Title: Identifying Small Molecules which Inhibit Autophagy: a Phenotypic Screen Using Image-Based High-Content Cell Analysis

Journal: Current Chemical Genomics and Translational Medicine

doi: 10.2174/2213988501408010003

Left: Dose response IC 50 values (M) of ~1,200 compounds which were designated Active in the Primary and Counter screen assays in EGFP-LC3-HeLa cells plotted vs. their cytotoxicity activity in the Secondary starvation assay in H1299 cells. So that they appear on the plot, compounds are set to 100 μM activity if inactive (red, n = ~450), and those with IC 50 values > 30 μM are set to 50 μM (n= ~50). For the two secondary assays in H1299 cells, compounds designated Active Specific (green) were compounds which gave IC 50 values in the starvation medium which were 3x or more lower than in the parallel assay in full growth medium (~400 compounds). Active Non-specific compounds (yellow) have similar IC 50 values in both formats. Right: Examples of compound dose responses obtained in the secondary assay (pink: starved; blue: full growth medium). ( A ): Active Non-specific, ( B ) and ( C ): Active Specific.
Figure Legend Snippet: Left: Dose response IC 50 values (M) of ~1,200 compounds which were designated Active in the Primary and Counter screen assays in EGFP-LC3-HeLa cells plotted vs. their cytotoxicity activity in the Secondary starvation assay in H1299 cells. So that they appear on the plot, compounds are set to 100 μM activity if inactive (red, n = ~450), and those with IC 50 values > 30 μM are set to 50 μM (n= ~50). For the two secondary assays in H1299 cells, compounds designated Active Specific (green) were compounds which gave IC 50 values in the starvation medium which were 3x or more lower than in the parallel assay in full growth medium (~400 compounds). Active Non-specific compounds (yellow) have similar IC 50 values in both formats. Right: Examples of compound dose responses obtained in the secondary assay (pink: starved; blue: full growth medium). ( A ): Active Non-specific, ( B ) and ( C ): Active Specific.

Techniques Used: Activity Assay

Flow cytometry analysis of the EGFP-LC3 HeLa cell population before clonal selection (left panel, blue line) and after (right panel, green line), where 8 % of the population were expressing EGFP-LC3, compared to WT HeLa cells (red line). Clone C10 (green line/green arrow) was produced by single-cell sorting of the mixed population, expanded, and selected because of its high fluorescent signal.
Figure Legend Snippet: Flow cytometry analysis of the EGFP-LC3 HeLa cell population before clonal selection (left panel, blue line) and after (right panel, green line), where 8 % of the population were expressing EGFP-LC3, compared to WT HeLa cells (red line). Clone C10 (green line/green arrow) was produced by single-cell sorting of the mixed population, expanded, and selected because of its high fluorescent signal.

Techniques Used: Flow Cytometry, Cytometry, Selection, Expressing, Produced, FACS

Images of HeLa-EGFP-LC3 cells after 2 hr starvation; cells were stained with Draq5 to indicate nuclei (red, Channel 1), EGFP-LC3 is shown in green (Channel 2). The algorithm Granularity Analysis Module GRN1 was applied to first identify cell cytoplasm by expanding out a set number of pixels from the nucleus boundary designated in Channel 1 (large box) and then identify EGFP-autphagosomes located within the cell mask, which are counted as “grains” (small boxes). Other measurements are also made, e.g. brightness of objects in each Channel; nuclear size and cytoplasm area. Typical images of the assay controls are shown; the left panel shows cells after 2 hr starvation and the right shows cells after 2 hr starvation in the presence of the inhibitor wortmannin.
Figure Legend Snippet: Images of HeLa-EGFP-LC3 cells after 2 hr starvation; cells were stained with Draq5 to indicate nuclei (red, Channel 1), EGFP-LC3 is shown in green (Channel 2). The algorithm Granularity Analysis Module GRN1 was applied to first identify cell cytoplasm by expanding out a set number of pixels from the nucleus boundary designated in Channel 1 (large box) and then identify EGFP-autphagosomes located within the cell mask, which are counted as “grains” (small boxes). Other measurements are also made, e.g. brightness of objects in each Channel; nuclear size and cytoplasm area. Typical images of the assay controls are shown; the left panel shows cells after 2 hr starvation and the right shows cells after 2 hr starvation in the presence of the inhibitor wortmannin.

Techniques Used: Staining

9) Product Images from "JCV agnoprotein-induced reduction in CXCL5/LIX secretion by oligodendrocytes is associated with activation of apoptotic signaling in neurons"

Article Title: JCV agnoprotein-induced reduction in CXCL5/LIX secretion by oligodendrocytes is associated with activation of apoptotic signaling in neurons

Journal: Journal of Cellular Physiology

doi: 10.1002/jcp.23065

Comparison of CM from agnoprotein-expressing CG4-Ol to CG4-Ol control for levels of the CXCL5/LIX chemokine
Figure Legend Snippet: Comparison of CM from agnoprotein-expressing CG4-Ol to CG4-Ol control for levels of the CXCL5/LIX chemokine

Techniques Used: Expressing

Effect of agnoprotein on GSK3β activity
Figure Legend Snippet: Effect of agnoprotein on GSK3β activity

Techniques Used: Activity Assay

Structural alterations in rat cortical neurons exposed to CM from CG4-Ol constitutively expressing JCV agnoprotein
Figure Legend Snippet: Structural alterations in rat cortical neurons exposed to CM from CG4-Ol constitutively expressing JCV agnoprotein

Techniques Used: Expressing

Neuronal survival after treatment with conditioned medium from agnoprotein-expressing CG4 cells
Figure Legend Snippet: Neuronal survival after treatment with conditioned medium from agnoprotein-expressing CG4 cells

Techniques Used: Expressing

10) Product Images from "The Cysteine-rich Domain of the Secreted Proprotein Convertases PC5A and PACE4 Functions as a Cell Surface Anchor and Interacts with Tissue Inhibitors of Metalloproteinases D⃞"

Article Title: The Cysteine-rich Domain of the Secreted Proprotein Convertases PC5A and PACE4 Functions as a Cell Surface Anchor and Interacts with Tissue Inhibitors of Metalloproteinases D⃞

Journal:

doi: 10.1091/mbc.E05-06-0504

PC5A binds to the C-terminal domain of TIMP-2 for its anchorage at the cell surface. (A) COS-1 cells were cotransfected at a DNA ratio of 1:3 with FL-PC5A-V5 and either empty pcDNA3 (Control), NT-TIMP-2, or CT-TIMP-2 expressing recombinants in pcDNA3.
Figure Legend Snippet: PC5A binds to the C-terminal domain of TIMP-2 for its anchorage at the cell surface. (A) COS-1 cells were cotransfected at a DNA ratio of 1:3 with FL-PC5A-V5 and either empty pcDNA3 (Control), NT-TIMP-2, or CT-TIMP-2 expressing recombinants in pcDNA3.

Techniques Used: Expressing

PC5A/TIMP-2 complex binds HSPGs at the cell surface and the CRD enhances processing of proEL by PC5A. (A) Displacement of cell surface PC5A by exogenous heparin or heparinase-I as revealed by confocal microscopy with Ab:V5, in COS-1 cells transfected
Figure Legend Snippet: PC5A/TIMP-2 complex binds HSPGs at the cell surface and the CRD enhances processing of proEL by PC5A. (A) Displacement of cell surface PC5A by exogenous heparin or heparinase-I as revealed by confocal microscopy with Ab:V5, in COS-1 cells transfected

Techniques Used: Confocal Microscopy, Transfection

Effect of protease inhibitors and overexpression of proteases on the C-terminal cleavage of PC5A. (A) V5-tagged WT PC5A and the R621A mutant were expressed with or without α1-PDX in HEK293 cells. Western blots were done on conditioned media using
Figure Legend Snippet: Effect of protease inhibitors and overexpression of proteases on the C-terminal cleavage of PC5A. (A) V5-tagged WT PC5A and the R621A mutant were expressed with or without α1-PDX in HEK293 cells. Western blots were done on conditioned media using

Techniques Used: Over Expression, Mutagenesis, Western Blot

TIMP-2 Is Required for the Cell Surface Localization of PC5A
Figure Legend Snippet: TIMP-2 Is Required for the Cell Surface Localization of PC5A

Techniques Used:

Cell surface colocalization of TIMP-2 and FL-PC5A. (A) Schematic representation of the 3 constructs used for the analysis by confocal microscopy. (B) Cell surface labeling of COS-1 cells expressing FL-PC5A, PC5A-ΔC, or PC5A-CRD with Ab:V5. (C)
Figure Legend Snippet: Cell surface colocalization of TIMP-2 and FL-PC5A. (A) Schematic representation of the 3 constructs used for the analysis by confocal microscopy. (B) Cell surface labeling of COS-1 cells expressing FL-PC5A, PC5A-ΔC, or PC5A-CRD with Ab:V5. (C)

Techniques Used: Construct, Confocal Microscopy, Labeling, Expressing

Coimmunoprecipitations of endogenous and overexpressed TIMP-2 with either FL-PC5A or PC5A-CRD. (A) HT1080 cells were transfected with either empty pIRES-EGFP + phCMV (Vectors), FL-PC5A + empty phCMV (Control), or FL-PC5A + TIMP-2 or TIMP-2-LP. The media
Figure Legend Snippet: Coimmunoprecipitations of endogenous and overexpressed TIMP-2 with either FL-PC5A or PC5A-CRD. (A) HT1080 cells were transfected with either empty pIRES-EGFP + phCMV (Vectors), FL-PC5A + empty phCMV (Control), or FL-PC5A + TIMP-2 or TIMP-2-LP. The media

Techniques Used: Transfection

PC5A interacts with all TIMP family members. (A) Effects of overexpressed TIMP-1, -3, and -4 and their TIMP-LP derivatives on the levels of coexpressed FL-PC5A. Cell extracts and media were resolved by SDS-PAGE and then revealed by Western blot with Ab:V5.
Figure Legend Snippet: PC5A interacts with all TIMP family members. (A) Effects of overexpressed TIMP-1, -3, and -4 and their TIMP-LP derivatives on the levels of coexpressed FL-PC5A. Cell extracts and media were resolved by SDS-PAGE and then revealed by Western blot with Ab:V5.

Techniques Used: SDS Page, Western Blot

Displacement of cell surface PC5A by exogenous TIMP-2 and cell surface localization of FL-PACE4 but not PACE4-ΔC. COS-1 cells were transfected with (A and B) FL-PC5A-V5, (C) FL-PACE4-V5, or (D) PACE4-ΔC-V5. Forty-eight hours, later the
Figure Legend Snippet: Displacement of cell surface PC5A by exogenous TIMP-2 and cell surface localization of FL-PACE4 but not PACE4-ΔC. COS-1 cells were transfected with (A and B) FL-PC5A-V5, (C) FL-PACE4-V5, or (D) PACE4-ΔC-V5. Forty-eight hours, later the

Techniques Used: Transfection

11) Product Images from "New PI(4,5)P2- and membrane proximal integrin-binding motifs in the talin head control ?3-integrin clustering"

Article Title: New PI(4,5)P2- and membrane proximal integrin-binding motifs in the talin head control ?3-integrin clustering

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200908134

Docking and MD analysis of the integrin–Tal interface. (A) Side view of the MP helix of β3-integrin docked to Tal-F3, revealing putative charge–charge interactions (dotted lines) of residues required for Tal-dependent integrin clustering (D 723 –K 324 ; E 726 –K 316 ; E 733 –K 364 ). (B) MD analysis starting from the model in A, after manual connection (magenta) to the Tal-F3–bound W 739 /NPLY 747 motif. Snapshot after 300 ps of MD analysis, showing the position maintained for another 1500 ps. (C) Details of peptide position at 300 ps, indicating amino acids involved in interactions between D 723 –K 324 ; E 726 –K 316 ; F 730 –L 325 (L 325 not depicted) and E 733 with S 365 , S 379 , and Q 381 . A PDB file of this model is available in the supplemental data. (D) Overlay and shifts in localization (arrows) of relevant amino acids between the NMR-derived model (dark green; Wegener et al., 2007 ) and the structure shown in C (light green).
Figure Legend Snippet: Docking and MD analysis of the integrin–Tal interface. (A) Side view of the MP helix of β3-integrin docked to Tal-F3, revealing putative charge–charge interactions (dotted lines) of residues required for Tal-dependent integrin clustering (D 723 –K 324 ; E 726 –K 316 ; E 733 –K 364 ). (B) MD analysis starting from the model in A, after manual connection (magenta) to the Tal-F3–bound W 739 /NPLY 747 motif. Snapshot after 300 ps of MD analysis, showing the position maintained for another 1500 ps. (C) Details of peptide position at 300 ps, indicating amino acids involved in interactions between D 723 –K 324 ; E 726 –K 316 ; F 730 –L 325 (L 325 not depicted) and E 733 with S 365 , S 379 , and Q 381 . A PDB file of this model is available in the supplemental data. (D) Overlay and shifts in localization (arrows) of relevant amino acids between the NMR-derived model (dark green; Wegener et al., 2007 ) and the structure shown in C (light green).

Techniques Used: Nuclear Magnetic Resonance, Derivative Assay

The K318A mutation in FL-Tal increases β3-integrin clustering. (A and B) Western blots of equivalent amounts of cell lysates probed with an anti-Tal mAb from wild-type (wt) or mutant mCherry– or EGFP–FL-Tal–transfected B16F1 cells. (C) Schematic view of Mn 2+ -induced integrin activation and association with wild-type or K318A FL-Tal. (D) Integrin clustering index (percentage of pixels with > 200 gray levels) of wild-type and K318A FL-Tal–transfected cells ( n = 4; SEM). (E and F) Representative TIRF images of Mn 2+ -stimulated B16F1 cells grown on serum-coated coverslips and double transfected with β3-EGFP-integrin (E and F) and wild-type (E) or K318A mCherry (mCh)–FL-Tal (F). Magnified views of the boxed areas are shown in E′–E‴ and F′–F‴. Bar, 20 µm.
Figure Legend Snippet: The K318A mutation in FL-Tal increases β3-integrin clustering. (A and B) Western blots of equivalent amounts of cell lysates probed with an anti-Tal mAb from wild-type (wt) or mutant mCherry– or EGFP–FL-Tal–transfected B16F1 cells. (C) Schematic view of Mn 2+ -induced integrin activation and association with wild-type or K318A FL-Tal. (D) Integrin clustering index (percentage of pixels with > 200 gray levels) of wild-type and K318A FL-Tal–transfected cells ( n = 4; SEM). (E and F) Representative TIRF images of Mn 2+ -stimulated B16F1 cells grown on serum-coated coverslips and double transfected with β3-EGFP-integrin (E and F) and wild-type (E) or K318A mCherry (mCh)–FL-Tal (F). Magnified views of the boxed areas are shown in E′–E‴ and F′–F‴. Bar, 20 µm.

Techniques Used: Mutagenesis, Western Blot, Transfection, Activation Assay

Complementation of focal adhesion formation by E726K β3-integrin and K316E FL-Tal. (A–L) Representative TIRF images of transiently transfected B16F1 cells cultured on serum-coated coverslips in the absence of Mn 2+ . Wild-type (wt; A and D) and E726K mutant (G and J) β3-EGFP-integrin were coexpressed with either wild-type (B and H) or K316E mutant (E and K) mCherry (mCh)–FL-Tal. The merged images demonstrate perfect colocalization in large focal adhesions in the wild-type/wild-type (C) and E726K/K316E (L) conditions. (E) In contrast, K316E FL-Tal was inefficiently recruited to focal adhesions in the presence of wild-type integrins. (G–I) Similarly, the E726K β3-integrin perturbed efficient cell spreading, causing irregular cell shapes and recruitment only to small focal adhesions. (M) Per cell quantification of the ratio of integrin to Tal fluorescence within focal adhesions. Each point corresponds to the mean of 5–15 contacts per cell. The horizontal bar represents the mean and SD of n > 20 cells. (N) Mean β3-EGFP-integrin fluorescence in focal adhesions, which is reduced for the E726K mutant integrin ( n > 20 cells; mean and SD of 300–400 contacts per condition). Bar, 20 µm.
Figure Legend Snippet: Complementation of focal adhesion formation by E726K β3-integrin and K316E FL-Tal. (A–L) Representative TIRF images of transiently transfected B16F1 cells cultured on serum-coated coverslips in the absence of Mn 2+ . Wild-type (wt; A and D) and E726K mutant (G and J) β3-EGFP-integrin were coexpressed with either wild-type (B and H) or K316E mutant (E and K) mCherry (mCh)–FL-Tal. The merged images demonstrate perfect colocalization in large focal adhesions in the wild-type/wild-type (C) and E726K/K316E (L) conditions. (E) In contrast, K316E FL-Tal was inefficiently recruited to focal adhesions in the presence of wild-type integrins. (G–I) Similarly, the E726K β3-integrin perturbed efficient cell spreading, causing irregular cell shapes and recruitment only to small focal adhesions. (M) Per cell quantification of the ratio of integrin to Tal fluorescence within focal adhesions. Each point corresponds to the mean of 5–15 contacts per cell. The horizontal bar represents the mean and SD of n > 20 cells. (N) Mean β3-EGFP-integrin fluorescence in focal adhesions, which is reduced for the E726K mutant integrin ( n > 20 cells; mean and SD of 300–400 contacts per condition). Bar, 20 µm.

Techniques Used: Transfection, Cell Culture, Mutagenesis, Fluorescence

Mutations of E 726 and E 733 affect integrin activation, Tal-H binding, and Tal-H–dependent integrin clustering. (A) β3-EGFP-integrin cytoplasmic tail sequence with critical amino acids involved in integrin activation. (B) Soluble integrin ligand binding capacity (integrin activation index; n > 3; SEM) of different integrin mutants. (C) GST–Tal-H pull-down of wild-type (wt) and mutant β3-EGFP-integrins from lysates of transiently transfected COS-7 cells, involving DelW739-T762, W739A/Y747A, D723A/E726A/E733A, and D723K/E726K and as controls, α6-EGFP-integrin and the high-affinity NPLY integrin mutation (SPLH) according to Wegener et al. (2007) . (D–I) Representative TIRF images (EGFP channel) of Mn 2+ -stimulated B16F1 cells cotransfected with wild-type ECFP–Tal-H (insets) and wild-type (D) or mutant β3-EGFP-integrin Y747A (E), F730A (F), E726K (G), E733K (H), or E726K/E733K (I) and cultured on serum-coated coverslips. (J) Mean cell surface reactivity with anti–β3-integrin mAb ( n > 3; SEM), as measured by FACS. Note that endogenous β3-integrin levels correspond to 22% of wild-type β3-EGFP-integrin–transfected cells. (K) Mean clustering index ( n > 25 cells; SD) taken from one representative out of three similar experiments. Bar, 20 µm.
Figure Legend Snippet: Mutations of E 726 and E 733 affect integrin activation, Tal-H binding, and Tal-H–dependent integrin clustering. (A) β3-EGFP-integrin cytoplasmic tail sequence with critical amino acids involved in integrin activation. (B) Soluble integrin ligand binding capacity (integrin activation index; n > 3; SEM) of different integrin mutants. (C) GST–Tal-H pull-down of wild-type (wt) and mutant β3-EGFP-integrins from lysates of transiently transfected COS-7 cells, involving DelW739-T762, W739A/Y747A, D723A/E726A/E733A, and D723K/E726K and as controls, α6-EGFP-integrin and the high-affinity NPLY integrin mutation (SPLH) according to Wegener et al. (2007) . (D–I) Representative TIRF images (EGFP channel) of Mn 2+ -stimulated B16F1 cells cotransfected with wild-type ECFP–Tal-H (insets) and wild-type (D) or mutant β3-EGFP-integrin Y747A (E), F730A (F), E726K (G), E733K (H), or E726K/E733K (I) and cultured on serum-coated coverslips. (J) Mean cell surface reactivity with anti–β3-integrin mAb ( n > 3; SEM), as measured by FACS. Note that endogenous β3-integrin levels correspond to 22% of wild-type β3-EGFP-integrin–transfected cells. (K) Mean clustering index ( n > 25 cells; SD) taken from one representative out of three similar experiments. Bar, 20 µm.

Techniques Used: Activation Assay, Binding Assay, Sequencing, Ligand Binding Assay, Mutagenesis, Transfection, Cell Culture, FACS

Mutations in the PI(4,5)P 2 -binding sites of Tal-H affect integrin clustering. (A–C) Representative TIRF images (EGFP channel) of Mn 2+ -stimulated B16F1 cells grown on serum-coated coverslips and double transfected with β3-EGFP-integrin and ECFP–Tal-H carrying PI(4,5)P 2 -binding mutations K320A/K322A (A), K322A/K324A (B), and K272A/K274Q/R277E (C). Magnified views of the boxed areas and ECFP–Tal-H expression are shown in the insets. (D and E) Averaged ( n > 25) intensity histograms (D) and mean clustering index ( n > 3; SEM; E) of β3-EGFP-integrin fluorescence as a function of Tal-H expression. The vertical dashed line in D represents the fluorescence intensity threshold ( > 200 12-bit gray levels) that was used to calculate the integrin clustering index. (F) Scheme of basic regions involved in Tal-R interaction, PI(4,5)P 2 binding, and integrin clustering. (G) Overlay of the proposed structure of the Tal-R–Tal-F3 complex (PDB ID 2KGX ; Goult et al., 2009 ) with the Tal-F2/3 structure (PDB ID 1MK7 ; García-Alvarez et al., 2003 ), indicating PI(4,5)P 2 (K 272 , K 274 , R 277 , K 322 , and K 324 ) as well as Tal-R–binding residues (K 318 , K 320 [hidden], K 322 [hidden], and K 324 ; Goult et al., 2009 ). wt, wild type. Bar, 20 µm.
Figure Legend Snippet: Mutations in the PI(4,5)P 2 -binding sites of Tal-H affect integrin clustering. (A–C) Representative TIRF images (EGFP channel) of Mn 2+ -stimulated B16F1 cells grown on serum-coated coverslips and double transfected with β3-EGFP-integrin and ECFP–Tal-H carrying PI(4,5)P 2 -binding mutations K320A/K322A (A), K322A/K324A (B), and K272A/K274Q/R277E (C). Magnified views of the boxed areas and ECFP–Tal-H expression are shown in the insets. (D and E) Averaged ( n > 25) intensity histograms (D) and mean clustering index ( n > 3; SEM; E) of β3-EGFP-integrin fluorescence as a function of Tal-H expression. The vertical dashed line in D represents the fluorescence intensity threshold ( > 200 12-bit gray levels) that was used to calculate the integrin clustering index. (F) Scheme of basic regions involved in Tal-R interaction, PI(4,5)P 2 binding, and integrin clustering. (G) Overlay of the proposed structure of the Tal-R–Tal-F3 complex (PDB ID 2KGX ; Goult et al., 2009 ) with the Tal-F2/3 structure (PDB ID 1MK7 ; García-Alvarez et al., 2003 ), indicating PI(4,5)P 2 (K 272 , K 274 , R 277 , K 322 , and K 324 ) as well as Tal-R–binding residues (K 318 , K 320 [hidden], K 322 [hidden], and K 324 ; Goult et al., 2009 ). wt, wild type. Bar, 20 µm.

Techniques Used: Binding Assay, Transfection, Expressing, Fluorescence

The F2 and F3 domains of Tal-H are required for β3-integrin clustering. (A and B) Scheme of the Tal-dependent β3-integrin clustering protocol (A) and ECFP-tagged Tal fragments (B). (C) Western blot with anti-EGFP antibodies of equivalent amounts of cell lysates (50% for ECFP) of transiently transfected B16F1 cells. (D–I) Representative TIRF images (EGFP channel) of Mn 2+ -stimulated B16F1 cells plated on serum-coated coverslips and double transfected with β3-EGFP-integrin (D–I) and ECFP–FL-Tal (D), ECFP–Tal-H (E), ECFP–Tal-F2/3 (F), ECFP–Tal-F3 (G), ECFP-tagged Tal-R (H), and ECFP only (I). Magnified views of the boxed areas and ECFP epifluorescence are shown in the insets. (J) Averaged ( n > 25) fluorescence intensity histograms of β3-EGFP-integrin transfected cells as shown in D–I. The dashed line represents the fluorescence intensity threshold ( > 200 12-bit gray levels) that was used to calculate the integrin clustering index (K). Histograms are from one representative experiment, whereas the integrin clustering index (K) is the mean of n > 3 (SEM) experiments. Bars: (D and E) 25 µm; (F–I) 20 µm.
Figure Legend Snippet: The F2 and F3 domains of Tal-H are required for β3-integrin clustering. (A and B) Scheme of the Tal-dependent β3-integrin clustering protocol (A) and ECFP-tagged Tal fragments (B). (C) Western blot with anti-EGFP antibodies of equivalent amounts of cell lysates (50% for ECFP) of transiently transfected B16F1 cells. (D–I) Representative TIRF images (EGFP channel) of Mn 2+ -stimulated B16F1 cells plated on serum-coated coverslips and double transfected with β3-EGFP-integrin (D–I) and ECFP–FL-Tal (D), ECFP–Tal-H (E), ECFP–Tal-F2/3 (F), ECFP–Tal-F3 (G), ECFP-tagged Tal-R (H), and ECFP only (I). Magnified views of the boxed areas and ECFP epifluorescence are shown in the insets. (J) Averaged ( n > 25) fluorescence intensity histograms of β3-EGFP-integrin transfected cells as shown in D–I. The dashed line represents the fluorescence intensity threshold ( > 200 12-bit gray levels) that was used to calculate the integrin clustering index (K). Histograms are from one representative experiment, whereas the integrin clustering index (K) is the mean of n > 3 (SEM) experiments. Bars: (D and E) 25 µm; (F–I) 20 µm.

Techniques Used: Western Blot, Transfection, Fluorescence

Complementation of integrin clustering by charge-inversion mutants. (A–D) Representative TIRF images (EGFP channel) of Mn 2+ -stimulated B16F1 cells cultured on serum-coated coverslips and coexpressing wild-type (wt; A and B) or E726K mutant (C and D) β3-EGFP-integrin together with wild-type (A and C) or K316E mutant (B and D) ECFP–Tal-H. Magnified views of the boxed areas and ECFP–Tal-H expression by epifluorescence are shown in the insets. Note the extensive integrin clustering in the wild-type/wild-type (A) and E726K/K316E condition (D). (E) Averaged ( n > 25) histograms of cells as shown in A–D. The dashed vertical line indicates the threshold used to calculate the integrin clustering index. (F) Mean integrin clustering index ( n > 3; SEM) of conditions as in A–D. Bar, 20 µm.
Figure Legend Snippet: Complementation of integrin clustering by charge-inversion mutants. (A–D) Representative TIRF images (EGFP channel) of Mn 2+ -stimulated B16F1 cells cultured on serum-coated coverslips and coexpressing wild-type (wt; A and B) or E726K mutant (C and D) β3-EGFP-integrin together with wild-type (A and C) or K316E mutant (B and D) ECFP–Tal-H. Magnified views of the boxed areas and ECFP–Tal-H expression by epifluorescence are shown in the insets. Note the extensive integrin clustering in the wild-type/wild-type (A) and E726K/K316E condition (D). (E) Averaged ( n > 25) histograms of cells as shown in A–D. The dashed vertical line indicates the threshold used to calculate the integrin clustering index. (F) Mean integrin clustering index ( n > 3; SEM) of conditions as in A–D. Bar, 20 µm.

Techniques Used: Cell Culture, Mutagenesis, Expressing

12) Product Images from "An expression system for screening of proteins for glycan and protein interactions"

Article Title: An expression system for screening of proteins for glycan and protein interactions

Journal: Analytical Biochemistry

doi: 10.1016/j.ab.2010.12.036

Binding of polymeric glycan probes to siglec-expressing CHO cells. Either nontransfected control cells (CHO) or CHO cells expressing siglec-7 (S7), siglec-8 (S8), or siglec-9 (S9) were incubated with biotinylated PAA probes carrying either lactose, Sia2,8Sia, SLe x , or 6′SU-SLe x and binding detected with streptavidin–APC. See Table 1 for carbohydrate sequences.
Figure Legend Snippet: Binding of polymeric glycan probes to siglec-expressing CHO cells. Either nontransfected control cells (CHO) or CHO cells expressing siglec-7 (S7), siglec-8 (S8), or siglec-9 (S9) were incubated with biotinylated PAA probes carrying either lactose, Sia2,8Sia, SLe x , or 6′SU-SLe x and binding detected with streptavidin–APC. See Table 1 for carbohydrate sequences.

Techniques Used: Binding Assay, Expressing, Incubation

Binding of biotinylated JAM-B–EGFP and JAM-C–EGFP chimeras to CHO cells expressing JAM-B and JAM-C. (A) Western blot of biotinylated JAM-C or JAM-B probed with streptavidin–alkaline phosphatase shows the presence of a single species for each protein at the expected molecular weight. (B) Wild-type CHO cells (black line) or CHO cells expressing JAM-B (red and green lines) were incubated with either biotinylated JAM-C–EGFP (black and green lines) or biotinylated JAM-B–EGFP (red line) at 1 μg/ml. Binding was detected with streptavidin–APC, and cells were analyzed by flow cytometry. (C) Wild-type CHO cells (black line) or CHO cells expressing JAM-C (red and green lines) were incubated with either biotinylated JAM-B–EGFP (black and green lines) or biotinylated JAM-C–EGFP (red line). Binding was detected with streptavidin–APC, and cells were analyzed by flow cytometry. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Figure Legend Snippet: Binding of biotinylated JAM-B–EGFP and JAM-C–EGFP chimeras to CHO cells expressing JAM-B and JAM-C. (A) Western blot of biotinylated JAM-C or JAM-B probed with streptavidin–alkaline phosphatase shows the presence of a single species for each protein at the expected molecular weight. (B) Wild-type CHO cells (black line) or CHO cells expressing JAM-B (red and green lines) were incubated with either biotinylated JAM-C–EGFP (black and green lines) or biotinylated JAM-B–EGFP (red line) at 1 μg/ml. Binding was detected with streptavidin–APC, and cells were analyzed by flow cytometry. (C) Wild-type CHO cells (black line) or CHO cells expressing JAM-C (red and green lines) were incubated with either biotinylated JAM-B–EGFP (black and green lines) or biotinylated JAM-C–EGFP (red line). Binding was detected with streptavidin–APC, and cells were analyzed by flow cytometry. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Techniques Used: Binding Assay, Expressing, Western Blot, Molecular Weight, Incubation, Flow Cytometry, Cytometry

Expression of EGFP fusion proteins in CHO cells measured by flow cytometry. Cell surface expression of EGFP fusion proteins was measured on living cells using biotinylated anti-GFP followed by streptavidin–APC and detected on the FL-4 channel (emission maximum 660 nm). Total endogenous GFP was detected on the FL-1 channel (emission maximum 509 nm). CHO cells were either nontransfected (CHO) or CHO cells expressing siglec-7 (S7), siglec-8 (S8), siglec-9 (S9), BACE, JAM-B, or JAM-C.
Figure Legend Snippet: Expression of EGFP fusion proteins in CHO cells measured by flow cytometry. Cell surface expression of EGFP fusion proteins was measured on living cells using biotinylated anti-GFP followed by streptavidin–APC and detected on the FL-4 channel (emission maximum 660 nm). Total endogenous GFP was detected on the FL-1 channel (emission maximum 509 nm). CHO cells were either nontransfected (CHO) or CHO cells expressing siglec-7 (S7), siglec-8 (S8), siglec-9 (S9), BACE, JAM-B, or JAM-C.

Techniques Used: Expressing, Flow Cytometry, Cytometry

13) Product Images from "The Modulation of CD40 Ligand Signaling by Transmembrane CD28 Splice Variant in Human T Cells"

Article Title: The Modulation of CD40 Ligand Signaling by Transmembrane CD28 Splice Variant in Human T Cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20031705

Poor induction of IL-2 promoter activity by cross-linking CD40L in D1.1 transfectant cells. CD28i-HA was induced (48 h) in D1.1 transfectant cells with doxicyclin treatment. Cells were loaded with an IL-2 promoter reporter plasmid for 6 h. Cells were then stimulated with PHA plus PMA in the presence of anti-CD40L Ab or anti-CD28 Ab for 6 h. Cell extracts were assayed for IL-2 promoter activity by luciferase assays. Results are representative of three experiments. Data are the average of triplicate cultures with SDs.
Figure Legend Snippet: Poor induction of IL-2 promoter activity by cross-linking CD40L in D1.1 transfectant cells. CD28i-HA was induced (48 h) in D1.1 transfectant cells with doxicyclin treatment. Cells were loaded with an IL-2 promoter reporter plasmid for 6 h. Cells were then stimulated with PHA plus PMA in the presence of anti-CD40L Ab or anti-CD28 Ab for 6 h. Cell extracts were assayed for IL-2 promoter activity by luciferase assays. Results are representative of three experiments. Data are the average of triplicate cultures with SDs.

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Luciferase

(a) Enhanced induction of NF-κB activation by CD40L cross-linking in CD28i-HA–overexpressing D1.1 cells. CD28i-HA–induced (48 h) or –noninduced D1.1 cell was loaded with NF-κB reporter plasmid. Cells were stimulated with anti-CD40L Ab for 6 h and then the cell extracts were measured for NF-κB activity by luciferase assay. Results are representative of three experiments. Data are the average of triplicate cultures with SDs. (b) Enhanced induction of NF-κB activation by CD40L cross-linking in HEK293 CD28i-HA transfectant cells. HEK293 cells were transfected with CD28i and CD40L, and were loaded with NF-κB reporter plasmid. Cells were stimulated with anti-CD40L Ab for 6 h and then the cell extracts were measured for NF-κB activity by luciferase assay. Results are representative of two experiments. Data are the average of triplicate cultures with SDs.
Figure Legend Snippet: (a) Enhanced induction of NF-κB activation by CD40L cross-linking in CD28i-HA–overexpressing D1.1 cells. CD28i-HA–induced (48 h) or –noninduced D1.1 cell was loaded with NF-κB reporter plasmid. Cells were stimulated with anti-CD40L Ab for 6 h and then the cell extracts were measured for NF-κB activity by luciferase assay. Results are representative of three experiments. Data are the average of triplicate cultures with SDs. (b) Enhanced induction of NF-κB activation by CD40L cross-linking in HEK293 CD28i-HA transfectant cells. HEK293 cells were transfected with CD28i and CD40L, and were loaded with NF-κB reporter plasmid. Cells were stimulated with anti-CD40L Ab for 6 h and then the cell extracts were measured for NF-κB activity by luciferase assay. Results are representative of two experiments. Data are the average of triplicate cultures with SDs.

Techniques Used: Activation Assay, Plasmid Preparation, Activity Assay, Luciferase, Transfection

(a) Tyrosine phosphorylation of CD28i induced by CD40L stimulation. CD28-HA–transfected D1.1 cells were serum starved for 6 h and incubated with anti-CD40L Ab for 30 min on ice. Anti–goat IgG was added to start stimulation at 37°C for 2, 5, and 10 min. Nonidet P-40 cell lysates of the stimulated cells were immunoprecipitated with anti-HA Ab. Immunoprecipitates were analyzed on Western blots. The top panel was blotted with anti-Phosphotyrosine (RC20; indicated as p-Tyr). The same membrane was reprobed with anti-HA Ab (indicated as CD28i-HA). L, Ig light chain; lane 1, nonstimulated control; lane 2, CD40L stimulated for 2 min; lane 3, CD40L stimulated for 5 min; lane 4, CD40L stimulated for 10 min. (b) Induction of CD28i-HA expression by doxicyclin in D1.1 transfectant cells. CD28i-HA (∼23 kD) was expressed in D1.1 cells by doxicyclin-inducible promoter and whole cell lysates were assayed by HA-specific Western blotting. (−), doxicyclin-nontreated cells; (+), doxicyclin-treated cells (0.5 μg/ml for 48 h). (c) Overexpression of CD28i-HA enhances the activation of JNK and PAK2 in CD40L-stimulated D1.1 cells. Cells were stimulated with anti-CD40L Ab for periods of time indicated on top. Whole cell lysates were characterized with Western blotting specific for active-form JNK (represented by p46), PAK2 (∼64 kD), or Akt (∼60 kD), and indicated as pJNK, pPAK2, and pAkt on the left of each panel. To measure the level of proteins, the assay membranes were reprobed with JNK-, PAK-, or Akt-specific Abs and indicated as JNK, PAK2, or Akt on the left of each panel. Dox (−), cells not treated with doxicyclin; Dox (+), cells treated with doxicyclin.
Figure Legend Snippet: (a) Tyrosine phosphorylation of CD28i induced by CD40L stimulation. CD28-HA–transfected D1.1 cells were serum starved for 6 h and incubated with anti-CD40L Ab for 30 min on ice. Anti–goat IgG was added to start stimulation at 37°C for 2, 5, and 10 min. Nonidet P-40 cell lysates of the stimulated cells were immunoprecipitated with anti-HA Ab. Immunoprecipitates were analyzed on Western blots. The top panel was blotted with anti-Phosphotyrosine (RC20; indicated as p-Tyr). The same membrane was reprobed with anti-HA Ab (indicated as CD28i-HA). L, Ig light chain; lane 1, nonstimulated control; lane 2, CD40L stimulated for 2 min; lane 3, CD40L stimulated for 5 min; lane 4, CD40L stimulated for 10 min. (b) Induction of CD28i-HA expression by doxicyclin in D1.1 transfectant cells. CD28i-HA (∼23 kD) was expressed in D1.1 cells by doxicyclin-inducible promoter and whole cell lysates were assayed by HA-specific Western blotting. (−), doxicyclin-nontreated cells; (+), doxicyclin-treated cells (0.5 μg/ml for 48 h). (c) Overexpression of CD28i-HA enhances the activation of JNK and PAK2 in CD40L-stimulated D1.1 cells. Cells were stimulated with anti-CD40L Ab for periods of time indicated on top. Whole cell lysates were characterized with Western blotting specific for active-form JNK (represented by p46), PAK2 (∼64 kD), or Akt (∼60 kD), and indicated as pJNK, pPAK2, and pAkt on the left of each panel. To measure the level of proteins, the assay membranes were reprobed with JNK-, PAK-, or Akt-specific Abs and indicated as JNK, PAK2, or Akt on the left of each panel. Dox (−), cells not treated with doxicyclin; Dox (+), cells treated with doxicyclin.

Techniques Used: Transfection, Incubation, Immunoprecipitation, Western Blot, Expressing, Over Expression, Activation Assay

(a) The colocalization of CD28i with CD40L monitored by confocal microscopy. CD40L + Jurkat cell subline, D1.1 cell, was transfected with EGFP or CD28i-EGFP as indicated. In A, cells were surface stained with PE anti-CD40L Ab, PE anti-CD28 Ab, PE anti-CD2 Ab, or PE anti-CD3 as indicated below. In B, green fluorescence by EGFP or CD28i-EGFP expression was shown. In C, green fluorescence and red fluorescence were merged. (b) Anti-CD40L Ab induced coendocytosis of CD40L and CD28i. D1.1 cell was transfected with CD28i-GFP and incubated with PE anti-CD40L Ab at 37°C (top) or 4°C (bottom) for 30 min. Then cells were assayed by confocal microscopy. In panel a, CD40L and CD28i are merged. The magnified views of coendocytosis or control are shown in panel b. Panel c shows CD40L and panel d shows CD28i-EGFP staining.
Figure Legend Snippet: (a) The colocalization of CD28i with CD40L monitored by confocal microscopy. CD40L + Jurkat cell subline, D1.1 cell, was transfected with EGFP or CD28i-EGFP as indicated. In A, cells were surface stained with PE anti-CD40L Ab, PE anti-CD28 Ab, PE anti-CD2 Ab, or PE anti-CD3 as indicated below. In B, green fluorescence by EGFP or CD28i-EGFP expression was shown. In C, green fluorescence and red fluorescence were merged. (b) Anti-CD40L Ab induced coendocytosis of CD40L and CD28i. D1.1 cell was transfected with CD28i-GFP and incubated with PE anti-CD40L Ab at 37°C (top) or 4°C (bottom) for 30 min. Then cells were assayed by confocal microscopy. In panel a, CD40L and CD28i are merged. The magnified views of coendocytosis or control are shown in panel b. Panel c shows CD40L and panel d shows CD28i-EGFP staining.

Techniques Used: Confocal Microscopy, Transfection, Staining, Fluorescence, Expressing, Incubation

Coimmunoprecipitation of CD28i with CD40L from 1% digitonin cell extracts. (a) Cell extracts from CD28i-HA–transfected D1.1 cells were prepared using 1% digitonin buffer and immunoprecipitated with anti-CD40L Ab, anti-CD28 Ab, or isotype control Ab. Immunoprecipitates were fractionated by SDS-PAGE. Western blot membranes were probed with anti-HA Ab. (b) The same extracts were immunoprecipitated with anti-HA Ab, anti-CD40L Ab, or isotype control Ab, and fractionated by SDS-PAGE. Subsequently, the CD40L-specific Western blotting was performed. (c and d) 1% digitonin cell extracts from 72-h PHA blasts of peripheral blood mononuclear cells were immunoprecipitated by CD28- or CD40L-specific Abs and then characterized by Western blotting specific to CD28 COOH terminus (c) (reference 16 ) or CD40L (d). Specific Abs and isotype control Ab used for immunoprecipitation were indicated under each lane. In b, the bracket shows the doublet of CD40L (reference 30 ). C-terminal in the figure stands for CD28 COOH terminus–specific Ab. Filled arrows indicate CD28i-HA in a, CD40L in b and d, and CD28i in c. H, Ig heavy chain; L, Ig light chain.
Figure Legend Snippet: Coimmunoprecipitation of CD28i with CD40L from 1% digitonin cell extracts. (a) Cell extracts from CD28i-HA–transfected D1.1 cells were prepared using 1% digitonin buffer and immunoprecipitated with anti-CD40L Ab, anti-CD28 Ab, or isotype control Ab. Immunoprecipitates were fractionated by SDS-PAGE. Western blot membranes were probed with anti-HA Ab. (b) The same extracts were immunoprecipitated with anti-HA Ab, anti-CD40L Ab, or isotype control Ab, and fractionated by SDS-PAGE. Subsequently, the CD40L-specific Western blotting was performed. (c and d) 1% digitonin cell extracts from 72-h PHA blasts of peripheral blood mononuclear cells were immunoprecipitated by CD28- or CD40L-specific Abs and then characterized by Western blotting specific to CD28 COOH terminus (c) (reference 16 ) or CD40L (d). Specific Abs and isotype control Ab used for immunoprecipitation were indicated under each lane. In b, the bracket shows the doublet of CD40L (reference 30 ). C-terminal in the figure stands for CD28 COOH terminus–specific Ab. Filled arrows indicate CD28i-HA in a, CD40L in b and d, and CD28i in c. H, Ig heavy chain; L, Ig light chain.

Techniques Used: Transfection, Immunoprecipitation, SDS Page, Western Blot

14) Product Images from "Short-term Oral Antibiotics Treatment Promotes Inflammatory Activation of Colonic Invariant Natural Killer T and Conventional CD4+ T Cells"

Article Title: Short-term Oral Antibiotics Treatment Promotes Inflammatory Activation of Colonic Invariant Natural Killer T and Conventional CD4+ T Cells

Journal: Frontiers in Medicine

doi: 10.3389/fmed.2018.00021

Antibiotic treatment influences colonic invariant natural killer T (iNKT) cell frequency and function (A–C) representative dot plots (A) , cumulative frequency (B) , and absolute numbers (C) of iNKT cells (upper panels) and CD4 + T cells (lower panels) in untreated mice (open circles), ABX-treated mice (closed circles), mice reconstituted with eubiotic fecal microbiota transplantation (FMT) (open squares), or with microbiota from DSS-treated mice (dysbiotic FMT, closed squares). (D) Absolute numbers of CD69 + cells among iNKT cells (upper panels) and CD4 + T cells (lower panels) in untreated mice (white bars), ABX-treated mice (light gray bars), mice reconstituted with eubiotic FMT (dark gray bars), or with microbiota from DSS-treated mice (dysbiotic FMT, black bars) (E) Cytokine production by iNKT cells (upper panels) and CD4 + T cells (lower panels) in untreated, ABX-treated, reconstituted with eubiotic or with dysbiotic FMT. Histograms normalized to 100% of production of total cytokines. Significance was determined using Kruskal–Wallis nonparametric test and expressed as mean SEM. Untreated n = 8, ABX-treated n = 10, reconstituted with eubiotic FMT n = 9, with dysbiotic FMT n = 11 or in DSS-treated n = 11 mice in four independent experiments. Outliers detected with Grubb’s test. P
Figure Legend Snippet: Antibiotic treatment influences colonic invariant natural killer T (iNKT) cell frequency and function (A–C) representative dot plots (A) , cumulative frequency (B) , and absolute numbers (C) of iNKT cells (upper panels) and CD4 + T cells (lower panels) in untreated mice (open circles), ABX-treated mice (closed circles), mice reconstituted with eubiotic fecal microbiota transplantation (FMT) (open squares), or with microbiota from DSS-treated mice (dysbiotic FMT, closed squares). (D) Absolute numbers of CD69 + cells among iNKT cells (upper panels) and CD4 + T cells (lower panels) in untreated mice (white bars), ABX-treated mice (light gray bars), mice reconstituted with eubiotic FMT (dark gray bars), or with microbiota from DSS-treated mice (dysbiotic FMT, black bars) (E) Cytokine production by iNKT cells (upper panels) and CD4 + T cells (lower panels) in untreated, ABX-treated, reconstituted with eubiotic or with dysbiotic FMT. Histograms normalized to 100% of production of total cytokines. Significance was determined using Kruskal–Wallis nonparametric test and expressed as mean SEM. Untreated n = 8, ABX-treated n = 10, reconstituted with eubiotic FMT n = 9, with dysbiotic FMT n = 11 or in DSS-treated n = 11 mice in four independent experiments. Outliers detected with Grubb’s test. P

Techniques Used: Mouse Assay, Transplantation Assay

T cell responses are affected by the origin of the transplanted microbiota. (A) Colonic expression levels of cxcl16 in mice reconstituted with eubiotic bacteria and unchallenged (white bars), reconstituted with eubiotic bacteria and DSS-treated (light gray bars), reconstituted with dysbiotic bacteria and DSS-treated (dark gray bars) and DSS-treated (black bars). (B) Absolute numbers of colonic invariant natural killer T (iNKT) cells (white bars) and CD4 + T cells (black bars) in the indicated experimental groups. (C,E) CD69 absolute numbers of colonic iNKT cells (C) and of colonic CD4 + T cells (E) . (D,F) Cytokine production by iNKT cells (D) and of CD4 + T cells (F) in the indicated experimental groups. Histograms normalized to 100% of production of total cytokines. Significance was determined by Kruskal–Wallis nonparametric test. Eubiotic fecal microbiota transplantation (FMT) + H 2 0 n = 6, eubiotic FMT + DSS n = 4, dysbiotic FMT + DSS n = 4, or DSS-treated n = 5 mice, two independent experiments. Outliers detected with Grubb’s test. P
Figure Legend Snippet: T cell responses are affected by the origin of the transplanted microbiota. (A) Colonic expression levels of cxcl16 in mice reconstituted with eubiotic bacteria and unchallenged (white bars), reconstituted with eubiotic bacteria and DSS-treated (light gray bars), reconstituted with dysbiotic bacteria and DSS-treated (dark gray bars) and DSS-treated (black bars). (B) Absolute numbers of colonic invariant natural killer T (iNKT) cells (white bars) and CD4 + T cells (black bars) in the indicated experimental groups. (C,E) CD69 absolute numbers of colonic iNKT cells (C) and of colonic CD4 + T cells (E) . (D,F) Cytokine production by iNKT cells (D) and of CD4 + T cells (F) in the indicated experimental groups. Histograms normalized to 100% of production of total cytokines. Significance was determined by Kruskal–Wallis nonparametric test. Eubiotic fecal microbiota transplantation (FMT) + H 2 0 n = 6, eubiotic FMT + DSS n = 4, dysbiotic FMT + DSS n = 4, or DSS-treated n = 5 mice, two independent experiments. Outliers detected with Grubb’s test. P

Techniques Used: Expressing, Mouse Assay, Transplantation Assay

Dysbiotic microbiota effects after antibiotic treatment on colonic invariant natural killer T (iNKT) cells are time-dependent. (A) Colonic expression levels of cxcl16 in mice treated with antibiotics and treated with DSS (white bars), transplanted with eubiotic bacteria and DSS-treated (light gray bars), transplanted with dysbiotic bacteria, and DSS-treated (dark gray bars) and DSS-treated (black bars). (B) Absolute numbers of colonic iNKT cells (white bars) and CD4 + T cells (black bars) in the indicated experimental groups. (C,E) CD69 absolute numbers of colonic iNKT cells (C) and of colonic CD4 + T cells (E) . (D,F) Cytokine production by iNKT cells (D) and CD4 + T cells (F) in the indicated experimental groups. Histograms normalized to 100% of production of total cytokines. Significance determined using Kruskal–Wallis nonparametric test and expressed as mean SEM. ABX + DSS n = 5, eubiotic FMT + DSS n = 6, dysbiotic FMT + DSS n = 7, DSS-treated n = 6 mice, two independent experiments. Outliers detected with Grubb’s test. P
Figure Legend Snippet: Dysbiotic microbiota effects after antibiotic treatment on colonic invariant natural killer T (iNKT) cells are time-dependent. (A) Colonic expression levels of cxcl16 in mice treated with antibiotics and treated with DSS (white bars), transplanted with eubiotic bacteria and DSS-treated (light gray bars), transplanted with dysbiotic bacteria, and DSS-treated (dark gray bars) and DSS-treated (black bars). (B) Absolute numbers of colonic iNKT cells (white bars) and CD4 + T cells (black bars) in the indicated experimental groups. (C,E) CD69 absolute numbers of colonic iNKT cells (C) and of colonic CD4 + T cells (E) . (D,F) Cytokine production by iNKT cells (D) and CD4 + T cells (F) in the indicated experimental groups. Histograms normalized to 100% of production of total cytokines. Significance determined using Kruskal–Wallis nonparametric test and expressed as mean SEM. ABX + DSS n = 5, eubiotic FMT + DSS n = 6, dysbiotic FMT + DSS n = 7, DSS-treated n = 6 mice, two independent experiments. Outliers detected with Grubb’s test. P

Techniques Used: Expressing, Mouse Assay

15) Product Images from "Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases"

Article Title: Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003772

PLP2 is required for K3 and K5 to ubiquitinate their substrate MHC-I. ( A ) Schematic representation of PLP2 topology. ( B–D ) K3 and K5 are unable to ubiquitinate MHC-I in the absence of PLP2. In ( B ), the indicated cell lines were pulse-labelled for 10 minutes with [ 35 S]-methionine, chased for 45 minutes, and MHC-I immunoprecipitated with the conformationally sensitive W6/32 mAb, and after dissociation and denaturation in 1% SDS, MHC-I molecules re-precipitated with the anti-MHC-I heavy chain mAb HC10, resolved by SDS-PAGE and analysed by autoradiography. In ( C ) and ( D ), MHC-I molecules were immunoprecipitated with the W6/32 mAb from the indicated cell types, and ubiquitinated MHC-I species were detected by immunoblot using the anti-ubiquitin antibody VU-1 (right panels) and the input cell lysates blotted for the indicated proteins (left panels).
Figure Legend Snippet: PLP2 is required for K3 and K5 to ubiquitinate their substrate MHC-I. ( A ) Schematic representation of PLP2 topology. ( B–D ) K3 and K5 are unable to ubiquitinate MHC-I in the absence of PLP2. In ( B ), the indicated cell lines were pulse-labelled for 10 minutes with [ 35 S]-methionine, chased for 45 minutes, and MHC-I immunoprecipitated with the conformationally sensitive W6/32 mAb, and after dissociation and denaturation in 1% SDS, MHC-I molecules re-precipitated with the anti-MHC-I heavy chain mAb HC10, resolved by SDS-PAGE and analysed by autoradiography. In ( C ) and ( D ), MHC-I molecules were immunoprecipitated with the W6/32 mAb from the indicated cell types, and ubiquitinated MHC-I species were detected by immunoblot using the anti-ubiquitin antibody VU-1 (right panels) and the input cell lysates blotted for the indicated proteins (left panels).

Techniques Used: Immunoprecipitation, SDS Page, Autoradiography

PLP2 is required for the export of a K3•MHC-I complex from the ER. ( A ) Validation of the cell lines used for the experiment. FLAG-K3 and PLP2 were immunoprecipitated from the indicated [ 35 S]-methionine pulse-labelled cell types, resolved by SDS-PAGE and analysed by autoradiography. ( B ) K3-bound MHC-I remains EndoH-sensitive in the absence of PLP2. The indicated cell types were [ 35 S]-methionine pulse-labelled for 12 min, chased for the indicated times and solubilised in 1% digitonin. FLAG-K3 was immunoprecipitated using the anti-FLAG M2 mAb, dissociated in 1% SDS, MHC-I molecules re-precipitated with the anti-MHC-I heavy chain mAb HC10 and then either EndoH (+) or mock digested (−) before the samples were resolved by SDS-PAGE and analysed by autoradiography. ( C ) Loss of PLP2 does not affect the normal MHC-I synthetic pathway. Free [ 35 S]-methionine labelled MHC-I molecules were immunoprecipitated from the post-M2 supernatants (from B) using the W6/32 mAb, EndoH (+) or mock digested (−), and analysed by SDS-PAGE and autoradiography. ( D ) PLP2 co-immunoprecipitates with K3 and K5. FLAG-K3 and K5 were immunoprecipitated from digitonin lysates of HeLa cells using the anti-FLAG M2 mAb, and co-immunoprecipitated PLP2 detected by immunoblot.
Figure Legend Snippet: PLP2 is required for the export of a K3•MHC-I complex from the ER. ( A ) Validation of the cell lines used for the experiment. FLAG-K3 and PLP2 were immunoprecipitated from the indicated [ 35 S]-methionine pulse-labelled cell types, resolved by SDS-PAGE and analysed by autoradiography. ( B ) K3-bound MHC-I remains EndoH-sensitive in the absence of PLP2. The indicated cell types were [ 35 S]-methionine pulse-labelled for 12 min, chased for the indicated times and solubilised in 1% digitonin. FLAG-K3 was immunoprecipitated using the anti-FLAG M2 mAb, dissociated in 1% SDS, MHC-I molecules re-precipitated with the anti-MHC-I heavy chain mAb HC10 and then either EndoH (+) or mock digested (−) before the samples were resolved by SDS-PAGE and analysed by autoradiography. ( C ) Loss of PLP2 does not affect the normal MHC-I synthetic pathway. Free [ 35 S]-methionine labelled MHC-I molecules were immunoprecipitated from the post-M2 supernatants (from B) using the W6/32 mAb, EndoH (+) or mock digested (−), and analysed by SDS-PAGE and autoradiography. ( D ) PLP2 co-immunoprecipitates with K3 and K5. FLAG-K3 and K5 were immunoprecipitated from digitonin lysates of HeLa cells using the anti-FLAG M2 mAb, and co-immunoprecipitated PLP2 detected by immunoblot.

Techniques Used: Immunoprecipitation, SDS Page, Autoradiography

16) Product Images from "Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling"

Article Title: Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0145451

Upregulation of candidate proteins upon BDNF treatment in astrocytes. (A) Neuron-glia cocultures (DIV 22), infected with LVGFAPEGFP-mMetRS L274G , were incubated with 4 mM ANL or 4 mM Met (Met) for 4 h with (BDNF) or without 50 ng/ml BDNF (CNTR). Cell lysates were tagged with a biotin-alkyne tag via 'click-chemistry'. Biotin-tagged proteins were affinity purified with NeutrAvidin agarose. ANL incorporation and subsequent biotin tagging allows enrichment of de novo synthesized proteins compared to the Met incorporation control (Met Eluate). Overall protein levels of Cx43 and Rpl10a are comparable among the three different conditions in the lysates (Input) representing the joined pool of both pre-existing and de novo synthesized proteins as well as in the unbound fraction (Sup). In contrast, BDNF application resulted in increased levels of de novo synthesized and thus biotin-tagged Cx43 (43 kDa; both lower bands represent unphosphorylated and monophosphorylated Cx43) and Rpl10a (25 kDa) on immunoblots after purification (Eluate; 3 independent experiments). (B) Neuron-glia cocultures (DIV 22) were treated with 50 ng/ml BDNF for 4 h. A positive immunocytochemical GM130 staining for the Golgi apparatus (ICC) was used as a mask to quantify Cx43 signal intensity. No difference in Cx43 signal intensities was found. Numbers at the X-axis indicate the number of cells included in the quantification (3 independent experiments, represented data are mean +/- SEM, student's t-test, p > 0.05). (C) Neuron-glia cocultures (DIV 22) were infected with LVGFAPEGFP and treated with 50 ng/ml BDNF for 4 h. Elevated Rpl10a positive signals were observed in GFAP-positive astrocytes by applying immunocytochemistry for Rpl10a and GFAP. Rpl10a signal intensities are color coded in the lower panel (scale bar = 10 μm). (D) Quantification of Rpl10a signal intensities, obtained as in (C), was done within the EGFP mask. A significant increase in Rpl10a signal intensity was observed. Numbers at the X-axis indicate the number of cells included in the quantification (5 independent experiments, represented data are mean +/- SEM, student's t-test, *: p
Figure Legend Snippet: Upregulation of candidate proteins upon BDNF treatment in astrocytes. (A) Neuron-glia cocultures (DIV 22), infected with LVGFAPEGFP-mMetRS L274G , were incubated with 4 mM ANL or 4 mM Met (Met) for 4 h with (BDNF) or without 50 ng/ml BDNF (CNTR). Cell lysates were tagged with a biotin-alkyne tag via 'click-chemistry'. Biotin-tagged proteins were affinity purified with NeutrAvidin agarose. ANL incorporation and subsequent biotin tagging allows enrichment of de novo synthesized proteins compared to the Met incorporation control (Met Eluate). Overall protein levels of Cx43 and Rpl10a are comparable among the three different conditions in the lysates (Input) representing the joined pool of both pre-existing and de novo synthesized proteins as well as in the unbound fraction (Sup). In contrast, BDNF application resulted in increased levels of de novo synthesized and thus biotin-tagged Cx43 (43 kDa; both lower bands represent unphosphorylated and monophosphorylated Cx43) and Rpl10a (25 kDa) on immunoblots after purification (Eluate; 3 independent experiments). (B) Neuron-glia cocultures (DIV 22) were treated with 50 ng/ml BDNF for 4 h. A positive immunocytochemical GM130 staining for the Golgi apparatus (ICC) was used as a mask to quantify Cx43 signal intensity. No difference in Cx43 signal intensities was found. Numbers at the X-axis indicate the number of cells included in the quantification (3 independent experiments, represented data are mean +/- SEM, student's t-test, p > 0.05). (C) Neuron-glia cocultures (DIV 22) were infected with LVGFAPEGFP and treated with 50 ng/ml BDNF for 4 h. Elevated Rpl10a positive signals were observed in GFAP-positive astrocytes by applying immunocytochemistry for Rpl10a and GFAP. Rpl10a signal intensities are color coded in the lower panel (scale bar = 10 μm). (D) Quantification of Rpl10a signal intensities, obtained as in (C), was done within the EGFP mask. A significant increase in Rpl10a signal intensity was observed. Numbers at the X-axis indicate the number of cells included in the quantification (5 independent experiments, represented data are mean +/- SEM, student's t-test, *: p

Techniques Used: Infection, Incubation, Affinity Purification, Synthesized, Western Blot, Purification, Staining, Immunocytochemistry

17) Product Images from "Human cytomegalovirus plasmid-based amplicon vector system for gene therapy"

Article Title: Human cytomegalovirus plasmid-based amplicon vector system for gene therapy

Journal: Genetic Vaccines and Therapy

doi: 10.1186/1479-0556-3-1

Flow cytometry analysis of human cord blood CD34 + cells infected with CMV amplicon containing stocks, virus, or uninfected cell control. TN9-8GF5 amplicon ( a,b ), CMV-EGFP (RC2.7EGFP) virus ( c ), CMV (Towne) infected ( d ), or control uninfected human cord blood CD34 cells ( e,f ), were stained 36 hours post-infection with PE-antiCD34 antibody ( a-e ), or were left unstained ( f ), and were analyzed for two-color cytometry analysis using a FACS Calibur instrument. The dot-plots are generated using Cell Quest software and reveal the EGFP + cells populations. Numbers in the upper right and lower right quadrants indicate percentage of the EGFP + CD34 + and EGFP + CD34 - cells respectively. A frequency lower than 0.01% is considered negative.
Figure Legend Snippet: Flow cytometry analysis of human cord blood CD34 + cells infected with CMV amplicon containing stocks, virus, or uninfected cell control. TN9-8GF5 amplicon ( a,b ), CMV-EGFP (RC2.7EGFP) virus ( c ), CMV (Towne) infected ( d ), or control uninfected human cord blood CD34 cells ( e,f ), were stained 36 hours post-infection with PE-antiCD34 antibody ( a-e ), or were left unstained ( f ), and were analyzed for two-color cytometry analysis using a FACS Calibur instrument. The dot-plots are generated using Cell Quest software and reveal the EGFP + cells populations. Numbers in the upper right and lower right quadrants indicate percentage of the EGFP + CD34 + and EGFP + CD34 - cells respectively. A frequency lower than 0.01% is considered negative.

Techniques Used: Flow Cytometry, Cytometry, Infection, Amplification, Staining, FACS, Generated, Software

Fluorescent Microscopic Analysis of TN9-8GF5 amplicon infected cells. Human fibroblast cells (HF) or human cord blood CD34 + cells were infected with TN9-8GF5 amplicon-containing stocks, or mock infected. Cells were observed at different time-points 24, 72 and 96 hrs post infection with TN9-8GF5 amplicon under the fluorescent microscope (Nikon TE2000 microscope). EGFP expressing fluorescent cells were observed in the TN9-8GF5 amplicon infected human fibroblast cells or human CD34+ cells at different time-points. Control uninfected cells were negative (not shown).
Figure Legend Snippet: Fluorescent Microscopic Analysis of TN9-8GF5 amplicon infected cells. Human fibroblast cells (HF) or human cord blood CD34 + cells were infected with TN9-8GF5 amplicon-containing stocks, or mock infected. Cells were observed at different time-points 24, 72 and 96 hrs post infection with TN9-8GF5 amplicon under the fluorescent microscope (Nikon TE2000 microscope). EGFP expressing fluorescent cells were observed in the TN9-8GF5 amplicon infected human fibroblast cells or human CD34+ cells at different time-points. Control uninfected cells were negative (not shown).

Techniques Used: Amplification, Infection, Microscopy, Expressing

18) Product Images from "The ER retention protein RER1 promotes alpha-synuclein degradation via the proteasome"

Article Title: The ER retention protein RER1 promotes alpha-synuclein degradation via the proteasome

Journal: PLoS ONE

doi: 10.1371/journal.pone.0184262

RER1 expression significantly reduces αSyn levels. (A) αSyn and RER1 were transiently overexpressed in HEK293. Cells were co-stained with αSyn (green) and RER1 (red) specific antibodies. The photos show that αSyn-immunoreactivity was reduced by wild type RER1 and less so by mutant RER1Δ25 expression. (B) Wild type RER1 expression significantly decreased αSyn levels (mean, 87.8 ±7.1%), but expression of RER1Δ25 resulted in significantly smaller effects on levels of αSyn (mean, 24.3 ±10.6%) (*p
Figure Legend Snippet: RER1 expression significantly reduces αSyn levels. (A) αSyn and RER1 were transiently overexpressed in HEK293. Cells were co-stained with αSyn (green) and RER1 (red) specific antibodies. The photos show that αSyn-immunoreactivity was reduced by wild type RER1 and less so by mutant RER1Δ25 expression. (B) Wild type RER1 expression significantly decreased αSyn levels (mean, 87.8 ±7.1%), but expression of RER1Δ25 resulted in significantly smaller effects on levels of αSyn (mean, 24.3 ±10.6%) (*p

Techniques Used: Expressing, Staining, Mutagenesis

Summary and putative model of RER1 effects on αSyn. 1) RER1 expression increases ER retrieval/retention of “immature” proteins in the cis-Golgi compartment which may contribute to ER retention of αSyn. 2) RER1 may indirectly retrieve αSyn back to the ER for degradation via the ERAD and proteasome (unfolded response system). 3) NEDD4 was found to interact with RER1. Although an E3 ligase, NEDD4 has been shown to reduce αSyn though the endosomal-lysosomal pathway [ 48 ]. RER1-mediated degradation of αSyn may also occur independent of ubiquitin via the 20S proteasome. 4) RER1 expression may act though maturation of an unknown protein (?) that mediates targeting and disposal of excess cytosolic αSyn via the proteasome (through an ubiquitin independent mechanism). ER, endoplasmic reticulum; ERAD, ER-associated degradation; ERGIC, ER-Golgi intermediate compartment.
Figure Legend Snippet: Summary and putative model of RER1 effects on αSyn. 1) RER1 expression increases ER retrieval/retention of “immature” proteins in the cis-Golgi compartment which may contribute to ER retention of αSyn. 2) RER1 may indirectly retrieve αSyn back to the ER for degradation via the ERAD and proteasome (unfolded response system). 3) NEDD4 was found to interact with RER1. Although an E3 ligase, NEDD4 has been shown to reduce αSyn though the endosomal-lysosomal pathway [ 48 ]. RER1-mediated degradation of αSyn may also occur independent of ubiquitin via the 20S proteasome. 4) RER1 expression may act though maturation of an unknown protein (?) that mediates targeting and disposal of excess cytosolic αSyn via the proteasome (through an ubiquitin independent mechanism). ER, endoplasmic reticulum; ERAD, ER-associated degradation; ERGIC, ER-Golgi intermediate compartment.

Techniques Used: Expressing, Activated Clotting Time Assay

Proteasome inhibition rescues αSyn levels. (A) 24h post transfection, cells were treated with 10 μM MG132 (MG) or 100 μM chloroquine (Chlor). MG132 treatment partially recovered RER1-mediated reduction of αSyn. In contrast, chloroquine did not rescue αSyn, but increased APP levels consistent with its effects on macroautophagy and the lysosome. (B) Chloroquine treatment blocks autophagy activity. Cells were treated with 10–100 μM Chlorquine for 24 h. Lipidated and sequesterted LC3-II increased by chloroquine treatment (top panel). Cells were transfected with GFP-LC3 and 24 h post transfection, cells were treated with 100 μM Chloroquine for 24 h. Diffused pattern of LC3-I decreased and punctated pattern of LC3-II increased by Chloroquine treatment (bottom panel; scale bar = 10 μm). (C) Cells were co-transfected with αSyn and either EGFP or RER1, and then 24h post transfection treated with DMSO, 100 nM Bafilomycin (Baf), 10 μM MG132 (MG), or 10 μM Eeyarestatin1 (Eer1). In cells co-transfected with EGFP control, MG132 did not increase αSyn levels compared to cells exposed to DMSO. In cells co-transfected with RER1, MG132 showed a similar partial recovery of RER1-mediated αSyn reduction (mean, 69.2%), whereas the macroautophagy and ERAD inhibitors, Bafilomycin and Eeyarestatin1, had no apparent effect (**p
Figure Legend Snippet: Proteasome inhibition rescues αSyn levels. (A) 24h post transfection, cells were treated with 10 μM MG132 (MG) or 100 μM chloroquine (Chlor). MG132 treatment partially recovered RER1-mediated reduction of αSyn. In contrast, chloroquine did not rescue αSyn, but increased APP levels consistent with its effects on macroautophagy and the lysosome. (B) Chloroquine treatment blocks autophagy activity. Cells were treated with 10–100 μM Chlorquine for 24 h. Lipidated and sequesterted LC3-II increased by chloroquine treatment (top panel). Cells were transfected with GFP-LC3 and 24 h post transfection, cells were treated with 100 μM Chloroquine for 24 h. Diffused pattern of LC3-I decreased and punctated pattern of LC3-II increased by Chloroquine treatment (bottom panel; scale bar = 10 μm). (C) Cells were co-transfected with αSyn and either EGFP or RER1, and then 24h post transfection treated with DMSO, 100 nM Bafilomycin (Baf), 10 μM MG132 (MG), or 10 μM Eeyarestatin1 (Eer1). In cells co-transfected with EGFP control, MG132 did not increase αSyn levels compared to cells exposed to DMSO. In cells co-transfected with RER1, MG132 showed a similar partial recovery of RER1-mediated αSyn reduction (mean, 69.2%), whereas the macroautophagy and ERAD inhibitors, Bafilomycin and Eeyarestatin1, had no apparent effect (**p

Techniques Used: Inhibition, Transfection, Activity Assay

RER1 effects are specific to αSyn. (A) RER1 overexpression decreased the levels of αSyn mutants (A30P, E46K, and A53T). (B) The levels of βSyn did not change with RER1 overexpression (p = 0.725) (n = 4/group). (C) Expression of αSyn Δ71–82 mutant which is unable to aggregate due to the lack of a corresponding middle hydrophobic region, is not significantly decreased by RER1 overexpression (F 2,15 = 2.214, p = 0.1438) (n = 6/group). (D) Overexpression of αSyn does not affect the maturation of APP or RER1 retrieval/retention function. C = control; R = RER1 transfected; G = EGFP
Figure Legend Snippet: RER1 effects are specific to αSyn. (A) RER1 overexpression decreased the levels of αSyn mutants (A30P, E46K, and A53T). (B) The levels of βSyn did not change with RER1 overexpression (p = 0.725) (n = 4/group). (C) Expression of αSyn Δ71–82 mutant which is unable to aggregate due to the lack of a corresponding middle hydrophobic region, is not significantly decreased by RER1 overexpression (F 2,15 = 2.214, p = 0.1438) (n = 6/group). (D) Overexpression of αSyn does not affect the maturation of APP or RER1 retrieval/retention function. C = control; R = RER1 transfected; G = EGFP

Techniques Used: Over Expression, Expressing, Mutagenesis, Transfection

RER1 colocalizes with αSyn in Lewy bodies. (A) Photomicrogaphs of tissue from control (left), and LB-positive tissues (right) show that RER1 colocalizes with αSyn in Lewy bodies. RER1 is detected in cell bodies, but appears enriched in round LB-like inclusions (arrows; see also enlargement in inset). (B) Photos show colocalization of both RER1 (green) and phosphorylated αSyn (pSer129: red) immunofluorescence in round, LB-like structures. Below are higher power images and confocal mapping of an inclusion positive for both RER1 and pSer129 immunoreactivity. Images were acquired on TCS SP2 AOBS Spectral Confocal Microscope (Leica) (B).
Figure Legend Snippet: RER1 colocalizes with αSyn in Lewy bodies. (A) Photomicrogaphs of tissue from control (left), and LB-positive tissues (right) show that RER1 colocalizes with αSyn in Lewy bodies. RER1 is detected in cell bodies, but appears enriched in round LB-like inclusions (arrows; see also enlargement in inset). (B) Photos show colocalization of both RER1 (green) and phosphorylated αSyn (pSer129: red) immunofluorescence in round, LB-like structures. Below are higher power images and confocal mapping of an inclusion positive for both RER1 and pSer129 immunoreactivity. Images were acquired on TCS SP2 AOBS Spectral Confocal Microscope (Leica) (B).

Techniques Used: Immunofluorescence, Microscopy

19) Product Images from "Nucleotide excision repair of abasic DNA lesions"

Article Title: Nucleotide excision repair of abasic DNA lesions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkz558

Reactivation of expression constructs containing AP lesions by complementation with XPA. ( A ) Flow cytometry expression analyses of constructs containing dG (blue colour), S-THF (amber) or AAF( N 2 )-dG (rose) at the analysed position in transcribed DNA strand of the EGFP gene. Fluorescence scatter plots show co-expression of EGFP with DsRed (as a marker for transfected cells). Cells were gated by DsRed expression to generate fluorescence distribution plots, which show S-THF and AAF( N 2 )-dG samples overlaid with a common dG reference sample. ( B ) Quantification of expression of constructs containing the specified AP lesions (S-THF or THF), relative to the dG reference (mean of six independent experiments ± SD; P -values calculated by the Student’s t -test). See also Supplementary Figure S2 .
Figure Legend Snippet: Reactivation of expression constructs containing AP lesions by complementation with XPA. ( A ) Flow cytometry expression analyses of constructs containing dG (blue colour), S-THF (amber) or AAF( N 2 )-dG (rose) at the analysed position in transcribed DNA strand of the EGFP gene. Fluorescence scatter plots show co-expression of EGFP with DsRed (as a marker for transfected cells). Cells were gated by DsRed expression to generate fluorescence distribution plots, which show S-THF and AAF( N 2 )-dG samples overlaid with a common dG reference sample. ( B ) Quantification of expression of constructs containing the specified AP lesions (S-THF or THF), relative to the dG reference (mean of six independent experiments ± SD; P -values calculated by the Student’s t -test). See also Supplementary Figure S2 .

Techniques Used: Expressing, Construct, Flow Cytometry, Cytometry, Fluorescence, Marker, Transfection

Impairment of transcription by BER-resistant AP lesion positioned at a specific nucleotide in the transcribed strand of the EGFP gene. ( A ) Structures of synthetic tetrahydrofuran (THF and S-THF) AP lesions and reactivity of BER enzymes towards the specified types of AP sites. ( B ) Characterization of reporter constructs containing deoxyguanine (dG) or the specified types of AP lesion at a defined nucleotide (*) in the transcribed DNA strand (TS). Scheme shows position for incorporation of synthetic oligonucleotides containing dG, THF or S-THF with respect to EGFP coding sequence (arrow) and transcription start (broken arrow). To demonstrate the presence of AP lesion, the obtained constructs were incubated with excess of APE1 and analysed by gel electrophoresis in the presence of ethidium bromide. See also Supplementary Figure S1 for more detail. ( C ) Flow cytometry analyses of expression of constructs containing specified modifications in transfected XP-A (GM04312) cells (a representative experiment). EGFP fluorescence distribution plots show expression data overlaid pairwise for each modification and the respective control constructs without modification. Bar chart on the right shows quantification of the EGFP expression, relative to the matched control constructs without the modifications.
Figure Legend Snippet: Impairment of transcription by BER-resistant AP lesion positioned at a specific nucleotide in the transcribed strand of the EGFP gene. ( A ) Structures of synthetic tetrahydrofuran (THF and S-THF) AP lesions and reactivity of BER enzymes towards the specified types of AP sites. ( B ) Characterization of reporter constructs containing deoxyguanine (dG) or the specified types of AP lesion at a defined nucleotide (*) in the transcribed DNA strand (TS). Scheme shows position for incorporation of synthetic oligonucleotides containing dG, THF or S-THF with respect to EGFP coding sequence (arrow) and transcription start (broken arrow). To demonstrate the presence of AP lesion, the obtained constructs were incubated with excess of APE1 and analysed by gel electrophoresis in the presence of ethidium bromide. See also Supplementary Figure S1 for more detail. ( C ) Flow cytometry analyses of expression of constructs containing specified modifications in transfected XP-A (GM04312) cells (a representative experiment). EGFP fluorescence distribution plots show expression data overlaid pairwise for each modification and the respective control constructs without modification. Bar chart on the right shows quantification of the EGFP expression, relative to the matched control constructs without the modifications.

Techniques Used: Construct, Sequencing, Incubation, Nucleic Acid Electrophoresis, Flow Cytometry, Cytometry, Expressing, Transfection, Fluorescence, Modification

Transcriptional mutagenesis at the BER-resistant abasic site in the template DNA and its suppression by NER. ( A ) Scheme of the reporter for detection of ribonucleotide misincorporation opposite to AP-lesion in the template DNA. Substitution of 613U in mRNA to any other ribonucleotide results in reversion to a fluorescent EGFP. ( B ) Flow cytometry assay for detection of the mRNA single nucleotide substitutions induced by the specified AP lesions (THF, S-THF) in the MRC-5 (group of panels on the left) and XP-A (group of panels on the right) cell lines. Fluorescence scatter plots show full data for individual samples from a representative experiment. The derived EGFP fluorescence distribution plots show overlaid data for EGFP construct without modification (green colour) and EGFP Q205* constructs without modification (blue) or with the indicated lesion (amber). The nature of the nucleotide/modification in the template DNA strand is indicated above the plots. Note the right shift of S-THF plots compared to dA.
Figure Legend Snippet: Transcriptional mutagenesis at the BER-resistant abasic site in the template DNA and its suppression by NER. ( A ) Scheme of the reporter for detection of ribonucleotide misincorporation opposite to AP-lesion in the template DNA. Substitution of 613U in mRNA to any other ribonucleotide results in reversion to a fluorescent EGFP. ( B ) Flow cytometry assay for detection of the mRNA single nucleotide substitutions induced by the specified AP lesions (THF, S-THF) in the MRC-5 (group of panels on the left) and XP-A (group of panels on the right) cell lines. Fluorescence scatter plots show full data for individual samples from a representative experiment. The derived EGFP fluorescence distribution plots show overlaid data for EGFP construct without modification (green colour) and EGFP Q205* constructs without modification (blue) or with the indicated lesion (amber). The nature of the nucleotide/modification in the template DNA strand is indicated above the plots. Note the right shift of S-THF plots compared to dA.

Techniques Used: Mutagenesis, Flow Cytometry, Cytometry, Fluorescence, Derivative Assay, Construct, Modification

20) Product Images from "Langerhans-type and monocyte-derived human dendritic cells have different susceptibilities to mRNA electroporation with distinct effects on maturation and activation: implications for immunogenicity in dendritic cell-based immunotherapy"

Article Title: Langerhans-type and monocyte-derived human dendritic cells have different susceptibilities to mRNA electroporation with distinct effects on maturation and activation: implications for immunogenicity in dendritic cell-based immunotherapy

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-11-166

mRNA electroporation induces the maturation and activation of LCs to a greater magnitude than for moDCs. Immature moDCs and LCs were electroporated with eGFP mRNA and then cultured with (+) or without (−) standard maturation-inducing inflammatory cytokines. After 24 hours, cells in each experimental group were compared with pre-electroporation controls by flow cytometry for the expression of phenotypic markers of DC maturation and activation, based on the upregulation of (A) CD83, (B) CD80, and (C) CD86, respectively. Representative dot plots of eGFP mRNA-electroporated moDCs and LCs from one of three independent experiments are shown in the top row. Pooled data for each experimental group (mean ± SD, n = 3 independent experiments) are shown in the bottom row (gray bar = pre-electroporation control, white bar = post-electroporation without inflammatory cytokines, black bar = post-electroporation with inflammatory cytokines). * P
Figure Legend Snippet: mRNA electroporation induces the maturation and activation of LCs to a greater magnitude than for moDCs. Immature moDCs and LCs were electroporated with eGFP mRNA and then cultured with (+) or without (−) standard maturation-inducing inflammatory cytokines. After 24 hours, cells in each experimental group were compared with pre-electroporation controls by flow cytometry for the expression of phenotypic markers of DC maturation and activation, based on the upregulation of (A) CD83, (B) CD80, and (C) CD86, respectively. Representative dot plots of eGFP mRNA-electroporated moDCs and LCs from one of three independent experiments are shown in the top row. Pooled data for each experimental group (mean ± SD, n = 3 independent experiments) are shown in the bottom row (gray bar = pre-electroporation control, white bar = post-electroporation without inflammatory cytokines, black bar = post-electroporation with inflammatory cytokines). * P

Techniques Used: Electroporation, Activation Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing

21) Product Images from "The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab"

Article Title: The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab

Journal: Biomolecules

doi: 10.3390/biom9100629

Expression of HER2 and epidermal growth factor receptor (EGFR) in 5637 and AU565 cells. ( a ) A representative sequence chromatogram showing the presence of two transcripts encoding wild-type HER2 and the S310F mutant in 5637 cells. ( b ) Flow cytometry analysis of two cancer cell lines assessing their reactivity to cetuximab, pertuzumab, and trastuzumab. The cells were incubated with individual antibody using the recombinant scFv-human Cκ fusion protein. The amount of bound antibody was determined using Allophycocyanin (APC)-labeled anti-human Cκ antibody.
Figure Legend Snippet: Expression of HER2 and epidermal growth factor receptor (EGFR) in 5637 and AU565 cells. ( a ) A representative sequence chromatogram showing the presence of two transcripts encoding wild-type HER2 and the S310F mutant in 5637 cells. ( b ) Flow cytometry analysis of two cancer cell lines assessing their reactivity to cetuximab, pertuzumab, and trastuzumab. The cells were incubated with individual antibody using the recombinant scFv-human Cκ fusion protein. The amount of bound antibody was determined using Allophycocyanin (APC)-labeled anti-human Cκ antibody.

Techniques Used: Expressing, Sequencing, Mutagenesis, Flow Cytometry, Cytometry, Incubation, Recombinant, Labeling

Single-molecular interaction analysis for the S310F mutant and EGFR heterodimerization. HEK293T cells were transfected with expression vectors encoding either EGFR-mCherry and wild-type HER2-eGFP or EGFR-mCherry and S310F HER2-eGFP fusion proteins. ( a ) After transfection, the cell surface expression of EGFR and HER2 or HER2 was checked by flow cytometry analysis using cetuximab, pertuzumab, and trastuzumab in the scFv-human Cκ format and APC-labeled anti-human Cκ antibody. ( b ) The lysates were subjected to a flow chamber coated with anti-mCherry antibody. Then, the summed intensity of eGFP spots was measured and plotted. Results represent the mean ± SD obtained from experiments performed in triplicate.
Figure Legend Snippet: Single-molecular interaction analysis for the S310F mutant and EGFR heterodimerization. HEK293T cells were transfected with expression vectors encoding either EGFR-mCherry and wild-type HER2-eGFP or EGFR-mCherry and S310F HER2-eGFP fusion proteins. ( a ) After transfection, the cell surface expression of EGFR and HER2 or HER2 was checked by flow cytometry analysis using cetuximab, pertuzumab, and trastuzumab in the scFv-human Cκ format and APC-labeled anti-human Cκ antibody. ( b ) The lysates were subjected to a flow chamber coated with anti-mCherry antibody. Then, the summed intensity of eGFP spots was measured and plotted. Results represent the mean ± SD obtained from experiments performed in triplicate.

Techniques Used: Mutagenesis, Transfection, Expressing, Flow Cytometry, Cytometry, Labeling

22) Product Images from "Raf-1 sets the threshold of Fas sensitivity by modulating Rok-? signaling"

Article Title: Raf-1 sets the threshold of Fas sensitivity by modulating Rok-? signaling

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200504137

Role of Raf-1 in Fas-mediated apoptosis: a working model. Fas binding to FasL stimulates DISC formation and internalization. Both of these processes depend on the linkage of Fas to the cytoskeleton. Phosphorylation of ezrin on T567 by Rok-α promotes Fas clustering but reduces DISC formation and internalization, generating a prolonged, albeit less efficient, Fas signal. In WT cells, formation of a Raf-1–Rok-α complex restrains Rok-α activity and ezrin phosphorylation. In addition, direct binding to Raf-1 prevents the dimerization and phosphorylation of the proapoptotic kinase MST-2 ( O'Neill et al., 2004 ). In the absence of Raf-1, Rok-α activity and ezrin phosphorylation generate a prolonged Fas signal, boosted by unrestrained MST-2 stimulation. White rods, Fas; gray rods, DISC components.
Figure Legend Snippet: Role of Raf-1 in Fas-mediated apoptosis: a working model. Fas binding to FasL stimulates DISC formation and internalization. Both of these processes depend on the linkage of Fas to the cytoskeleton. Phosphorylation of ezrin on T567 by Rok-α promotes Fas clustering but reduces DISC formation and internalization, generating a prolonged, albeit less efficient, Fas signal. In WT cells, formation of a Raf-1–Rok-α complex restrains Rok-α activity and ezrin phosphorylation. In addition, direct binding to Raf-1 prevents the dimerization and phosphorylation of the proapoptotic kinase MST-2 ( O'Neill et al., 2004 ). In the absence of Raf-1, Rok-α activity and ezrin phosphorylation generate a prolonged Fas signal, boosted by unrestrained MST-2 stimulation. White rods, Fas; gray rods, DISC components.

Techniques Used: Binding Assay, Activity Assay, Microscale Thermophoresis

Heterozygosity at the lpr or gld locus prevents fetal liver apoptosis and embryonic lethality as a result of c-raf-1 ablation. (A) Heterozygosity at the lpr locus prevents fetal liver apoptosis in Raf-1 KO embryos. Parasagittal section of E11.5 livers from WT, Raf-1 KO, and Raf-1 KO; lpr/ + embryos. Note the presence of Fas clusters in Raf-1 KO fetal livers and the reduction in Fas staining in the Raf-1 KO; lpr/ + liver. Apoptotic (TUNEL + ) cells are absent in Raf-1 KO; lpr/ +. (B) Increased Fas surface expression in KO fetal liver cells. Fas expression (solid lines) was determined by FACS analysis as described in Fig. 1 B. Dashed lines, isotype controls. (C) Comparison of a Raf-1 KO; lpr/ + pup (right), a control littermate (left), and eyes open at birth phenotype of the Raf-1 KO; lpr/ + pup (right). The experiments were performed exclusively with F2 animals.
Figure Legend Snippet: Heterozygosity at the lpr or gld locus prevents fetal liver apoptosis and embryonic lethality as a result of c-raf-1 ablation. (A) Heterozygosity at the lpr locus prevents fetal liver apoptosis in Raf-1 KO embryos. Parasagittal section of E11.5 livers from WT, Raf-1 KO, and Raf-1 KO; lpr/ + embryos. Note the presence of Fas clusters in Raf-1 KO fetal livers and the reduction in Fas staining in the Raf-1 KO; lpr/ + liver. Apoptotic (TUNEL + ) cells are absent in Raf-1 KO; lpr/ +. (B) Increased Fas surface expression in KO fetal liver cells. Fas expression (solid lines) was determined by FACS analysis as described in Fig. 1 B. Dashed lines, isotype controls. (C) Comparison of a Raf-1 KO; lpr/ + pup (right), a control littermate (left), and eyes open at birth phenotype of the Raf-1 KO; lpr/ + pup (right). The experiments were performed exclusively with F2 animals.

Techniques Used: Staining, TUNEL Assay, Expressing, FACS

Interfering with Rok-α and ezrin restores normal sensitivity to Fas-induced apoptosis in Raf-1–deficient fibroblasts. (A and B) Transfection with DN Rok-α (eG–Rok-α KD) prevents ezrin hyperphosphorylation (A) and Fas clustering (B) in Raf-1 KO cells. Reduced ezrin phosphorylation and lack of Fas clustering were observed in 89 ± 3% of the cells transfected with eG–Rok-α KD. (C–E) Silencing Rok-α expression reduces Fas sensitivity, Fas clustering, and ezrin hyperphosphorylation in KO cells. (C) Expression of Rok-α was assessed by immunoblotting 72 h after transfection with scrambled (SCR) or Rok-α siRNA. The related kinase Rok-β is shown as a specificity control and tubulin as a loading control. (D) KO and WT MEFs were transfected with Rok-α or SCR siRNA. Apoptosis was induced with 50 ng/ml αFas (5 μg/ml Chx for 22 h) and detected as described in Fig. 1 A. The values represent the mean ± SD (error bars) of triplicates. Two independent transfections are shown. *, P
Figure Legend Snippet: Interfering with Rok-α and ezrin restores normal sensitivity to Fas-induced apoptosis in Raf-1–deficient fibroblasts. (A and B) Transfection with DN Rok-α (eG–Rok-α KD) prevents ezrin hyperphosphorylation (A) and Fas clustering (B) in Raf-1 KO cells. Reduced ezrin phosphorylation and lack of Fas clustering were observed in 89 ± 3% of the cells transfected with eG–Rok-α KD. (C–E) Silencing Rok-α expression reduces Fas sensitivity, Fas clustering, and ezrin hyperphosphorylation in KO cells. (C) Expression of Rok-α was assessed by immunoblotting 72 h after transfection with scrambled (SCR) or Rok-α siRNA. The related kinase Rok-β is shown as a specificity control and tubulin as a loading control. (D) KO and WT MEFs were transfected with Rok-α or SCR siRNA. Apoptosis was induced with 50 ng/ml αFas (5 μg/ml Chx for 22 h) and detected as described in Fig. 1 A. The values represent the mean ± SD (error bars) of triplicates. Two independent transfections are shown. *, P

Techniques Used: Transfection, Expressing

Endogenous Raf-1 coimmunoprecipitates with Rok-α upon Fas stimulation, and Raf-1 kinase activity is dispensable for the regulation of Fas surface expression. (A) Rok-α is mislocalized in unstimulated and αFas-treated KO fibroblasts. WT and KO fibroblasts were treated with αFas as described in Fig. 2 A. The subcellular localization of Rok-α was determined by immunofluorescence. Arrowheads indicate Rok-α staining in the blebs, which was observed in 66 ± 2% of stimulated KO cells. (B) Fas stimulates the formation of a Raf-1–Rok-α complex. WT MEFs were stimulated with 2 μg/ml αFas, and Raf-1 IPs were prepared at the indicated times. The presence of Raf-1 and Rok-α in the IP (top) and in the input (bottom) was detected by immunoblotting. B, lysates incubated with protein A–Sepharose beads only. (C and D) Expression of KC or KD Raf-1 restores normal Fas expression, ezrin phosphorylation/distribution, and sensitivity to Fas-induced apoptosis in Raf-1 −/− MEFs. (C) The amount of Fas and Raf-1 in whole cell lysates was determined by immunoblotting. Molecular mass markers are shown in kilodaltons on the left. (D) Fas surface expression and ezrin phosphorylation/distribution were analyzed by immunofluorescence. The pictures shown are representative of 90 ± 1% KC and 87 ± 4% KD cells. (E) Sensitivity to αFas or TNFα-induced apoptosis was determined as described in Fig. 1 A. The values represent the mean ± SD (error bars) of at least three independent clones, each assessed in at least two independent experiments. *, P
Figure Legend Snippet: Endogenous Raf-1 coimmunoprecipitates with Rok-α upon Fas stimulation, and Raf-1 kinase activity is dispensable for the regulation of Fas surface expression. (A) Rok-α is mislocalized in unstimulated and αFas-treated KO fibroblasts. WT and KO fibroblasts were treated with αFas as described in Fig. 2 A. The subcellular localization of Rok-α was determined by immunofluorescence. Arrowheads indicate Rok-α staining in the blebs, which was observed in 66 ± 2% of stimulated KO cells. (B) Fas stimulates the formation of a Raf-1–Rok-α complex. WT MEFs were stimulated with 2 μg/ml αFas, and Raf-1 IPs were prepared at the indicated times. The presence of Raf-1 and Rok-α in the IP (top) and in the input (bottom) was detected by immunoblotting. B, lysates incubated with protein A–Sepharose beads only. (C and D) Expression of KC or KD Raf-1 restores normal Fas expression, ezrin phosphorylation/distribution, and sensitivity to Fas-induced apoptosis in Raf-1 −/− MEFs. (C) The amount of Fas and Raf-1 in whole cell lysates was determined by immunoblotting. Molecular mass markers are shown in kilodaltons on the left. (D) Fas surface expression and ezrin phosphorylation/distribution were analyzed by immunofluorescence. The pictures shown are representative of 90 ± 1% KC and 87 ± 4% KD cells. (E) Sensitivity to αFas or TNFα-induced apoptosis was determined as described in Fig. 1 A. The values represent the mean ± SD (error bars) of at least three independent clones, each assessed in at least two independent experiments. *, P

Techniques Used: Activity Assay, Expressing, Immunofluorescence, Staining, Incubation, Clone Assay

Selective hypersensitivity of Raf-1–deficient MEFs to Fas activation correlates with increased Fas expression. (A) Raf-1 KO MEFs are hypersensitive toward apoptosis induced by an agonistic Fas antibody or by FasL, but not by TNFα. MEFs were treated either with αFas, with recombinant FLAG-tagged FasL cross-linked with 1 μg/ml α-FLAG M2 antibody, or with recombinant mouse TNFα at the concentrations indicated for 22 h in the presence of 5 μg/ml Chx and 0.5% FCS. Cell death was determined by CytoTox 96 assay. The values represent the mean ± SD (error bars) of three independent cell lines. *, P
Figure Legend Snippet: Selective hypersensitivity of Raf-1–deficient MEFs to Fas activation correlates with increased Fas expression. (A) Raf-1 KO MEFs are hypersensitive toward apoptosis induced by an agonistic Fas antibody or by FasL, but not by TNFα. MEFs were treated either with αFas, with recombinant FLAG-tagged FasL cross-linked with 1 μg/ml α-FLAG M2 antibody, or with recombinant mouse TNFα at the concentrations indicated for 22 h in the presence of 5 μg/ml Chx and 0.5% FCS. Cell death was determined by CytoTox 96 assay. The values represent the mean ± SD (error bars) of three independent cell lines. *, P

Techniques Used: Activation Assay, Expressing, Recombinant

23) Product Images from "A double helical motif in OCIAD2 is essential for its localization, interactions and STAT3 activation"

Article Title: A double helical motif in OCIAD2 is essential for its localization, interactions and STAT3 activation

Journal: Scientific Reports

doi: 10.1038/s41598-018-25667-3

OCIAD2 localizes to endosomes and mitochondria. ( A – G ) Confocal images and co-localization plots of OCIAD2_FL-EGFP transfected HEK293 cells showing localization of OCIAD2 with various markers detected by immunofluorescence staining (red). ( A ) endogenous OCIAD2; ( B ) cytochrome oxidase subunit IV (CoxIV); ( C ) early endosome, Rab5; ( D ) late endosome, Rab7; ( E ) recycling endosome, Rab11; ( F ) lysosome, Lysotracker; ( G ) Golgi, GM130. Insets (A, B and C) show magnified view of the boxed region. Scale bars = 5 µm in C, D, E and 10 µm in A, B, F and G.
Figure Legend Snippet: OCIAD2 localizes to endosomes and mitochondria. ( A – G ) Confocal images and co-localization plots of OCIAD2_FL-EGFP transfected HEK293 cells showing localization of OCIAD2 with various markers detected by immunofluorescence staining (red). ( A ) endogenous OCIAD2; ( B ) cytochrome oxidase subunit IV (CoxIV); ( C ) early endosome, Rab5; ( D ) late endosome, Rab7; ( E ) recycling endosome, Rab11; ( F ) lysosome, Lysotracker; ( G ) Golgi, GM130. Insets (A, B and C) show magnified view of the boxed region. Scale bars = 5 µm in C, D, E and 10 µm in A, B, F and G.

Techniques Used: Transfection, Immunofluorescence, Staining

Phylogenetic analysis of the OCIAD proteins. ( A ) Schematic representation of the mouse OCIAD1/Asrij and OCIAD2 protein organization. ( B ) Multiple sequence alignment of OCIAD1/Asrij and OCIAD2 protein sequences (using MUSCLE) from mouse and human showing conserved regions. Dark shading shows amino acids identical in all sequences and light shading indicates similar amino acids. Numbers at the end of each sequence indicate amino acid positions. ( C ) Multiple sequence alignment of the predicted mouse, rat and human OCIAD2 open reading frames (using MUSCLE). ( D ) Evolutionary history of the OCIA domain proteins. The evolutionary history was inferred using the Maximum Likelihood method (see Methods). The tree with the highest log likelihood is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches, in green. The tree is a topology only tree and nodes with a bootstrap value less than 45 have been collapsed and shown as polytomies. Our analysis involved 106 amino acid sequences from 58 unique species. All positions containing gaps and missing data were eliminated, leaving 93 positions in the final dataset. Gene duplications are inferred using the method described in 23 . Three gene duplication events (diamonds) were identified in the tree. Open diamonds mark duplication nodes with low bootstrap values and blue diamond marks duplication node with highest bootstrap value (47). Given that there is only one copy of OCIA-domain containing proteins in invertebrates, these have been referred to as OCIAD. The extra copy of OCIAD1 protein present in Danio rerio and Clupea harengus has been listed as OCIAD1-like protein in the tree. ( E ) Sequence conservation analysis of vertebrate OCIAD1 (red line) and OCIAD2 (blue line) with respect to a conserved protein Histone H3 (black line) across different taxa. The dotted lines represent best-fit whereas solid lines represent the original values.
Figure Legend Snippet: Phylogenetic analysis of the OCIAD proteins. ( A ) Schematic representation of the mouse OCIAD1/Asrij and OCIAD2 protein organization. ( B ) Multiple sequence alignment of OCIAD1/Asrij and OCIAD2 protein sequences (using MUSCLE) from mouse and human showing conserved regions. Dark shading shows amino acids identical in all sequences and light shading indicates similar amino acids. Numbers at the end of each sequence indicate amino acid positions. ( C ) Multiple sequence alignment of the predicted mouse, rat and human OCIAD2 open reading frames (using MUSCLE). ( D ) Evolutionary history of the OCIA domain proteins. The evolutionary history was inferred using the Maximum Likelihood method (see Methods). The tree with the highest log likelihood is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) are shown next to the branches, in green. The tree is a topology only tree and nodes with a bootstrap value less than 45 have been collapsed and shown as polytomies. Our analysis involved 106 amino acid sequences from 58 unique species. All positions containing gaps and missing data were eliminated, leaving 93 positions in the final dataset. Gene duplications are inferred using the method described in 23 . Three gene duplication events (diamonds) were identified in the tree. Open diamonds mark duplication nodes with low bootstrap values and blue diamond marks duplication node with highest bootstrap value (47). Given that there is only one copy of OCIA-domain containing proteins in invertebrates, these have been referred to as OCIAD. The extra copy of OCIAD1 protein present in Danio rerio and Clupea harengus has been listed as OCIAD1-like protein in the tree. ( E ) Sequence conservation analysis of vertebrate OCIAD1 (red line) and OCIAD2 (blue line) with respect to a conserved protein Histone H3 (black line) across different taxa. The dotted lines represent best-fit whereas solid lines represent the original values.

Techniques Used: Sequencing

Knockdown of OCIAD2 retards migration of HEK293 cells. ( A ) Western Blotting confirming ociad2 knockdown (KD) in OCIAD2_ shRNA (1, 2 and 3) transfected HEK293 cells as compared to non-silencing (NS) shRNA. Graph shows relative OCIAD2 levels in NS and KD. n = 4 ( B ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in NS and KD. Data are representative of three independent experiments and graph shows relative STAT3/pSTAT3 ratio upon ociad2 KD. Protein size markers (in kDa) are indicated to the left. Full-length blots are presented in Supplementary Figure S6 . n = 4 ( C ) Flow cytometry analysis of Ki-67 + population in ociad2 KD HEK293 cells. Graph shows results obtained from four independent experiments (mean ± SEM). ( D ) Reduced migration of ociad2 KD cells observed in wound healing assays. Monolayers of control and ociad2 knockdown HEK293 cells were wounded and the degree of recovery was measured at 0, 12 and 24 hours post-wounding. Representative phase and fluorescence images, 4× magnification. Measurement and estimation of wound recovery was based on the initial wound size. Graph shows quantification of results obtained from three independent experiments (mean ± SEM) with at least 5 fields analyzed per sample per experiment. ( E ) Representative images (4× magnification) showing reduced migration of ociad2 KD HEK293 cells after 48 hours in a trans-well migration assays. Graphs represent quantification of cell migration (absorbance at 570 nm) obtained from two independent trans-well experiments performed in two technical replicates (mean ± SEM). * p
Figure Legend Snippet: Knockdown of OCIAD2 retards migration of HEK293 cells. ( A ) Western Blotting confirming ociad2 knockdown (KD) in OCIAD2_ shRNA (1, 2 and 3) transfected HEK293 cells as compared to non-silencing (NS) shRNA. Graph shows relative OCIAD2 levels in NS and KD. n = 4 ( B ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in NS and KD. Data are representative of three independent experiments and graph shows relative STAT3/pSTAT3 ratio upon ociad2 KD. Protein size markers (in kDa) are indicated to the left. Full-length blots are presented in Supplementary Figure S6 . n = 4 ( C ) Flow cytometry analysis of Ki-67 + population in ociad2 KD HEK293 cells. Graph shows results obtained from four independent experiments (mean ± SEM). ( D ) Reduced migration of ociad2 KD cells observed in wound healing assays. Monolayers of control and ociad2 knockdown HEK293 cells were wounded and the degree of recovery was measured at 0, 12 and 24 hours post-wounding. Representative phase and fluorescence images, 4× magnification. Measurement and estimation of wound recovery was based on the initial wound size. Graph shows quantification of results obtained from three independent experiments (mean ± SEM) with at least 5 fields analyzed per sample per experiment. ( E ) Representative images (4× magnification) showing reduced migration of ociad2 KD HEK293 cells after 48 hours in a trans-well migration assays. Graphs represent quantification of cell migration (absorbance at 570 nm) obtained from two independent trans-well experiments performed in two technical replicates (mean ± SEM). * p

Techniques Used: Migration, Western Blot, shRNA, Transfection, Flow Cytometry, Cytometry, Fluorescence

Intron-exon structure of the ociad1/2 genes and comparison of their transcript levels in human tissues and cell lines. ( A ) Intron-exon structures of the genes coding for OCIAD1 and OCIAD2 in human, mouse and zebra fish. Intron-exon structure for the longest transcript was obtained from the Ensemble database (Accession numbers: ENST00000381473.7, ENSMUST00000031038.10, ENSDART00000103365.4, ENST00000508632.5, ENSMUST00000087195.8 and ENSDART00000164503.1). Colored boxes correspond to exons, grey-color indicates untranslated regions. Black solid lines represent introns. The size of introns and exons in nucleotides is mentioned. Introns are not drawn to scale. Exons coding for the double helical domain of OCIAD proteins have been indicated. ( B ) Comparison of transcript levels of ociad1 and ociad2 across different human tissues and cell lines as per data obtained from the Vertebrate Alternative Splicing and Transcription Database (VastDB, http://vastdb.crg.eu/ ).
Figure Legend Snippet: Intron-exon structure of the ociad1/2 genes and comparison of their transcript levels in human tissues and cell lines. ( A ) Intron-exon structures of the genes coding for OCIAD1 and OCIAD2 in human, mouse and zebra fish. Intron-exon structure for the longest transcript was obtained from the Ensemble database (Accession numbers: ENST00000381473.7, ENSMUST00000031038.10, ENSDART00000103365.4, ENST00000508632.5, ENSMUST00000087195.8 and ENSDART00000164503.1). Colored boxes correspond to exons, grey-color indicates untranslated regions. Black solid lines represent introns. The size of introns and exons in nucleotides is mentioned. Introns are not drawn to scale. Exons coding for the double helical domain of OCIAD proteins have been indicated. ( B ) Comparison of transcript levels of ociad1 and ociad2 across different human tissues and cell lines as per data obtained from the Vertebrate Alternative Splicing and Transcription Database (VastDB, http://vastdb.crg.eu/ ).

Techniques Used: Fluorescence In Situ Hybridization

The double helical motif of OCIAD2 is necessary for interaction with OCIAD1. ( A ) Schematic representation of full-length (FL), N-terminal (N), hydrophobic region (Hph) and C-terminal (C) fragments of OCIAD2 generated for this study. Numbers indicate amino acid positions. Hx: predicted alpha helix. ( B – H ) Confocal images of HEK293 cells transfected with OCIAD2 reporter constructs, showing localization of ectopic OCIAD2-reporter with endogenous OCIAD2 ( B – D ) or endogenous OCIAD1 ( E – H ) as indicated, detected by fluorescence immunostaining with respective antibodies. Insets show magnified view of the boxed region. Co-localization plots are to the right of each panel. Scale bar: ( B – D ): 10 µm; ( E – H ): 5 µm. ( I – O ) Cell lysates from untransfected HEK293 cells (I) or those expressing various OCIAD2 (J, K, L, O) or OCIAD1/Asrij ( M , N ) constructs as indicated on the top of each panel, were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) with antibodies as indicated. Protein size markers (in kDa) are indicated to the left. At least two independent immunoprecipitation experiments with two technical replicates each were performed and similar results were obtained. Full-length blots are presented in Supplementary Figure S6 .
Figure Legend Snippet: The double helical motif of OCIAD2 is necessary for interaction with OCIAD1. ( A ) Schematic representation of full-length (FL), N-terminal (N), hydrophobic region (Hph) and C-terminal (C) fragments of OCIAD2 generated for this study. Numbers indicate amino acid positions. Hx: predicted alpha helix. ( B – H ) Confocal images of HEK293 cells transfected with OCIAD2 reporter constructs, showing localization of ectopic OCIAD2-reporter with endogenous OCIAD2 ( B – D ) or endogenous OCIAD1 ( E – H ) as indicated, detected by fluorescence immunostaining with respective antibodies. Insets show magnified view of the boxed region. Co-localization plots are to the right of each panel. Scale bar: ( B – D ): 10 µm; ( E – H ): 5 µm. ( I – O ) Cell lysates from untransfected HEK293 cells (I) or those expressing various OCIAD2 (J, K, L, O) or OCIAD1/Asrij ( M , N ) constructs as indicated on the top of each panel, were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) with antibodies as indicated. Protein size markers (in kDa) are indicated to the left. At least two independent immunoprecipitation experiments with two technical replicates each were performed and similar results were obtained. Full-length blots are presented in Supplementary Figure S6 .

Techniques Used: Generated, Transfection, Construct, Fluorescence, Immunostaining, Expressing, Immunoprecipitation

Comparison of the genomic organization for ociad1 and ociad2 across different species. Schematics depict genomic region encompassing ociad1 (red) and ociad2 (blue) and flanking genes fryl , cwh43 or dcun1d4 as indicated. Chromosome numbers and position of genes on sense or antisense strand are indicated. Direction of arrows indicates direction of transcription. Phyla are grouped into colored boxes.
Figure Legend Snippet: Comparison of the genomic organization for ociad1 and ociad2 across different species. Schematics depict genomic region encompassing ociad1 (red) and ociad2 (blue) and flanking genes fryl , cwh43 or dcun1d4 as indicated. Chromosome numbers and position of genes on sense or antisense strand are indicated. Direction of arrows indicates direction of transcription. Phyla are grouped into colored boxes.

Techniques Used:

The double helical motif of OCIAD2 interacts with STAT3 and promotes its activation. ( A ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in HEK293 cells transfected with various OCIAD2 constructs as indicated. UT: untransfected, V: empty vector transfected; FL: OCIAD2_FL-EGFP; N: OCIAD2_N-EGFP; Hph: OCIAD2_Hph-dsRed and C: OCIAD2_C-dsRed. Graph represents pSTAT3/STAT3 ratio obtained upon overexpression of various OCIAD2 constructs relative to control (shown as a dotted line). n = 4 ( B ) Validation of transfection of FLAG-STAT3 construct into HEK293 cells by Western Blotting. ( C ) FLAG-STAT3 transfected HEK293 cell lysate subjected to immunoprecipitation with anti-FLAG antibody and assessed for interaction with OCIAD1 and OCIAD2. ( D – G ) HEK293 cell lysates expressing various OCIAD2 constructs (FL, N, Hph and C) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) to probe for interaction with STAT3. Protein size markers (in kDa) are indicated to the left. Data for STAT3/pSTAT3 Western Blotting are representative of three independent experiments and * p
Figure Legend Snippet: The double helical motif of OCIAD2 interacts with STAT3 and promotes its activation. ( A ) Western Blotting for detection of STAT3 and pSTAT3 (Y705) levels in HEK293 cells transfected with various OCIAD2 constructs as indicated. UT: untransfected, V: empty vector transfected; FL: OCIAD2_FL-EGFP; N: OCIAD2_N-EGFP; Hph: OCIAD2_Hph-dsRed and C: OCIAD2_C-dsRed. Graph represents pSTAT3/STAT3 ratio obtained upon overexpression of various OCIAD2 constructs relative to control (shown as a dotted line). n = 4 ( B ) Validation of transfection of FLAG-STAT3 construct into HEK293 cells by Western Blotting. ( C ) FLAG-STAT3 transfected HEK293 cell lysate subjected to immunoprecipitation with anti-FLAG antibody and assessed for interaction with OCIAD1 and OCIAD2. ( D – G ) HEK293 cell lysates expressing various OCIAD2 constructs (FL, N, Hph and C) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) to probe for interaction with STAT3. Protein size markers (in kDa) are indicated to the left. Data for STAT3/pSTAT3 Western Blotting are representative of three independent experiments and * p

Techniques Used: Activation Assay, Western Blot, Transfection, Construct, Plasmid Preparation, Over Expression, Immunoprecipitation, Expressing

24) Product Images from "Decreased CD4 expression by polarized T helper 2 cells contributes to suboptimal TCR-induced phosphorylation and reduced Ca2+ signaling"

Article Title: Decreased CD4 expression by polarized T helper 2 cells contributes to suboptimal TCR-induced phosphorylation and reduced Ca2+ signaling

Journal: European journal of immunology

doi: 10.1002/eji.200526064

Th1-like pattern of proximal TCR-induced protein tyrosine phosphorylation in Th2 cells with restored CD4 expression. (A) Polarized Th1 and Th2 5C.C7 T cells were infected with recombinant retrovirus encoding either EGFP or a mouse CD4-EGFP fusion protein.
Figure Legend Snippet: Th1-like pattern of proximal TCR-induced protein tyrosine phosphorylation in Th2 cells with restored CD4 expression. (A) Polarized Th1 and Th2 5C.C7 T cells were infected with recombinant retrovirus encoding either EGFP or a mouse CD4-EGFP fusion protein.

Techniques Used: Expressing, Infection, Recombinant

Alteration of ζ chain phosphorylation pattern in Th1 cells with disrupted CD4 function. Polarized 5C.C7 Th1 cells were exposed to antigen-presenting cells pre-pulsed with 30 μM PCC88–104 peptide in the absence or the presence of
Figure Legend Snippet: Alteration of ζ chain phosphorylation pattern in Th1 cells with disrupted CD4 function. Polarized 5C.C7 Th1 cells were exposed to antigen-presenting cells pre-pulsed with 30 μM PCC88–104 peptide in the absence or the presence of

Techniques Used:

Selective decrease in CD4 surface membrane expression on polarized Th2 cells as compared to Th1 cells. Polarized 5C.C7 T cells were stained for CD3ε, CD4, CD28, and CD45 (T200) expression and analyzed by flow cytometry. The numbers below each
Figure Legend Snippet: Selective decrease in CD4 surface membrane expression on polarized Th2 cells as compared to Th1 cells. Polarized 5C.C7 T cells were stained for CD3ε, CD4, CD28, and CD45 (T200) expression and analyzed by flow cytometry. The numbers below each

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry

Th1-like pattern of TCR-induced intracellular Ca 2+ elevation in Th2 cells with restored CD4 expression. (A) Polarized Th1 and Th2 5C.C7 T cells were loaded with the indicator dye Indo-1 and analyzed for changes in intracellular Ca 2+ by FACS following
Figure Legend Snippet: Th1-like pattern of TCR-induced intracellular Ca 2+ elevation in Th2 cells with restored CD4 expression. (A) Polarized Th1 and Th2 5C.C7 T cells were loaded with the indicator dye Indo-1 and analyzed for changes in intracellular Ca 2+ by FACS following

Techniques Used: Expressing, FACS

25) Product Images from "Avidity and Bystander Suppressive Capacity of Human Regulatory T Cells Expressing De Novo Autoreactive T-Cell Receptors in Type 1 Diabetes"

Article Title: Avidity and Bystander Suppressive Capacity of Human Regulatory T Cells Expressing De Novo Autoreactive T-Cell Receptors in Type 1 Diabetes

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.01313

Serial activation increases transduction efficiency. (A) Primary CD4 + T cells remain untransduced (Mock) or transduced with lentivirus (LV TD) expressing T-cell receptor (TCR) clones 4.13 or R164 were activated with α-CD3/α-CD28 coated beads on day 0 (D0). Cells were restimulated with artificial APC (aAPCs; K562 cell line expressing HLA-DR4) and GAD 555–567 peptide for an additional two rounds on day 9 (D9) and day 16 (D16). IL-2 (30 IU/mL) was given every 2–3 days. Transduction efficiency was detected by fluorescence-activated cell sorting (FACS) every 4 days after stimulation (D4, D13, and D20). (B) TCR Vβ5.1 and enhanced green fluorescent protein (eGFP) reporter were assessed by flow cytometry on day 4 (D4, top), day 13 (D13, middle), and day 20 (D20, bottom). At each time point, a portion of untransduced cells were positive for TCR Vβ5.1, but no eGFP was observed. TCR Vβ5.1 and eGFP positivity was observed for 4.13 and R164 transduced cells at each time point, and the proportion of dual positive cells increased with time.
Figure Legend Snippet: Serial activation increases transduction efficiency. (A) Primary CD4 + T cells remain untransduced (Mock) or transduced with lentivirus (LV TD) expressing T-cell receptor (TCR) clones 4.13 or R164 were activated with α-CD3/α-CD28 coated beads on day 0 (D0). Cells were restimulated with artificial APC (aAPCs; K562 cell line expressing HLA-DR4) and GAD 555–567 peptide for an additional two rounds on day 9 (D9) and day 16 (D16). IL-2 (30 IU/mL) was given every 2–3 days. Transduction efficiency was detected by fluorescence-activated cell sorting (FACS) every 4 days after stimulation (D4, D13, and D20). (B) TCR Vβ5.1 and enhanced green fluorescent protein (eGFP) reporter were assessed by flow cytometry on day 4 (D4, top), day 13 (D13, middle), and day 20 (D20, bottom). At each time point, a portion of untransduced cells were positive for TCR Vβ5.1, but no eGFP was observed. TCR Vβ5.1 and eGFP positivity was observed for 4.13 and R164 transduced cells at each time point, and the proportion of dual positive cells increased with time.

Techniques Used: Activation Assay, Transduction, Expressing, Fluorescence, FACS, Flow Cytometry, Cytometry

Verifying transfection and activation of Jurkat cells expressing T-cell receptor (TCR) clones. (A) Lentiviral constructs were designed containing the TCR α- and TCR β-chain genes (TRA and TRB, respectively) for known glutamic acid decarboxylase (GAD)-reactive clones (R164 and 4.13, additional clone information is listed in Table 1 ). The TRA and TRB coding regions were joined by a multicystronic P2A element, and TRB was linked by a multicystronic T2A element to an enhanced green fluorescent protein (eGFP) reporter. Amino-acid sequences for the complementarity determining region 3 (CDR3) are shown for both clones. (B) Jurkat T cells were untransduced (Mock; top), transduced with lentivirus expressing the R164 TCR (middle), and lentivirus expressing the 4.13 TCR (bottom), and expression was confirmed by flow cytometry. Double positivity for TCR Va12.1 and Vβ5.1, which is comparable between both clones, and eGFP indicates successful transduction. Untransduced cells were negative for both markers. (C) Mock (top), 4.13 (middle), and R164 (bottom) TCR-transduced cells were unstimulated (black), stimulated with plate bound anti-CD3 (p.b. α-CD3, blue), irrelevant antigen influenza hemagglutinin (HA 306–318 , orange), or cognate antigen (GAD 555–567 , green). TCR expression (left panels) was comparable across all unstimulated, HA-stimulated, and GAD-stimulated cells, and p.b. α-CD3 stimulation induced TCR downregulation. p.b. α-CD3 stimulation also induced the highest level of CD69 expression (right panels), and unstimulated cells exhibited low CD69 expression. These observations were comparable across mock, R164, or 4.13 transduced cells.
Figure Legend Snippet: Verifying transfection and activation of Jurkat cells expressing T-cell receptor (TCR) clones. (A) Lentiviral constructs were designed containing the TCR α- and TCR β-chain genes (TRA and TRB, respectively) for known glutamic acid decarboxylase (GAD)-reactive clones (R164 and 4.13, additional clone information is listed in Table 1 ). The TRA and TRB coding regions were joined by a multicystronic P2A element, and TRB was linked by a multicystronic T2A element to an enhanced green fluorescent protein (eGFP) reporter. Amino-acid sequences for the complementarity determining region 3 (CDR3) are shown for both clones. (B) Jurkat T cells were untransduced (Mock; top), transduced with lentivirus expressing the R164 TCR (middle), and lentivirus expressing the 4.13 TCR (bottom), and expression was confirmed by flow cytometry. Double positivity for TCR Va12.1 and Vβ5.1, which is comparable between both clones, and eGFP indicates successful transduction. Untransduced cells were negative for both markers. (C) Mock (top), 4.13 (middle), and R164 (bottom) TCR-transduced cells were unstimulated (black), stimulated with plate bound anti-CD3 (p.b. α-CD3, blue), irrelevant antigen influenza hemagglutinin (HA 306–318 , orange), or cognate antigen (GAD 555–567 , green). TCR expression (left panels) was comparable across all unstimulated, HA-stimulated, and GAD-stimulated cells, and p.b. α-CD3 stimulation induced TCR downregulation. p.b. α-CD3 stimulation also induced the highest level of CD69 expression (right panels), and unstimulated cells exhibited low CD69 expression. These observations were comparable across mock, R164, or 4.13 transduced cells.

Techniques Used: Transfection, Activation Assay, Expressing, Construct, Clone Assay, Transduction, Flow Cytometry, Cytometry

Verification of T-cell receptor (TCR) overexpression and stability of transfected regulatory T cells (Tregs). (A) CD25 + CD127 lo/− Tregs (top and middle) and CD25 lo/− CD127 + CD4 + CD45RA + naïve conventional T cells (Tconv, bottom) were purified from adult peripheral blood by fluorescent-activated cell sorting (FACS). (B) Tregs were activated with α-CD3/α-CD28 coated beads on day 0 (D0). Lentiviral transduction (LV TD) was performed on day 2 (D2). Successfully transduced cells expressing enhanced green fluorescent protein (eGFP) were FACS-purified on day 19 (D19) for further analysis. (C) Treg (top) and Tconv cells (bottom) were untransduced (Mock; black), transduced with lentivirus containing the expression vector for the GAD-reactive TCR clone 4.13 (blue) or R164 (red), and eGFP expression among TCR transduced cells was confirmed by flow cytometry. (D) Mock (left), 4.13 (middle), and R164 (right) transduced Tregs (top) and Tconv cells (bottom) were stained for the TCRVα12.1 and TCRVβ5.1 chains that comprise the TCR clones 4.13 and R164, and overexpression was confirmed for TCR transduced cells. (E) Cell activation markers OX40 and CD25 were measured after co-culturing T cells with HLA-DR4 expressing K562 artificial antigen-presenting cells (aAPCs) loaded with GAD GAD 555–567 peptide for 1 day. (F) The majority of Tregs in all conditions (non-transduced, 4.13, or R164) maintain FOXP3 expression (top left). Low frequency of FOXP3 expression was observed in Tconv (bottom left).
Figure Legend Snippet: Verification of T-cell receptor (TCR) overexpression and stability of transfected regulatory T cells (Tregs). (A) CD25 + CD127 lo/− Tregs (top and middle) and CD25 lo/− CD127 + CD4 + CD45RA + naïve conventional T cells (Tconv, bottom) were purified from adult peripheral blood by fluorescent-activated cell sorting (FACS). (B) Tregs were activated with α-CD3/α-CD28 coated beads on day 0 (D0). Lentiviral transduction (LV TD) was performed on day 2 (D2). Successfully transduced cells expressing enhanced green fluorescent protein (eGFP) were FACS-purified on day 19 (D19) for further analysis. (C) Treg (top) and Tconv cells (bottom) were untransduced (Mock; black), transduced with lentivirus containing the expression vector for the GAD-reactive TCR clone 4.13 (blue) or R164 (red), and eGFP expression among TCR transduced cells was confirmed by flow cytometry. (D) Mock (left), 4.13 (middle), and R164 (right) transduced Tregs (top) and Tconv cells (bottom) were stained for the TCRVα12.1 and TCRVβ5.1 chains that comprise the TCR clones 4.13 and R164, and overexpression was confirmed for TCR transduced cells. (E) Cell activation markers OX40 and CD25 were measured after co-culturing T cells with HLA-DR4 expressing K562 artificial antigen-presenting cells (aAPCs) loaded with GAD GAD 555–567 peptide for 1 day. (F) The majority of Tregs in all conditions (non-transduced, 4.13, or R164) maintain FOXP3 expression (top left). Low frequency of FOXP3 expression was observed in Tconv (bottom left).

Techniques Used: Over Expression, Transfection, Purification, FACS, Transduction, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry, Staining, Clone Assay, Activation Assay

Regulatory T-cell (Treg) suppression is optimal with activation. (A) Antigen-specific suppression by 4.13 Tregs was tested on 4.13 T-cell receptor (TCR) transduced conventional T cells (Tconv) in vitro (left). Bystander suppression by 4.13 Tregs was assessed on CD8 + T cells expressing the melanoma antigen recognized by T cells 1 (MART-1) TCR, with (middle) or without (right) Treg activation. (B) Tregs were isolated from adult peripheral blood and transduced to express glutamic acid decarboxylase (GAD) 4.13 TCR. Transduced Tregs were sorted, labeled with cell proliferation dye (CPD) eFluor670, and plated in decreasing proportions with GAD 4.13 TCR transduced CD4 + responder T cells (Tresp) (Ag-specific) or MART-1 transduced CD8 + Tresp (Bystander) stained with cell trace violet (CTV) dye. For Ag-specific suppression, GAD 4.13 Tresp and Treg were activated with cognate GAD 555–567 peptide presented by CD3-depleted peripheral blood mononuclear cell (PBMC) from an HLA-DR4 individual. For bystander suppression, MART-1 CD8 + Tresp and GAD 4.13 Tregs were activated with Melan-A 27–35 with or without GAD 555–567 peptide, again presented by CD3-depleted PBMC from an HLA-DR4 individual. Cell proliferation was evaluated via dye dilution for Tresp (top) and Tregs (bottom). Tresp proliferation decreased as the Treg to Tresp ratio increased only when Tregs were activated, and suppression was most effective when Treg activation was antigen-specific. Unactivated Tregs exhibited little to no proliferation. (C) Suppression was evaluated by Tresp division index (left) and percent (%) suppression (right). Tresp division index was significantly lower and percent suppression of Tresp proliferation was significantly greater in antigen-specific settings (Ag-specific, black) followed by bystander suppression when Tregs were activated (red). Two-way analysis of variance (ANOVA) (* P
Figure Legend Snippet: Regulatory T-cell (Treg) suppression is optimal with activation. (A) Antigen-specific suppression by 4.13 Tregs was tested on 4.13 T-cell receptor (TCR) transduced conventional T cells (Tconv) in vitro (left). Bystander suppression by 4.13 Tregs was assessed on CD8 + T cells expressing the melanoma antigen recognized by T cells 1 (MART-1) TCR, with (middle) or without (right) Treg activation. (B) Tregs were isolated from adult peripheral blood and transduced to express glutamic acid decarboxylase (GAD) 4.13 TCR. Transduced Tregs were sorted, labeled with cell proliferation dye (CPD) eFluor670, and plated in decreasing proportions with GAD 4.13 TCR transduced CD4 + responder T cells (Tresp) (Ag-specific) or MART-1 transduced CD8 + Tresp (Bystander) stained with cell trace violet (CTV) dye. For Ag-specific suppression, GAD 4.13 Tresp and Treg were activated with cognate GAD 555–567 peptide presented by CD3-depleted peripheral blood mononuclear cell (PBMC) from an HLA-DR4 individual. For bystander suppression, MART-1 CD8 + Tresp and GAD 4.13 Tregs were activated with Melan-A 27–35 with or without GAD 555–567 peptide, again presented by CD3-depleted PBMC from an HLA-DR4 individual. Cell proliferation was evaluated via dye dilution for Tresp (top) and Tregs (bottom). Tresp proliferation decreased as the Treg to Tresp ratio increased only when Tregs were activated, and suppression was most effective when Treg activation was antigen-specific. Unactivated Tregs exhibited little to no proliferation. (C) Suppression was evaluated by Tresp division index (left) and percent (%) suppression (right). Tresp division index was significantly lower and percent suppression of Tresp proliferation was significantly greater in antigen-specific settings (Ag-specific, black) followed by bystander suppression when Tregs were activated (red). Two-way analysis of variance (ANOVA) (* P

Techniques Used: Activation Assay, In Vitro, Expressing, Isolation, Labeling, Staining

26) Product Images from "A versatile T cell-based assay to assess therapeutic antigen-specific PD-1-targeted approaches"

Article Title: A versatile T cell-based assay to assess therapeutic antigen-specific PD-1-targeted approaches

Journal: Oncotarget

doi: 10.18632/oncotarget.25591

Validation of antigen-specific TCR function of transfected 2D3 and PD-1 + 2D3 cells ( A – B ) Activation profiles of freshly used or thawed WT1-specific TCR mRNA-electroporated PD-1 − and PD-1 + 2D3 cells left unstimulated (-) versus 24 hours co-culture with unloaded (T2 -pept ) and WT1 peptide-pulsed (T2 +pept ) stimulator cells at a ratio of 2:1. Comparable results were obtained with gp100 TCR-positive PD-1 − and PD-1 + 2D3 cells. (A) Representative example of TCR activation-mediated eGFP expression within the viable CD8 + cell population as assessed with flow cytometry (freshly used WT1 TCR mRNA-electroporated PD-1 + 2D3 cells). (B) The left graph shows the mean percentage (± SEM) WT1-specific TCR activation-mediated eGFP expression from 2–8 replicate experiments. The right panel depicts the mean amount (± SEM) of secreted granzyme B determined with ELISA in cell-free 24-hour culture supernatant of 10 5 effector cells for 2–4 replicate experiments. Data information: * P ≤ 0.05, ** P
Figure Legend Snippet: Validation of antigen-specific TCR function of transfected 2D3 and PD-1 + 2D3 cells ( A – B ) Activation profiles of freshly used or thawed WT1-specific TCR mRNA-electroporated PD-1 − and PD-1 + 2D3 cells left unstimulated (-) versus 24 hours co-culture with unloaded (T2 -pept ) and WT1 peptide-pulsed (T2 +pept ) stimulator cells at a ratio of 2:1. Comparable results were obtained with gp100 TCR-positive PD-1 − and PD-1 + 2D3 cells. (A) Representative example of TCR activation-mediated eGFP expression within the viable CD8 + cell population as assessed with flow cytometry (freshly used WT1 TCR mRNA-electroporated PD-1 + 2D3 cells). (B) The left graph shows the mean percentage (± SEM) WT1-specific TCR activation-mediated eGFP expression from 2–8 replicate experiments. The right panel depicts the mean amount (± SEM) of secreted granzyme B determined with ELISA in cell-free 24-hour culture supernatant of 10 5 effector cells for 2–4 replicate experiments. Data information: * P ≤ 0.05, ** P

Techniques Used: Transfection, Activation Assay, Co-Culture Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

Efficiency of PD-1 transduction, TCR mRNA electroporation and cryopreservation of 2D3 cells ( A ) Representative flow cytometry T-cell receptor (TCRαβ) and programmed death-1 (PD-1) protein surface expression profiles and corresponding isotype controls of non-transduced PD-1 − 2D3 and PD-1-transduced (PD-1 + ) 2D3 cells 24 hours after TCR mRNA electroporation (fresh; 10-14 replicates) and after thawing of TCR mRNA-electroporated cells (cryo; 6 replicates). ( B ) Percentage viability and recovery upon TCR mRNA electroporation of PD-1 − and PD-1 + 2D3 cells. Data information: in (B), means are depicted. * P ≤ 0.05 (Student’s t -test). Abbreviations: cryo, transfected effector cells were cryopreserved prior to co-culture; fresh, stimulator cells were co-cultured immediately following transfection; ns, not significant; PD-1, programmed death-1 protein; TCR, T-cell receptor.
Figure Legend Snippet: Efficiency of PD-1 transduction, TCR mRNA electroporation and cryopreservation of 2D3 cells ( A ) Representative flow cytometry T-cell receptor (TCRαβ) and programmed death-1 (PD-1) protein surface expression profiles and corresponding isotype controls of non-transduced PD-1 − 2D3 and PD-1-transduced (PD-1 + ) 2D3 cells 24 hours after TCR mRNA electroporation (fresh; 10-14 replicates) and after thawing of TCR mRNA-electroporated cells (cryo; 6 replicates). ( B ) Percentage viability and recovery upon TCR mRNA electroporation of PD-1 − and PD-1 + 2D3 cells. Data information: in (B), means are depicted. * P ≤ 0.05 (Student’s t -test). Abbreviations: cryo, transfected effector cells were cryopreserved prior to co-culture; fresh, stimulator cells were co-cultured immediately following transfection; ns, not significant; PD-1, programmed death-1 protein; TCR, T-cell receptor.

Techniques Used: Transduction, Electroporation, Flow Cytometry, Cytometry, Expressing, Transfection, Co-Culture Assay, Cell Culture

TCR + PD-1 + 2D3 cells as a model assay for evaluation of involvement of PD-1 signaling in cell-mediated antigen-specific T-cell activation ( A – C ) WT1 (A, B) and gp100 (A, C) specific T-cell activation expressed as percentage viable CD8 + eGFP + PD-1 − and PD-1 + 2D3 cells (± SEM) after 24 hours co-culture with different PD-L1 + stimulator cells. Neutralizing antibody against PD-1 (αPD-1; A, B; 15 µg/mL in WT1 model (A, B), 5 µg/mL in gp100 model (A)) or PD-L1 (αPD-L1; C) was added to cells 1 hour prior to co-culture to verify PD-1-mediated signaling, where indicated. (A) PD-1-dependent stimulating capacity of two differently generated peptide-pulsed mature monocyte-derived dendritic cells (WT1 (4 DC donors tested in two independent experiments); gp100 (4 DC donors in four independent experiments)). (B)Impact of induced PD-L1 expression on peptide-pulsed THP-1 leukemic cells on WT1-specific T-cell activation (6 replicate experiments). (C) gp100-specific PD-1 + T cell-activating capacity of peptide-pulsed wild-type or stably transduced PD-L1 + MCF-7 breast carcinoma cells (4 replicate experiments). Data information: in A, the horizontal line represents the median percentage eGFP expression ( n = 4). * P ≤ 0.05, ** P
Figure Legend Snippet: TCR + PD-1 + 2D3 cells as a model assay for evaluation of involvement of PD-1 signaling in cell-mediated antigen-specific T-cell activation ( A – C ) WT1 (A, B) and gp100 (A, C) specific T-cell activation expressed as percentage viable CD8 + eGFP + PD-1 − and PD-1 + 2D3 cells (± SEM) after 24 hours co-culture with different PD-L1 + stimulator cells. Neutralizing antibody against PD-1 (αPD-1; A, B; 15 µg/mL in WT1 model (A, B), 5 µg/mL in gp100 model (A)) or PD-L1 (αPD-L1; C) was added to cells 1 hour prior to co-culture to verify PD-1-mediated signaling, where indicated. (A) PD-1-dependent stimulating capacity of two differently generated peptide-pulsed mature monocyte-derived dendritic cells (WT1 (4 DC donors tested in two independent experiments); gp100 (4 DC donors in four independent experiments)). (B)Impact of induced PD-L1 expression on peptide-pulsed THP-1 leukemic cells on WT1-specific T-cell activation (6 replicate experiments). (C) gp100-specific PD-1 + T cell-activating capacity of peptide-pulsed wild-type or stably transduced PD-L1 + MCF-7 breast carcinoma cells (4 replicate experiments). Data information: in A, the horizontal line represents the median percentage eGFP expression ( n = 4). * P ≤ 0.05, ** P

Techniques Used: Activation Assay, Co-Culture Assay, Generated, Derivative Assay, Expressing, Stable Transfection

27) Product Images from "Nanoliposomes for Safe and Efficient Therapeutic mRNA Delivery: A Step Toward Nanotheranostics in Inflammatory and Cardiovascular Diseases as well as Cancer"

Article Title: Nanoliposomes for Safe and Efficient Therapeutic mRNA Delivery: A Step Toward Nanotheranostics in Inflammatory and Cardiovascular Diseases as well as Cancer

Journal: Nanotheranostics

doi: 10.7150/ntno.19449

Flow cytometry demonstrating the transfection efficiency of CD39 mRNA nanoplexes in media without FBS in CHO cells. Significant transfection efficiency could be seen 24 h after transfection with CD39 mRNA using 1 μl (A) and 2.5 μl (B) nanoliposomes using an anti-CD39-FITC antibody in flow cytometry. The bar graphs depict the % of protein expressing cells, as well as the median fluorescence intensity AU. The different groups were compared using repeated ANOVA measures with Bonferroni post-tests. (C) Representative fluorescence histograms are shown underneath the bar graphs (mean ± SD, **p
Figure Legend Snippet: Flow cytometry demonstrating the transfection efficiency of CD39 mRNA nanoplexes in media without FBS in CHO cells. Significant transfection efficiency could be seen 24 h after transfection with CD39 mRNA using 1 μl (A) and 2.5 μl (B) nanoliposomes using an anti-CD39-FITC antibody in flow cytometry. The bar graphs depict the % of protein expressing cells, as well as the median fluorescence intensity AU. The different groups were compared using repeated ANOVA measures with Bonferroni post-tests. (C) Representative fluorescence histograms are shown underneath the bar graphs (mean ± SD, **p

Techniques Used: Flow Cytometry, Cytometry, Transfection, Expressing, Fluorescence

CD39 mRNA nanoplexes show long-term transfection in A549 cells in flow cytometry. CD39 mRNA and 5 μl of nanoplexes were transfected in A549 cells. The efficiency was analyzed in flow cytometry 120 h (A) and 168 h (B) after transfection using an anti-CD39-FITC antibody. The bar graphs depict the % of protein expressing cells. These assays were analyzed using repeated ANOVA measures with Bonferroni post-tests (mean ± SD, ***p > 0.001).
Figure Legend Snippet: CD39 mRNA nanoplexes show long-term transfection in A549 cells in flow cytometry. CD39 mRNA and 5 μl of nanoplexes were transfected in A549 cells. The efficiency was analyzed in flow cytometry 120 h (A) and 168 h (B) after transfection using an anti-CD39-FITC antibody. The bar graphs depict the % of protein expressing cells. These assays were analyzed using repeated ANOVA measures with Bonferroni post-tests (mean ± SD, ***p > 0.001).

Techniques Used: Transfection, Flow Cytometry, Cytometry, Expressing

CD39 mRNA nanoplexes demonstrating no influence on cell morphology, growth, or transfection efficiency in HEK293 cells 72 h after transfection in flow cytometry. (A) Representative microscope pictures show no influence on morphology or growth of 5 μl of CD39 mRNA nanoplexes after transfection on HEK293 cells after 24 h and 72 h. The transfection efficiency (%) (B) and mean fluorescence intensity (AU) (C) were evaluated additionally in flow cytometry 72 h after transfection using an anti-CD39-FITC antibody. The flow cytometry assay was analyzed using repeated ANOVA measures with Bonferroni post-tests (mean ± SD, ***p > 0.001).
Figure Legend Snippet: CD39 mRNA nanoplexes demonstrating no influence on cell morphology, growth, or transfection efficiency in HEK293 cells 72 h after transfection in flow cytometry. (A) Representative microscope pictures show no influence on morphology or growth of 5 μl of CD39 mRNA nanoplexes after transfection on HEK293 cells after 24 h and 72 h. The transfection efficiency (%) (B) and mean fluorescence intensity (AU) (C) were evaluated additionally in flow cytometry 72 h after transfection using an anti-CD39-FITC antibody. The flow cytometry assay was analyzed using repeated ANOVA measures with Bonferroni post-tests (mean ± SD, ***p > 0.001).

Techniques Used: Transfection, Flow Cytometry, Cytometry, Microscopy, Fluorescence

mRNA generation and size measurement of the DC-Cholesterol/DOPE nanoliposomes. (A) Electrophoresis with 1% agarose-TBE gel: CD39 mRNA (1533 bp). eGFP mRNA (993 bp) after purification and 0.5-10 kB RNA ladder. (B) Size distribution curve of the DC-Cholesterol/DOPE NLps 1 day after generation measured with a Zetazizer spectrometer exhibit an average of about 110 nm. (C) Comparison of size distribution of liposomes 1 day and 30 days after generation showing no difference. The two groups were compared using Student t-tests (mean ± SD, n=5).
Figure Legend Snippet: mRNA generation and size measurement of the DC-Cholesterol/DOPE nanoliposomes. (A) Electrophoresis with 1% agarose-TBE gel: CD39 mRNA (1533 bp). eGFP mRNA (993 bp) after purification and 0.5-10 kB RNA ladder. (B) Size distribution curve of the DC-Cholesterol/DOPE NLps 1 day after generation measured with a Zetazizer spectrometer exhibit an average of about 110 nm. (C) Comparison of size distribution of liposomes 1 day and 30 days after generation showing no difference. The two groups were compared using Student t-tests (mean ± SD, n=5).

Techniques Used: Electrophoresis, Purification

28) Product Images from "Retrovirally transduced murine T lymphocytes expressing FasL mediate effective killing of prostate cancer cells"

Article Title: Retrovirally transduced murine T lymphocytes expressing FasL mediate effective killing of prostate cancer cells

Journal: Cancer gene therapy

doi: 10.1038/cgt.2008.96

Reduction of T-cell transgene expression over time in culture is slowed by IL-15. ( a ) Transgene expression on T cells stimulated with anti-CD3/CD28 beads and transduced with oncoretroviruses is shown relative to expression levels at day 3, as measured by flow cytometry. Data are representative of two independent experiments. ( b ) Where the percentage of eGFP-expressing T cells remains stable by day 3 post-transduction, the MFI of transduced cells is decreased with time. MFI is measured by flow cytometry and is calculated on events falling in the gated area R4. ( c ) Transduced cells have a reduced proliferative capacity compared with non-transduced control cells over time, as measured by 3 H-thymidine incorporation assays. Cells (10 5 ) were plated per well on day 3 and growth rates were assessed on day 3, day 4, and day 5 post-transduction. Transduced cells were 46% eGFP positive on day 3. Data are representative of two experiments, each with n = 3. ( d ) When ncFasL T cells are cultured with IL-15 rather than IL-2, loss of ncFasL transgene expression is slowed.
Figure Legend Snippet: Reduction of T-cell transgene expression over time in culture is slowed by IL-15. ( a ) Transgene expression on T cells stimulated with anti-CD3/CD28 beads and transduced with oncoretroviruses is shown relative to expression levels at day 3, as measured by flow cytometry. Data are representative of two independent experiments. ( b ) Where the percentage of eGFP-expressing T cells remains stable by day 3 post-transduction, the MFI of transduced cells is decreased with time. MFI is measured by flow cytometry and is calculated on events falling in the gated area R4. ( c ) Transduced cells have a reduced proliferative capacity compared with non-transduced control cells over time, as measured by 3 H-thymidine incorporation assays. Cells (10 5 ) were plated per well on day 3 and growth rates were assessed on day 3, day 4, and day 5 post-transduction. Transduced cells were 46% eGFP positive on day 3. Data are representative of two experiments, each with n = 3. ( d ) When ncFasL T cells are cultured with IL-15 rather than IL-2, loss of ncFasL transgene expression is slowed.

Techniques Used: Expressing, Transduction, Flow Cytometry, Cytometry, Cell Culture

Optimization of T-cell transduction. T cells were transduced with E86-ncFasL virus under various conditions and transduction efficiency determined. ( a ) Representative plots showing ncFasL-expression on T cells following transduction with oncoretroviral supernatant in the presence of protamine sulfate with: no additional manipulation; transduction performed on fibronectin-coated plates; transduction by spinoculation at 1000 g for 1 h, 32 °C; or by co-culture with E86 ncFasL virus-producing cells for 3 days. With the exception of co-culture transduced cells, T cells were transduced two times. Filled histograms represent anti-FasL-labeled cells, outline histograms represent matched isotype control staining. ( b ) Representative flow cytometry plots showing FasL expression on non-transduced (NT) T cells or T cells following one round of transduction with: unmanipulated retroviral supernatant; concentrated retroviral supernatant; or concentrated retroviral supernatant with a 3 h incubation on ice. In parts ( a ) and ( b ), T cells were stimulated with anti-CD3/CD28 beads. ( c ) Effect of the T-cell stimulation method on transduction efficiency. T cells were stimulated for 48 h with 5 μg ml −1 ConA, 2.5 μg ml −1 PHA, or anti-CD3/CD28 beads at an initial bead-to-cell ratio of 3:1, and subjected to retroviral transduction with concentrated supernatant and incubation on ice. Transgene expression levels are shown on day 3 post-transduction for E86-eGFP, E86-FasL, E86-ncFasL and E86-ncFasL/c-FLIP transduced T cells, as determined by flow cytometry.
Figure Legend Snippet: Optimization of T-cell transduction. T cells were transduced with E86-ncFasL virus under various conditions and transduction efficiency determined. ( a ) Representative plots showing ncFasL-expression on T cells following transduction with oncoretroviral supernatant in the presence of protamine sulfate with: no additional manipulation; transduction performed on fibronectin-coated plates; transduction by spinoculation at 1000 g for 1 h, 32 °C; or by co-culture with E86 ncFasL virus-producing cells for 3 days. With the exception of co-culture transduced cells, T cells were transduced two times. Filled histograms represent anti-FasL-labeled cells, outline histograms represent matched isotype control staining. ( b ) Representative flow cytometry plots showing FasL expression on non-transduced (NT) T cells or T cells following one round of transduction with: unmanipulated retroviral supernatant; concentrated retroviral supernatant; or concentrated retroviral supernatant with a 3 h incubation on ice. In parts ( a ) and ( b ), T cells were stimulated with anti-CD3/CD28 beads. ( c ) Effect of the T-cell stimulation method on transduction efficiency. T cells were stimulated for 48 h with 5 μg ml −1 ConA, 2.5 μg ml −1 PHA, or anti-CD3/CD28 beads at an initial bead-to-cell ratio of 3:1, and subjected to retroviral transduction with concentrated supernatant and incubation on ice. Transgene expression levels are shown on day 3 post-transduction for E86-eGFP, E86-FasL, E86-ncFasL and E86-ncFasL/c-FLIP transduced T cells, as determined by flow cytometry.

Techniques Used: Transduction, Expressing, Co-Culture Assay, Labeling, Staining, Flow Cytometry, Cytometry, Incubation, Cell Stimulation

29) Product Images from "Novel Gene Therapy Viral Vector Using Non-Oncogenic Lymphotropic Herpesvirus"

Article Title: Novel Gene Therapy Viral Vector Using Non-Oncogenic Lymphotropic Herpesvirus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0056027

Two-color flow cytometric characterization of surface marker and EGFP expression of PBMCs infected with H6R28LEP. PBMCs were activated by culture with IL-2 and plate-bound anti-CD3 monoclonal antibody for 3 d, infected with H6R28LEP, and underwent fluorescence-activated cell sorting (FACS) analysis at 4 d after infection. A: Positivity of CD4 and EGFP in H6R28LEP-infected PBMCs. B: Positivity of CD8 and EGFP under the same experimental situation as for panel A. C: Positivity of CD19 and EGFP in unstimulated H6R28LEP-infected PBMCs. D: Positivity of CD34 and EGFP in unstimulated H6R28LEP-infected CBMCs. The percentage of cells in each quadrant of the FACS profile is shown in the diagram beneath each panel. Results are representative of at least three independent experiments.
Figure Legend Snippet: Two-color flow cytometric characterization of surface marker and EGFP expression of PBMCs infected with H6R28LEP. PBMCs were activated by culture with IL-2 and plate-bound anti-CD3 monoclonal antibody for 3 d, infected with H6R28LEP, and underwent fluorescence-activated cell sorting (FACS) analysis at 4 d after infection. A: Positivity of CD4 and EGFP in H6R28LEP-infected PBMCs. B: Positivity of CD8 and EGFP under the same experimental situation as for panel A. C: Positivity of CD19 and EGFP in unstimulated H6R28LEP-infected PBMCs. D: Positivity of CD34 and EGFP in unstimulated H6R28LEP-infected CBMCs. The percentage of cells in each quadrant of the FACS profile is shown in the diagram beneath each panel. Results are representative of at least three independent experiments.

Techniques Used: Flow Cytometry, Marker, Expressing, Infection, Fluorescence, FACS

30) Product Images from "A versatile T cell-based assay to assess therapeutic antigen-specific PD-1-targeted approaches"

Article Title: A versatile T cell-based assay to assess therapeutic antigen-specific PD-1-targeted approaches

Journal: Oncotarget

doi: 10.18632/oncotarget.25591

Validation of antigen-specific TCR function of transfected 2D3 and PD-1 + 2D3 cells ( A – B ) Activation profiles of freshly used or thawed WT1-specific TCR mRNA-electroporated PD-1 − and PD-1 + 2D3 cells left unstimulated (-) versus 24 hours co-culture with unloaded (T2 -pept ) and WT1 peptide-pulsed (T2 +pept ) stimulator cells at a ratio of 2:1. Comparable results were obtained with gp100 TCR-positive PD-1 − and PD-1 + 2D3 cells. (A) Representative example of TCR activation-mediated eGFP expression within the viable CD8 + cell population as assessed with flow cytometry (freshly used WT1 TCR mRNA-electroporated PD-1 + 2D3 cells). (B) The left graph shows the mean percentage (± SEM) WT1-specific TCR activation-mediated eGFP expression from 2–8 replicate experiments. The right panel depicts the mean amount (± SEM) of secreted granzyme B determined with ELISA in cell-free 24-hour culture supernatant of 10 5 effector cells for 2–4 replicate experiments. Data information: * P ≤ 0.05, ** P
Figure Legend Snippet: Validation of antigen-specific TCR function of transfected 2D3 and PD-1 + 2D3 cells ( A – B ) Activation profiles of freshly used or thawed WT1-specific TCR mRNA-electroporated PD-1 − and PD-1 + 2D3 cells left unstimulated (-) versus 24 hours co-culture with unloaded (T2 -pept ) and WT1 peptide-pulsed (T2 +pept ) stimulator cells at a ratio of 2:1. Comparable results were obtained with gp100 TCR-positive PD-1 − and PD-1 + 2D3 cells. (A) Representative example of TCR activation-mediated eGFP expression within the viable CD8 + cell population as assessed with flow cytometry (freshly used WT1 TCR mRNA-electroporated PD-1 + 2D3 cells). (B) The left graph shows the mean percentage (± SEM) WT1-specific TCR activation-mediated eGFP expression from 2–8 replicate experiments. The right panel depicts the mean amount (± SEM) of secreted granzyme B determined with ELISA in cell-free 24-hour culture supernatant of 10 5 effector cells for 2–4 replicate experiments. Data information: * P ≤ 0.05, ** P

Techniques Used: Transfection, Activation Assay, Co-Culture Assay, Expressing, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

Efficiency of PD-1 transduction, TCR mRNA electroporation and cryopreservation of 2D3 cells ( A ) Representative flow cytometry T-cell receptor (TCRαβ) and programmed death-1 (PD-1) protein surface expression profiles and corresponding isotype controls of non-transduced PD-1 − 2D3 and PD-1-transduced (PD-1 + ) 2D3 cells 24 hours after TCR mRNA electroporation (fresh; 10-14 replicates) and after thawing of TCR mRNA-electroporated cells (cryo; 6 replicates). ( B ) Percentage viability and recovery upon TCR mRNA electroporation of PD-1 − and PD-1 + 2D3 cells. Data information: in (B), means are depicted. * P ≤ 0.05 (Student’s t -test). Abbreviations: cryo, transfected effector cells were cryopreserved prior to co-culture; fresh, stimulator cells were co-cultured immediately following transfection; ns, not significant; PD-1, programmed death-1 protein; TCR, T-cell receptor.
Figure Legend Snippet: Efficiency of PD-1 transduction, TCR mRNA electroporation and cryopreservation of 2D3 cells ( A ) Representative flow cytometry T-cell receptor (TCRαβ) and programmed death-1 (PD-1) protein surface expression profiles and corresponding isotype controls of non-transduced PD-1 − 2D3 and PD-1-transduced (PD-1 + ) 2D3 cells 24 hours after TCR mRNA electroporation (fresh; 10-14 replicates) and after thawing of TCR mRNA-electroporated cells (cryo; 6 replicates). ( B ) Percentage viability and recovery upon TCR mRNA electroporation of PD-1 − and PD-1 + 2D3 cells. Data information: in (B), means are depicted. * P ≤ 0.05 (Student’s t -test). Abbreviations: cryo, transfected effector cells were cryopreserved prior to co-culture; fresh, stimulator cells were co-cultured immediately following transfection; ns, not significant; PD-1, programmed death-1 protein; TCR, T-cell receptor.

Techniques Used: Transduction, Electroporation, Flow Cytometry, Cytometry, Expressing, Transfection, Co-Culture Assay, Cell Culture

TCR + PD-1 + 2D3 cells as a model assay for evaluation of involvement of PD-1 signaling in cell-mediated antigen-specific T-cell activation ( A – C ) WT1 (A, B) and gp100 (A, C) specific T-cell activation expressed as percentage viable CD8 + eGFP + PD-1 − and PD-1 + 2D3 cells (± SEM) after 24 hours co-culture with different PD-L1 + stimulator cells. Neutralizing antibody against PD-1 (αPD-1; A, B; 15 µg/mL in WT1 model (A, B), 5 µg/mL in gp100 model (A)) or PD-L1 (αPD-L1; C) was added to cells 1 hour prior to co-culture to verify PD-1-mediated signaling, where indicated. (A) PD-1-dependent stimulating capacity of two differently generated peptide-pulsed mature monocyte-derived dendritic cells (WT1 (4 DC donors tested in two independent experiments); gp100 (4 DC donors in four independent experiments)). (B)Impact of induced PD-L1 expression on peptide-pulsed THP-1 leukemic cells on WT1-specific T-cell activation (6 replicate experiments). (C) gp100-specific PD-1 + T cell-activating capacity of peptide-pulsed wild-type or stably transduced PD-L1 + MCF-7 breast carcinoma cells (4 replicate experiments). Data information: in A, the horizontal line represents the median percentage eGFP expression ( n = 4). * P ≤ 0.05, ** P
Figure Legend Snippet: TCR + PD-1 + 2D3 cells as a model assay for evaluation of involvement of PD-1 signaling in cell-mediated antigen-specific T-cell activation ( A – C ) WT1 (A, B) and gp100 (A, C) specific T-cell activation expressed as percentage viable CD8 + eGFP + PD-1 − and PD-1 + 2D3 cells (± SEM) after 24 hours co-culture with different PD-L1 + stimulator cells. Neutralizing antibody against PD-1 (αPD-1; A, B; 15 µg/mL in WT1 model (A, B), 5 µg/mL in gp100 model (A)) or PD-L1 (αPD-L1; C) was added to cells 1 hour prior to co-culture to verify PD-1-mediated signaling, where indicated. (A) PD-1-dependent stimulating capacity of two differently generated peptide-pulsed mature monocyte-derived dendritic cells (WT1 (4 DC donors tested in two independent experiments); gp100 (4 DC donors in four independent experiments)). (B)Impact of induced PD-L1 expression on peptide-pulsed THP-1 leukemic cells on WT1-specific T-cell activation (6 replicate experiments). (C) gp100-specific PD-1 + T cell-activating capacity of peptide-pulsed wild-type or stably transduced PD-L1 + MCF-7 breast carcinoma cells (4 replicate experiments). Data information: in A, the horizontal line represents the median percentage eGFP expression ( n = 4). * P ≤ 0.05, ** P

Techniques Used: Activation Assay, Co-Culture Assay, Generated, Derivative Assay, Expressing, Stable Transfection

31) Product Images from "Histamine receptor H1 is required for TCR-mediated p38 MAPK activation and optimal IFN-? production in mice"

Article Title: Histamine receptor H1 is required for TCR-mediated p38 MAPK activation and optimal IFN-? production in mice

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI32792

Activation of p38 MAPK by TCR ligation requires H 1 R signals. ( A ) Purified CD4 + T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and nuclear extracts were prepared and analyzed for NF-κB DNA binding by EMSA. ( B ) CD4 + T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-STAT1 (P-STAT1) and total STAT1 by Western blot analysis. Actin was used as loading control. ( C ) CD4 + T cells from WT and H1RKO mice were treated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-p38 MAPK and total p38 by Western blot analysis. ( D ) CD4 + T cells from WT and H1RKO mice were activated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-ERK and total ERK by Western blot analysis. ( E ) CD4 + T cells from WT, H1RKO, and HIRKO-Tg3 mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were analyzed for phospho-p38, total p38, and actin by Western blotting. Data are representative of at least 2 independent experiments.
Figure Legend Snippet: Activation of p38 MAPK by TCR ligation requires H 1 R signals. ( A ) Purified CD4 + T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and nuclear extracts were prepared and analyzed for NF-κB DNA binding by EMSA. ( B ) CD4 + T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-STAT1 (P-STAT1) and total STAT1 by Western blot analysis. Actin was used as loading control. ( C ) CD4 + T cells from WT and H1RKO mice were treated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-p38 MAPK and total p38 by Western blot analysis. ( D ) CD4 + T cells from WT and H1RKO mice were activated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-ERK and total ERK by Western blot analysis. ( E ) CD4 + T cells from WT, H1RKO, and HIRKO-Tg3 mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were analyzed for phospho-p38, total p38, and actin by Western blotting. Data are representative of at least 2 independent experiments.

Techniques Used: Activation Assay, Ligation, Purification, Mouse Assay, Binding Assay, Western Blot

Hrh1 is downregulated upon activation in CD4 + T cells. ( A ) CD4 + T cells from WT and H1RKO mice were stimulated in the presence of anti-CD3 and anti-CD28 mAbs for 16 h and then retrovirally transduced with pEGZ-HA-H 1 R or with empty pEGZ control plasmids. Transduced, sorted EGFP + cells were then restimulated with anti-CD3 mAb, and 24 h later, the supernatants were harvested for determination of IFN-γ by ELISA in triplicate. Data are representative of 2 independent experiments. *** P
Figure Legend Snippet: Hrh1 is downregulated upon activation in CD4 + T cells. ( A ) CD4 + T cells from WT and H1RKO mice were stimulated in the presence of anti-CD3 and anti-CD28 mAbs for 16 h and then retrovirally transduced with pEGZ-HA-H 1 R or with empty pEGZ control plasmids. Transduced, sorted EGFP + cells were then restimulated with anti-CD3 mAb, and 24 h later, the supernatants were harvested for determination of IFN-γ by ELISA in triplicate. Data are representative of 2 independent experiments. *** P

Techniques Used: Activation Assay, Mouse Assay, Transduction, Enzyme-linked Immunosorbent Assay

Transgenic expression of H 1 R in H1RKO CD4 + T cells complements IFN-γ production. ( A ) Hrh1 transgene expression was analyzed by RT-PCR in CD4 + T cells from WT and H1RKO mice and the 2 independent lines of H 1 R transgenic mice crossed with H1RKO mice, HIRKO-Tg1 and HIRKO-Tg3. ( B ) CD4 + T cells were stained with anti-HA mAb (red) and visualized by confocal microscopy. Nuclear stain Topro (blue) is shown. ( C ) CD4 + T cells were activated with anti-CD3 and anti-CD28 mAbs for 72 h, and IFN-γ was determined by ELISA. Data are expressed as IFN-γ production relative to that by WT cells (set as 100%). ( D ) CD4 + T cells from WT, H1RKO, and HIRKO-Tg3 mice were stimulated as in C for the indicated periods of time, and IFN-γ was determined by ELISA. F = 55.1, P
Figure Legend Snippet: Transgenic expression of H 1 R in H1RKO CD4 + T cells complements IFN-γ production. ( A ) Hrh1 transgene expression was analyzed by RT-PCR in CD4 + T cells from WT and H1RKO mice and the 2 independent lines of H 1 R transgenic mice crossed with H1RKO mice, HIRKO-Tg1 and HIRKO-Tg3. ( B ) CD4 + T cells were stained with anti-HA mAb (red) and visualized by confocal microscopy. Nuclear stain Topro (blue) is shown. ( C ) CD4 + T cells were activated with anti-CD3 and anti-CD28 mAbs for 72 h, and IFN-γ was determined by ELISA. Data are expressed as IFN-γ production relative to that by WT cells (set as 100%). ( D ) CD4 + T cells from WT, H1RKO, and HIRKO-Tg3 mice were stimulated as in C for the indicated periods of time, and IFN-γ was determined by ELISA. F = 55.1, P

Techniques Used: Transgenic Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Activation of p38 MAPK by TCR ligation is mediated by histamine/H 1 R binding. ( A ) CD4 + T cells from WT and H1RKO mice were treated with histamine (10 –7 M) for the indicated periods of time in the histamine-free medium. Whole-cell extracts were used to analyze phospho-p38, total p38, and actin by Western blotting. ( B ) CD4 + T cells were isolated from WT and H1RKO mice and stimulated with anti-CD3 and anti-CD28 mAbs in the histamine free-medium for the indicated periods of time. CD4 + T cells stimulated in medium containing 10 –7 M histamine (Hist) are shown as positive control for p38 MAPK activation. Phospho-p38, total p38, and actin are shown. ( C ) CD4 + T cells from WT and H1RKO mice were incubated with anti-CD3 and anti-CD28 mAbs, 10 –7 M histamine, or both in the histamine-free medium for 30 minutes, and whole-cell lysates were analyzed for phospho-p38, total p-38, and actin by Western blotting. ( D ) CD4 + T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were analyzed for T-bet expression by Western blot. Actin is shown as loading control. ( E ) Purified CD4 + T cells from WT, H1RKO, and H1RKO-MKK6 Glu Tg mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and supernatants were analyzed for IFN-γ production by ELISA in triplicate. F = 21.7, P
Figure Legend Snippet: Activation of p38 MAPK by TCR ligation is mediated by histamine/H 1 R binding. ( A ) CD4 + T cells from WT and H1RKO mice were treated with histamine (10 –7 M) for the indicated periods of time in the histamine-free medium. Whole-cell extracts were used to analyze phospho-p38, total p38, and actin by Western blotting. ( B ) CD4 + T cells were isolated from WT and H1RKO mice and stimulated with anti-CD3 and anti-CD28 mAbs in the histamine free-medium for the indicated periods of time. CD4 + T cells stimulated in medium containing 10 –7 M histamine (Hist) are shown as positive control for p38 MAPK activation. Phospho-p38, total p38, and actin are shown. ( C ) CD4 + T cells from WT and H1RKO mice were incubated with anti-CD3 and anti-CD28 mAbs, 10 –7 M histamine, or both in the histamine-free medium for 30 minutes, and whole-cell lysates were analyzed for phospho-p38, total p-38, and actin by Western blotting. ( D ) CD4 + T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were analyzed for T-bet expression by Western blot. Actin is shown as loading control. ( E ) Purified CD4 + T cells from WT, H1RKO, and H1RKO-MKK6 Glu Tg mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and supernatants were analyzed for IFN-γ production by ELISA in triplicate. F = 21.7, P

Techniques Used: Activation Assay, Ligation, Binding Assay, Mouse Assay, Western Blot, Isolation, Positive Control, Incubation, Expressing, Purification, Enzyme-linked Immunosorbent Assay

H 1 R is required for IFN-γ production by CD4 + T cells. Purified CD4 + T cells from WT and H1RKO mice were activated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) mAbs either ( A ) in the presence of IL-12 (4 ng/ml) and anti–IL-4 mAb (10 μg/ml), ( B ) in the presence of IL-4 (30 ng/ml) and anti–IFN-γ mAb (10 μg/ml), or ( C ) in the presence of TGF-β (1 ng/ml), IL-6 (30 ng/ml), and anti–IFN-γ (10 μg/ml) and anti-IL4 mAbs (10 μg/ml). After 4 days, the cells were restimulated with anti-CD3 mAb (5 μg/ml) for 24 h. Production of ( A ) IFN-γ, ( B ) IL-4, and ( C ) IL-17 was determined by ELISA in triplicate. * P
Figure Legend Snippet: H 1 R is required for IFN-γ production by CD4 + T cells. Purified CD4 + T cells from WT and H1RKO mice were activated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) mAbs either ( A ) in the presence of IL-12 (4 ng/ml) and anti–IL-4 mAb (10 μg/ml), ( B ) in the presence of IL-4 (30 ng/ml) and anti–IFN-γ mAb (10 μg/ml), or ( C ) in the presence of TGF-β (1 ng/ml), IL-6 (30 ng/ml), and anti–IFN-γ (10 μg/ml) and anti-IL4 mAbs (10 μg/ml). After 4 days, the cells were restimulated with anti-CD3 mAb (5 μg/ml) for 24 h. Production of ( A ) IFN-γ, ( B ) IL-4, and ( C ) IL-17 was determined by ELISA in triplicate. * P

Techniques Used: Purification, Mouse Assay, Enzyme-linked Immunosorbent Assay

32) Product Images from "Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid"

Article Title: Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20070602

RA production by LpDC is requisite for optimal Foxp3 + T reg cell conversion. Foxp3 − CD4 T cells were cocultured with DC at a 10:1 ratio as described in Fig 4. (A) Cells were stained for α 4 β 7 and assessed for eGFP fluorescence (Foxp3) by flow cytometry. Results are representative of three independent experiments. (B) The dose responsiveness of all-trans RA on eGFP (Foxp3) expression by CD4 T cells cocultured with SpDC was determined by flow cytometry. Error bars represent the SDs of the means of three individual samples from one experiment. Statistical significance was determined using the Student's t test. *, P
Figure Legend Snippet: RA production by LpDC is requisite for optimal Foxp3 + T reg cell conversion. Foxp3 − CD4 T cells were cocultured with DC at a 10:1 ratio as described in Fig 4. (A) Cells were stained for α 4 β 7 and assessed for eGFP fluorescence (Foxp3) by flow cytometry. Results are representative of three independent experiments. (B) The dose responsiveness of all-trans RA on eGFP (Foxp3) expression by CD4 T cells cocultured with SpDC was determined by flow cytometry. Error bars represent the SDs of the means of three individual samples from one experiment. Statistical significance was determined using the Student's t test. *, P

Techniques Used: Staining, Fluorescence, Flow Cytometry, Cytometry, Expressing

33) Product Images from "Activation of NF-κB after chronic ethanol intake and haemorrhagic shock/resuscitation in mice"

Article Title: Activation of NF-κB after chronic ethanol intake and haemorrhagic shock/resuscitation in mice

Journal: British Journal of Pharmacology

doi: 10.1111/bph.12224

The EtOH-diet increases expression of CD11b on PMNL cell surface and induces cis -NF-κB EGFP transcriptional activity at 2 h following H/R. Representative FACS dot plots are given. In (A), whole leukocyte population was analysed and PMNL were gated
Figure Legend Snippet: The EtOH-diet increases expression of CD11b on PMNL cell surface and induces cis -NF-κB EGFP transcriptional activity at 2 h following H/R. Representative FACS dot plots are given. In (A), whole leukocyte population was analysed and PMNL were gated

Techniques Used: Expressing, Activity Assay, FACS

34) Product Images from "CRISPR-Cas3 induces broad and unidirectional genome editing in human cells"

Article Title: CRISPR-Cas3 induces broad and unidirectional genome editing in human cells

Journal: Nature Communications

doi: 10.1038/s41467-019-13226-x

CRISPR-Cas3 facilitates a large deletion at endogenous targeted loci in 293T cells. a Schematic of the CRISPR-Cas3 system targeting human EMX1 and CCR5 loci. Primer sets (red arrows) used for a 3.7 kb PCR product of EMX1 and a 9.7 kb PCR product of CCR5 are shown. b Electrophoresis of the PCR products. CRISPR-Cas3 targeting AAG PAM or ATG PAM, but not TTT PAM, mediates deletions (see Supplementary Fig. 5a ). c Comparison of the editing efficiency between Cas3 (red) and Cas9 (blue) via NGS of the PCR amplicons at various target sites (see Supplementary Table 3 ). d Cas3-mediated DNA deletion patterns via microarray-based capture sequencing at EMX1 and CCR5 loci in 293T cells. The deletions (blue bars) are aligned at the starting point of the distal end. e Microarray-based capture sequencing with Cas3/crRNAs (#1–6) targeting a 1-Mb region at the CCR5 loci (see Supplementary Fig. 11 for EMX1 loci). Cas3-mediated DNA deletion patterns are aligned with human genome assembly hg38. Dual crRNA (#1 and #5 or #6) are used for sticking in the interval region. f A surrogate reporter assay using mCherry-2A-EGFP plasmids to characterize Cas3-mediated knockouts in f or knock-ins in g . crRNA #1 (red arrows)/sgRNA (blue arrows) were designed at EGFP-coding sequences, and crRNAs/sgRNAs #2 and #3 were outside the coding sequences (see Supplementary Table 9 ). Data are presented as mean ± SD. * P
Figure Legend Snippet: CRISPR-Cas3 facilitates a large deletion at endogenous targeted loci in 293T cells. a Schematic of the CRISPR-Cas3 system targeting human EMX1 and CCR5 loci. Primer sets (red arrows) used for a 3.7 kb PCR product of EMX1 and a 9.7 kb PCR product of CCR5 are shown. b Electrophoresis of the PCR products. CRISPR-Cas3 targeting AAG PAM or ATG PAM, but not TTT PAM, mediates deletions (see Supplementary Fig. 5a ). c Comparison of the editing efficiency between Cas3 (red) and Cas9 (blue) via NGS of the PCR amplicons at various target sites (see Supplementary Table 3 ). d Cas3-mediated DNA deletion patterns via microarray-based capture sequencing at EMX1 and CCR5 loci in 293T cells. The deletions (blue bars) are aligned at the starting point of the distal end. e Microarray-based capture sequencing with Cas3/crRNAs (#1–6) targeting a 1-Mb region at the CCR5 loci (see Supplementary Fig. 11 for EMX1 loci). Cas3-mediated DNA deletion patterns are aligned with human genome assembly hg38. Dual crRNA (#1 and #5 or #6) are used for sticking in the interval region. f A surrogate reporter assay using mCherry-2A-EGFP plasmids to characterize Cas3-mediated knockouts in f or knock-ins in g . crRNA #1 (red arrows)/sgRNA (blue arrows) were designed at EGFP-coding sequences, and crRNAs/sgRNAs #2 and #3 were outside the coding sequences (see Supplementary Table 9 ). Data are presented as mean ± SD. * P

Techniques Used: CRISPR, Polymerase Chain Reaction, Electrophoresis, Next-Generation Sequencing, Microarray, Sequencing, Reporter Assay

35) Product Images from "A Novel Role of Cab45-G in Mediating Cell Migration in Cancer Cells"

Article Title: A Novel Role of Cab45-G in Mediating Cell Migration in Cancer Cells

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.11037

Overexpression of Cab45-G altered the secretion of ECM proteins. A, Hela cells were transfected with SP-EGFP or SP-EGFP-Cab45-G. The protein levels in the cell lysate and culture medium were determined by Western blot analysis. In SP-EGFP-Cab45G overexpressing HeLa cells, secretion of fibronectin (promote metastasis) to cell medium was increased, while fibulin-1 (inhibit metastasis) decreased. B, Quantification of the ratio of fibronectin secretion. The relative mRNA and protein expression levels were analyzed using the ImageJ image analysis software (NIH, Bethesda, MD). The data are presented as the means ± SEM and were normalized relative to the control cells. ** P
Figure Legend Snippet: Overexpression of Cab45-G altered the secretion of ECM proteins. A, Hela cells were transfected with SP-EGFP or SP-EGFP-Cab45-G. The protein levels in the cell lysate and culture medium were determined by Western blot analysis. In SP-EGFP-Cab45G overexpressing HeLa cells, secretion of fibronectin (promote metastasis) to cell medium was increased, while fibulin-1 (inhibit metastasis) decreased. B, Quantification of the ratio of fibronectin secretion. The relative mRNA and protein expression levels were analyzed using the ImageJ image analysis software (NIH, Bethesda, MD). The data are presented as the means ± SEM and were normalized relative to the control cells. ** P

Techniques Used: Over Expression, Transfection, Western Blot, Expressing, Software

36) Product Images from "Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation"

Article Title: Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation

Journal: Nature Communications

doi: 10.1038/ncomms11730

Depletion of NRON reactivates HIV-1 viruses in latently infected CD4 + T lymphocytes. ( a ) Primary resting CD4 + T lymphocytes were nucleofected with HIV-1 promoter reporter system plasmids, pcDNA3.1-Tat-HA and siRNAs against NRON or nonspecific control. The promoter activity was determined with dual-luciferase reporter assay at 48 h after transfection ( n =3). ( b ) Tat and control GFP were detected by western blotting on NRON knockdown in nucleofected primary resting CD4 + T lymphocytes. Numbers indicated the fold change related to the control. ( c ) The latently infected cells were transfected with NRON siRNAs or nonspecific control, or were transfected with siRNAs in combination with the treatment of SAHA, and detected by FACS at 48–72 h post transfection. The GFP+ ratio indicated the reactivation level ( d ; n =3). ( e ) Resting CD4 + T lymphocytes isolated from HIV-1-infected individuals on suppressive cART were transfected with siRNAs in combination with the treatment of SAHA. After 48 h, HIV-1 virion-associated RNAs in the supernatants were isolated and detected with real-time qRT–PCR ( n =3). ( f ) The intracellular HIV-1 RNA and NRON RNA expression levels were detected in resting CD4 + T lymphocytes isolated from HIV-1-infected individuals on suppressive cART ( n =20), and the correlation between the HIV-1 RNA and NRON RNA levels was shown. The simple linear regression analysis was performed and linear regression line was shown. Data in a , d , e show mean±s.d. (error bars). Results in b represent three independent experiments. * P
Figure Legend Snippet: Depletion of NRON reactivates HIV-1 viruses in latently infected CD4 + T lymphocytes. ( a ) Primary resting CD4 + T lymphocytes were nucleofected with HIV-1 promoter reporter system plasmids, pcDNA3.1-Tat-HA and siRNAs against NRON or nonspecific control. The promoter activity was determined with dual-luciferase reporter assay at 48 h after transfection ( n =3). ( b ) Tat and control GFP were detected by western blotting on NRON knockdown in nucleofected primary resting CD4 + T lymphocytes. Numbers indicated the fold change related to the control. ( c ) The latently infected cells were transfected with NRON siRNAs or nonspecific control, or were transfected with siRNAs in combination with the treatment of SAHA, and detected by FACS at 48–72 h post transfection. The GFP+ ratio indicated the reactivation level ( d ; n =3). ( e ) Resting CD4 + T lymphocytes isolated from HIV-1-infected individuals on suppressive cART were transfected with siRNAs in combination with the treatment of SAHA. After 48 h, HIV-1 virion-associated RNAs in the supernatants were isolated and detected with real-time qRT–PCR ( n =3). ( f ) The intracellular HIV-1 RNA and NRON RNA expression levels were detected in resting CD4 + T lymphocytes isolated from HIV-1-infected individuals on suppressive cART ( n =20), and the correlation between the HIV-1 RNA and NRON RNA levels was shown. The simple linear regression analysis was performed and linear regression line was shown. Data in a , d , e show mean±s.d. (error bars). Results in b represent three independent experiments. * P

Techniques Used: Infection, Activity Assay, Luciferase, Reporter Assay, Transfection, Western Blot, FACS, Isolation, Quantitative RT-PCR, RNA Expression

37) Product Images from "The Armadillo (Dasypus novemcinctus): A Witness but Not a Functional Example for the Emergence of the Butyrophilin 3/Vγ9Vδ2 System in Placental Mammals"

Article Title: The Armadillo (Dasypus novemcinctus): A Witness but Not a Functional Example for the Emergence of the Butyrophilin 3/Vγ9Vδ2 System in Placental Mammals

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00265

Surface expression of a functional armadillo Vδ2 T cell receptor (TCR) chain. (A) Armadillo Vδ2 TCR chains (pMSCV-IRES-mCherry FP armadillo Vδ2 cl7 or cl9) were retrovirally transduced into TCR-negative murine cell lines (BW58 r/mCD28). The human Vγ9 TCR MOP chain (pEGN huVγ9, GenBank: KC170727.1) was co-transduced and TCR surface expression was confirmed with flow cytometry stainings of the human Vγ9 chain, Vδ2 chain, and mouse CD3. Dotplots of Vγ9 ( Y -axis, log) and CD3 ( X -axis, log) co-stainings are shown and geometric means of Vγ9, Vδ2, and CD3 stainings are indicated. (B) BW58 r/mCD28 cells and TCR transductants were cultured for 22 h in 96-well plates coated with α-mCD3 mAb or with RAJI-RT1B 1 cells in the presence of increasing amounts of HMBPP. Mean + SEM of three independent experiments is shown for each cell line. Stimulation of hu/huTCR with 10 µg/ml α-mCD3 mAb resulted in 651 pg/ml (SEM: 129).
Figure Legend Snippet: Surface expression of a functional armadillo Vδ2 T cell receptor (TCR) chain. (A) Armadillo Vδ2 TCR chains (pMSCV-IRES-mCherry FP armadillo Vδ2 cl7 or cl9) were retrovirally transduced into TCR-negative murine cell lines (BW58 r/mCD28). The human Vγ9 TCR MOP chain (pEGN huVγ9, GenBank: KC170727.1) was co-transduced and TCR surface expression was confirmed with flow cytometry stainings of the human Vγ9 chain, Vδ2 chain, and mouse CD3. Dotplots of Vγ9 ( Y -axis, log) and CD3 ( X -axis, log) co-stainings are shown and geometric means of Vγ9, Vδ2, and CD3 stainings are indicated. (B) BW58 r/mCD28 cells and TCR transductants were cultured for 22 h in 96-well plates coated with α-mCD3 mAb or with RAJI-RT1B 1 cells in the presence of increasing amounts of HMBPP. Mean + SEM of three independent experiments is shown for each cell line. Stimulation of hu/huTCR with 10 µg/ml α-mCD3 mAb resulted in 651 pg/ml (SEM: 129).

Techniques Used: Expressing, Functional Assay, Flow Cytometry, Cytometry, Cell Culture

38) Product Images from "Transdifferentiation of Human Hair Follicle Mesenchymal Stem Cells into Red Blood Cells by OCT4"

Article Title: Transdifferentiation of Human Hair Follicle Mesenchymal Stem Cells into Red Blood Cells by OCT4

Journal: Stem Cells International

doi: 10.1155/2015/389628

Transdifferentiation of hHFMSCs into erythrocytes without passing by stage of HSC. (a) Cell stained with monoclonal antibody against HSC marker CD34, myeloid progenitor marker CD45, monocyte-macrophage marker CD14, and granulocytic marker CD15. (b) Expression of hematopoietic progenitor markers CD34 and CD45 was detected after hematopoietic stimulation by flow cytometric analysis. On days 3 and 5 after hHFMSC OCT4 cells were induced by hematopoietic medium, cells were analyzed by staining with fluorochrome-conjugated monoclonal antibodies PE-anti-CD45 and PE-Cy5-anti-CD34. (c) CFU assay images and (d) quantitative analysis of CFU formation ( n = 3). Erythroid blast forming units, BFU-E; erythroid CFU, CFU-E; monocytic CFU, CFU-M; granulocytic CFU, CFU-G; megakaryocytic CFU, CFU-Mk. n.d., undetected.
Figure Legend Snippet: Transdifferentiation of hHFMSCs into erythrocytes without passing by stage of HSC. (a) Cell stained with monoclonal antibody against HSC marker CD34, myeloid progenitor marker CD45, monocyte-macrophage marker CD14, and granulocytic marker CD15. (b) Expression of hematopoietic progenitor markers CD34 and CD45 was detected after hematopoietic stimulation by flow cytometric analysis. On days 3 and 5 after hHFMSC OCT4 cells were induced by hematopoietic medium, cells were analyzed by staining with fluorochrome-conjugated monoclonal antibodies PE-anti-CD45 and PE-Cy5-anti-CD34. (c) CFU assay images and (d) quantitative analysis of CFU formation ( n = 3). Erythroid blast forming units, BFU-E; erythroid CFU, CFU-E; monocytic CFU, CFU-M; granulocytic CFU, CFU-G; megakaryocytic CFU, CFU-Mk. n.d., undetected.

Techniques Used: Staining, Marker, Expressing, Flow Cytometry, Colony-forming Unit Assay

39) Product Images from "Primary Cell Model for Activation-Inducible Human Immunodeficiency Virus ▿"

Article Title: Primary Cell Model for Activation-Inducible Human Immunodeficiency Virus ▿

Journal:

doi: 10.1128/JVI.02838-06

Effects of thymocyte maturation on the ability to induce expression of dormant HIV. (A) Luciferase activity of CD4 + CD8 + thymocytes infected with NLEGFPLuc at an MOI of 0.1 and cultured for the indicated times before 5 × 10 5 cells
Figure Legend Snippet: Effects of thymocyte maturation on the ability to induce expression of dormant HIV. (A) Luciferase activity of CD4 + CD8 + thymocytes infected with NLEGFPLuc at an MOI of 0.1 and cultured for the indicated times before 5 × 10 5 cells

Techniques Used: Expressing, Luciferase, Activity Assay, Infection, Cell Culture

Reverse transcription and integration of NLEGFPLuc in infected CD4 + CD8 + thymocytes. (A) Luciferase assay of cells infected and cultured for 10 days as described in the legend of Fig. . AZT was added at the indicated time
Figure Legend Snippet: Reverse transcription and integration of NLEGFPLuc in infected CD4 + CD8 + thymocytes. (A) Luciferase assay of cells infected and cultured for 10 days as described in the legend of Fig. . AZT was added at the indicated time

Techniques Used: Infection, Luciferase, Cell Culture

Assessment of viral gene expression. (A) Luciferase activity of CD4 + CD8 + thymocytes cultured for 10 days as described in the legend of Fig. . Cells (10 5 ) were harvested from noninfected and infected cultures on the days
Figure Legend Snippet: Assessment of viral gene expression. (A) Luciferase activity of CD4 + CD8 + thymocytes cultured for 10 days as described in the legend of Fig. . Cells (10 5 ) were harvested from noninfected and infected cultures on the days

Techniques Used: Expressing, Luciferase, Activity Assay, Cell Culture, Infection

40) Product Images from "Langerhans-type and monocyte-derived human dendritic cells have different susceptibilities to mRNA electroporation with distinct effects on maturation and activation: implications for immunogenicity in dendritic cell-based immunotherapy"

Article Title: Langerhans-type and monocyte-derived human dendritic cells have different susceptibilities to mRNA electroporation with distinct effects on maturation and activation: implications for immunogenicity in dendritic cell-based immunotherapy

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-11-166

mRNA electroporation induces the maturation and activation of LCs to a greater magnitude than for moDCs. Immature moDCs and LCs were electroporated with eGFP mRNA and then cultured with (+) or without (−) standard maturation-inducing inflammatory cytokines. After 24 hours, cells in each experimental group were compared with pre-electroporation controls by flow cytometry for the expression of phenotypic markers of DC maturation and activation, based on the upregulation of (A) CD83, (B) CD80, and (C) CD86, respectively. Representative dot plots of eGFP mRNA-electroporated moDCs and LCs from one of three independent experiments are shown in the top row. Pooled data for each experimental group (mean ± SD, n = 3 independent experiments) are shown in the bottom row (gray bar = pre-electroporation control, white bar = post-electroporation without inflammatory cytokines, black bar = post-electroporation with inflammatory cytokines). * P
Figure Legend Snippet: mRNA electroporation induces the maturation and activation of LCs to a greater magnitude than for moDCs. Immature moDCs and LCs were electroporated with eGFP mRNA and then cultured with (+) or without (−) standard maturation-inducing inflammatory cytokines. After 24 hours, cells in each experimental group were compared with pre-electroporation controls by flow cytometry for the expression of phenotypic markers of DC maturation and activation, based on the upregulation of (A) CD83, (B) CD80, and (C) CD86, respectively. Representative dot plots of eGFP mRNA-electroporated moDCs and LCs from one of three independent experiments are shown in the top row. Pooled data for each experimental group (mean ± SD, n = 3 independent experiments) are shown in the bottom row (gray bar = pre-electroporation control, white bar = post-electroporation without inflammatory cytokines, black bar = post-electroporation with inflammatory cytokines). * P

Techniques Used: Electroporation, Activation Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing

Related Articles

Transduction:

Article Title: Establishment of a bioluminescent canine B-cell lymphoma xenograft model for monitoring tumor progression and treatment response in preclinical studies
Article Snippet: .. At 72h, transduction efficiency was assessed by FACS and GFP positive cells were sorted, using FACSAria IIu sorter (BD Biosciences), and maintained in the same culture medium supplemented with gentamycin 50 μg/ml (Gibco) for 7 days to avoid contamination. .. After 4 weeks in culture, CLBL-1GFP+luciferase+ cells were subjected to a second sort to ensure a stable GFP expressing cell line and cultured as previously described above.

Flow Cytometry:

Article Title: BCG Vaccination Prevents Reactivation of Latent Lymphatic Murine Tuberculosis Independently of CD4+ T Cells
Article Snippet: .. Flow Cytometry Viable, red blood cell-depleted single splenocytes were stained with mAb (all from BD Pharmingen) against CD4 (RM4-5), CD8α (53-6.7), CD3 (500A2), CD44 (1M7) and NKp46 (29A1.4), CD69 (H1.2F3), CD103 (M290), CD62L (MEL-14). .. After washing the cells, samples were analyzed using a FortessaX20 analyzer (BD Biosciences, CA).

Article Title: HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge
Article Snippet: .. Rectal Biopsy samples were digested with 200 U ml− 1 Collagenase-IV (Worthington) and 0.03% DNAse-I (Life) for two hours before mechanical disruption with a syringe and needle followed by washing with RPMI supplemented with 10% FBS and stained for phenotypic markers, CD3 (BD, SP34-2, Cat# 558124, 1:200), CD4 (BD, SK3, Custom, 1:2000 dilution), CD8 (BD, RPA-T8, Custom, 1:600 dilution), CD45RA (BD, 5H9, Cat# 561216, 1:40 dilution), CCR7 (BD, 150503, Cat# 562381, 1:50 dilution), CCR5 (BD, 3A9, Cat# 742913, 1:40 dilution), HLA-DR (BD, G46-6, Custom, 1:500 dilution), and LIVE/DEAD for flow cytometry analysis. .. Rhesus macaque tissue digestion and flow cytometry Uninfected rhesus macaques scheduled for necropsy were euthanized and their buccal tissue, sublingual tissue, and submandibular, submental, and inguinal lymph nodes were collected.

Article Title: Anticancer effects and underlying mechanism of Colchicine on human gastric cancer cell lines in vitro and in vivo
Article Snippet: .. Flow cytometric analysis of apoptosis The percentage of cells undergoing apoptosis was quantitated with an Annexin V-FITC apoptosis detection kit (BD Bioscience, CA, U.S.A.) according to the manufacturer’s instructions. .. Briefly, following Colchicine incubation for 24 h, the cells were washed twice with cold PBS, mixed with binding buffer (200 μl) at a concentration of 1 × 106 cells/ml, and added with 5 μl of annexin V-FITC and 5 μl of PI in the dark at room temperature for 5 min. At the end of the incubation period, the cells were then transferred into a flow cytometry tube and analyzed on an FACScan flow cytometer (Becton & Dickinson Co., U.S.A.).

Article Title: Roscovitine Suppresses CD4+ T Cells and T Cell-Mediated Experimental Uveitis
Article Snippet: .. Incorporated BrdU and total DNA levels were determined by flow cytometry to quantify the CD4+ lymphocytes in different cell cycle phases in accordance with the manufacturer's instructions (BD Biosciences). .. Annexin V and Propidium Iodide Staining Cell apoptosis and death were detected by double staining for annexin V and propidium iodide (PI) (eBioscience).

Immunohistochemistry:

Article Title: Chemokine Receptors CCR6 and CXCR3 Are Necessary for CD4+ T Cell Mediated Ocular Surface Disease in Experimental Dry Eye Disease
Article Snippet: .. Immunohistochemistry Immunohistochemistry was performed to detect and count the number of cells in the cornea and conjunctival epithelium that stained positively for CD4 (clone H129.9, 10 µg/mL, BD Bioscience, San Diego, CA) and appropriate biotinylated secondary antibody (BD Pharmingen) and Vectastain Elite ABC using NovaRed reagents (Vector, Burlingame, CA) as previously described . .. Secondary antibody alone and appropriate anti-rat isotype (BD Biosciences) controls were also examined.

Cell Culture:

Article Title: Myeloid Dendritic Cells Induce HIV-1 Latency in Non-proliferating CD4+ T Cells
Article Snippet: .. Transwell/transfer experiments DC were cultured with resting CD4+ T cells in the presence and absence of 0.4 µm cell culture inserts (BD, Franklin Lakes, NJ) with DC in the top chamber and resting CD4+ T cells in the lower chamber. .. Following 24 hours of culture, both the DC and the CD4+ T cells were infected as described above.

Cytometry:

Article Title: BCG Vaccination Prevents Reactivation of Latent Lymphatic Murine Tuberculosis Independently of CD4+ T Cells
Article Snippet: .. Flow Cytometry Viable, red blood cell-depleted single splenocytes were stained with mAb (all from BD Pharmingen) against CD4 (RM4-5), CD8α (53-6.7), CD3 (500A2), CD44 (1M7) and NKp46 (29A1.4), CD69 (H1.2F3), CD103 (M290), CD62L (MEL-14). .. After washing the cells, samples were analyzed using a FortessaX20 analyzer (BD Biosciences, CA).

Article Title: HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge
Article Snippet: .. Rectal Biopsy samples were digested with 200 U ml− 1 Collagenase-IV (Worthington) and 0.03% DNAse-I (Life) for two hours before mechanical disruption with a syringe and needle followed by washing with RPMI supplemented with 10% FBS and stained for phenotypic markers, CD3 (BD, SP34-2, Cat# 558124, 1:200), CD4 (BD, SK3, Custom, 1:2000 dilution), CD8 (BD, RPA-T8, Custom, 1:600 dilution), CD45RA (BD, 5H9, Cat# 561216, 1:40 dilution), CCR7 (BD, 150503, Cat# 562381, 1:50 dilution), CCR5 (BD, 3A9, Cat# 742913, 1:40 dilution), HLA-DR (BD, G46-6, Custom, 1:500 dilution), and LIVE/DEAD for flow cytometry analysis. .. Rhesus macaque tissue digestion and flow cytometry Uninfected rhesus macaques scheduled for necropsy were euthanized and their buccal tissue, sublingual tissue, and submandibular, submental, and inguinal lymph nodes were collected.

Article Title: Roscovitine Suppresses CD4+ T Cells and T Cell-Mediated Experimental Uveitis
Article Snippet: .. Incorporated BrdU and total DNA levels were determined by flow cytometry to quantify the CD4+ lymphocytes in different cell cycle phases in accordance with the manufacturer's instructions (BD Biosciences). .. Annexin V and Propidium Iodide Staining Cell apoptosis and death were detected by double staining for annexin V and propidium iodide (PI) (eBioscience).

Staining:

Article Title: A nonhuman primate toxicology and immunogenicity study evaluating aerosol delivery of AERAS-402/Ad35 vaccine
Article Snippet: .. Cells were then washed and stained for phenotypic markers CD14 (Pacific Blue), CD16 (Pacific Blue) and CD4 (PerCP-Cy5.5) (All BD). .. Cells were then washed and then fixed and permeabilized with BD Cytofix/Cytoperm (BD) and then stained for CD3 (APC-Cy7), CD8 (APC), IFN-γ (FITC), IL-2 (PE), and TNF (PE-Cy7) (All BD).

Article Title: BCG Vaccination Prevents Reactivation of Latent Lymphatic Murine Tuberculosis Independently of CD4+ T Cells
Article Snippet: .. Flow Cytometry Viable, red blood cell-depleted single splenocytes were stained with mAb (all from BD Pharmingen) against CD4 (RM4-5), CD8α (53-6.7), CD3 (500A2), CD44 (1M7) and NKp46 (29A1.4), CD69 (H1.2F3), CD103 (M290), CD62L (MEL-14). .. After washing the cells, samples were analyzed using a FortessaX20 analyzer (BD Biosciences, CA).

Article Title: HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge
Article Snippet: .. Rectal Biopsy samples were digested with 200 U ml− 1 Collagenase-IV (Worthington) and 0.03% DNAse-I (Life) for two hours before mechanical disruption with a syringe and needle followed by washing with RPMI supplemented with 10% FBS and stained for phenotypic markers, CD3 (BD, SP34-2, Cat# 558124, 1:200), CD4 (BD, SK3, Custom, 1:2000 dilution), CD8 (BD, RPA-T8, Custom, 1:600 dilution), CD45RA (BD, 5H9, Cat# 561216, 1:40 dilution), CCR7 (BD, 150503, Cat# 562381, 1:50 dilution), CCR5 (BD, 3A9, Cat# 742913, 1:40 dilution), HLA-DR (BD, G46-6, Custom, 1:500 dilution), and LIVE/DEAD for flow cytometry analysis. .. Rhesus macaque tissue digestion and flow cytometry Uninfected rhesus macaques scheduled for necropsy were euthanized and their buccal tissue, sublingual tissue, and submandibular, submental, and inguinal lymph nodes were collected.

Article Title: Chemokine Receptors CCR6 and CXCR3 Are Necessary for CD4+ T Cell Mediated Ocular Surface Disease in Experimental Dry Eye Disease
Article Snippet: .. Immunohistochemistry Immunohistochemistry was performed to detect and count the number of cells in the cornea and conjunctival epithelium that stained positively for CD4 (clone H129.9, 10 µg/mL, BD Bioscience, San Diego, CA) and appropriate biotinylated secondary antibody (BD Pharmingen) and Vectastain Elite ABC using NovaRed reagents (Vector, Burlingame, CA) as previously described . .. Secondary antibody alone and appropriate anti-rat isotype (BD Biosciences) controls were also examined.

FACS:

Article Title: Establishment of a bioluminescent canine B-cell lymphoma xenograft model for monitoring tumor progression and treatment response in preclinical studies
Article Snippet: .. At 72h, transduction efficiency was assessed by FACS and GFP positive cells were sorted, using FACSAria IIu sorter (BD Biosciences), and maintained in the same culture medium supplemented with gentamycin 50 μg/ml (Gibco) for 7 days to avoid contamination. .. After 4 weeks in culture, CLBL-1GFP+luciferase+ cells were subjected to a second sort to ensure a stable GFP expressing cell line and cultured as previously described above.

Recombinase Polymerase Amplification:

Article Title: HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge
Article Snippet: .. Rectal Biopsy samples were digested with 200 U ml− 1 Collagenase-IV (Worthington) and 0.03% DNAse-I (Life) for two hours before mechanical disruption with a syringe and needle followed by washing with RPMI supplemented with 10% FBS and stained for phenotypic markers, CD3 (BD, SP34-2, Cat# 558124, 1:200), CD4 (BD, SK3, Custom, 1:2000 dilution), CD8 (BD, RPA-T8, Custom, 1:600 dilution), CD45RA (BD, 5H9, Cat# 561216, 1:40 dilution), CCR7 (BD, 150503, Cat# 562381, 1:50 dilution), CCR5 (BD, 3A9, Cat# 742913, 1:40 dilution), HLA-DR (BD, G46-6, Custom, 1:500 dilution), and LIVE/DEAD for flow cytometry analysis. .. Rhesus macaque tissue digestion and flow cytometry Uninfected rhesus macaques scheduled for necropsy were euthanized and their buccal tissue, sublingual tissue, and submandibular, submental, and inguinal lymph nodes were collected.

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    Becton Dickinson egfp expression
    Reactivation of expression constructs containing AP lesions by complementation with XPA. ( A ) Flow <t>cytometry</t> expression analyses of constructs containing dG (blue colour), S-THF (amber) or AAF( N 2 )-dG (rose) at the analysed position in transcribed DNA strand of the <t>EGFP</t> gene. Fluorescence scatter plots show co-expression of EGFP with DsRed (as a marker for transfected cells). Cells were gated by DsRed expression to generate fluorescence distribution plots, which show S-THF and AAF( N 2 )-dG samples overlaid with a common dG reference sample. ( B ) Quantification of expression of constructs containing the specified AP lesions (S-THF or THF), relative to the dG reference (mean of six independent experiments ± SD; P -values calculated by the Student’s t -test). See also Supplementary Figure S2 .
    Egfp Expression, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson antibody expression egfp expression
    ( a ) hCMV-MIE sequence (left) and list of mutants (right). Positions where C to G mutations were introduced are marked with an asterisk. Transcription factor binding sites are underlined. CpG sites are highlighted in grey. hCMV-MIE variants are coded with a three letter identifier referring to the nucleotides at positions −508, −179 and −41. DNA positions are numbered relative to the transcription start site. (b) Transient expression of SEAP driven by hCMV-MIE mutants. Expression levels were normalised to unmutated hCMV-MIE (CCC). Error bars represent SD of eight biological replicates. Two independent experiments were performed. Potential effects of the hCMV-MIE mutations within the same experiment were tested with Tukey HSD (α = 0.05). Significant differences between the promoter mutants and CCC within the same experiment are marked with the respective p-value. (c) <t>eGFP</t> expression of permanently transfected <t>CHO</t> cell pools 34 days (upper panel) and 87 days (lower panel) after transfection. eGFP expression was quantified by FACS and the geometrical mean of the fluorescence intensity (gMFI) was plotted for each cell pool. The identity of the promoter variants and the number (N) of independent cell pools is indicated below the x-axis. The upper and the lower ends of the boxes represent the first and the third quartile of each group. The ends of the whiskers represent the lowest and highest values still within the 1.5fold interquartile range. The grey line indicates the overall mean of the gMFI values. (d) eGFP expression of permanently transfected CHO cell pools 42 days (left panel) and 69 days (right panel) after transfection; all further labelling is identical to ( c ). (e) Percentage methylation levels of hCMV-MIE variants at each CpG cytosine 42 days (upper panels) and 69 days (lower panels) after transfection. Methylation levels are plotted against DNA positions numbered 5′ to 3′. The positions of C-508, C-179, and C-41 are highlighted.
    Antibody Expression Egfp Expression, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reactivation of expression constructs containing AP lesions by complementation with XPA. ( A ) Flow cytometry expression analyses of constructs containing dG (blue colour), S-THF (amber) or AAF( N 2 )-dG (rose) at the analysed position in transcribed DNA strand of the EGFP gene. Fluorescence scatter plots show co-expression of EGFP with DsRed (as a marker for transfected cells). Cells were gated by DsRed expression to generate fluorescence distribution plots, which show S-THF and AAF( N 2 )-dG samples overlaid with a common dG reference sample. ( B ) Quantification of expression of constructs containing the specified AP lesions (S-THF or THF), relative to the dG reference (mean of six independent experiments ± SD; P -values calculated by the Student’s t -test). See also Supplementary Figure S2 .

    Journal: Nucleic Acids Research

    Article Title: Nucleotide excision repair of abasic DNA lesions

    doi: 10.1093/nar/gkz558

    Figure Lengend Snippet: Reactivation of expression constructs containing AP lesions by complementation with XPA. ( A ) Flow cytometry expression analyses of constructs containing dG (blue colour), S-THF (amber) or AAF( N 2 )-dG (rose) at the analysed position in transcribed DNA strand of the EGFP gene. Fluorescence scatter plots show co-expression of EGFP with DsRed (as a marker for transfected cells). Cells were gated by DsRed expression to generate fluorescence distribution plots, which show S-THF and AAF( N 2 )-dG samples overlaid with a common dG reference sample. ( B ) Quantification of expression of constructs containing the specified AP lesions (S-THF or THF), relative to the dG reference (mean of six independent experiments ± SD; P -values calculated by the Student’s t -test). See also Supplementary Figure S2 .

    Article Snippet: At 24 h post transfection, cells were fixed for quantitative determination of EGFP expression by flow cytometry using FACSCalibur™ and the CellQuest™ Pro software (Beckton Dickinson GmbH, Heidelberg, Germany), as described in detail previously ( ).

    Techniques: Expressing, Construct, Flow Cytometry, Cytometry, Fluorescence, Marker, Transfection

    Impairment of transcription by BER-resistant AP lesion positioned at a specific nucleotide in the transcribed strand of the EGFP gene. ( A ) Structures of synthetic tetrahydrofuran (THF and S-THF) AP lesions and reactivity of BER enzymes towards the specified types of AP sites. ( B ) Characterization of reporter constructs containing deoxyguanine (dG) or the specified types of AP lesion at a defined nucleotide (*) in the transcribed DNA strand (TS). Scheme shows position for incorporation of synthetic oligonucleotides containing dG, THF or S-THF with respect to EGFP coding sequence (arrow) and transcription start (broken arrow). To demonstrate the presence of AP lesion, the obtained constructs were incubated with excess of APE1 and analysed by gel electrophoresis in the presence of ethidium bromide. See also Supplementary Figure S1 for more detail. ( C ) Flow cytometry analyses of expression of constructs containing specified modifications in transfected XP-A (GM04312) cells (a representative experiment). EGFP fluorescence distribution plots show expression data overlaid pairwise for each modification and the respective control constructs without modification. Bar chart on the right shows quantification of the EGFP expression, relative to the matched control constructs without the modifications.

    Journal: Nucleic Acids Research

    Article Title: Nucleotide excision repair of abasic DNA lesions

    doi: 10.1093/nar/gkz558

    Figure Lengend Snippet: Impairment of transcription by BER-resistant AP lesion positioned at a specific nucleotide in the transcribed strand of the EGFP gene. ( A ) Structures of synthetic tetrahydrofuran (THF and S-THF) AP lesions and reactivity of BER enzymes towards the specified types of AP sites. ( B ) Characterization of reporter constructs containing deoxyguanine (dG) or the specified types of AP lesion at a defined nucleotide (*) in the transcribed DNA strand (TS). Scheme shows position for incorporation of synthetic oligonucleotides containing dG, THF or S-THF with respect to EGFP coding sequence (arrow) and transcription start (broken arrow). To demonstrate the presence of AP lesion, the obtained constructs were incubated with excess of APE1 and analysed by gel electrophoresis in the presence of ethidium bromide. See also Supplementary Figure S1 for more detail. ( C ) Flow cytometry analyses of expression of constructs containing specified modifications in transfected XP-A (GM04312) cells (a representative experiment). EGFP fluorescence distribution plots show expression data overlaid pairwise for each modification and the respective control constructs without modification. Bar chart on the right shows quantification of the EGFP expression, relative to the matched control constructs without the modifications.

    Article Snippet: At 24 h post transfection, cells were fixed for quantitative determination of EGFP expression by flow cytometry using FACSCalibur™ and the CellQuest™ Pro software (Beckton Dickinson GmbH, Heidelberg, Germany), as described in detail previously ( ).

    Techniques: Construct, Sequencing, Incubation, Nucleic Acid Electrophoresis, Flow Cytometry, Cytometry, Expressing, Transfection, Fluorescence, Modification

    Transcriptional mutagenesis at the BER-resistant abasic site in the template DNA and its suppression by NER. ( A ) Scheme of the reporter for detection of ribonucleotide misincorporation opposite to AP-lesion in the template DNA. Substitution of 613U in mRNA to any other ribonucleotide results in reversion to a fluorescent EGFP. ( B ) Flow cytometry assay for detection of the mRNA single nucleotide substitutions induced by the specified AP lesions (THF, S-THF) in the MRC-5 (group of panels on the left) and XP-A (group of panels on the right) cell lines. Fluorescence scatter plots show full data for individual samples from a representative experiment. The derived EGFP fluorescence distribution plots show overlaid data for EGFP construct without modification (green colour) and EGFP Q205* constructs without modification (blue) or with the indicated lesion (amber). The nature of the nucleotide/modification in the template DNA strand is indicated above the plots. Note the right shift of S-THF plots compared to dA.

    Journal: Nucleic Acids Research

    Article Title: Nucleotide excision repair of abasic DNA lesions

    doi: 10.1093/nar/gkz558

    Figure Lengend Snippet: Transcriptional mutagenesis at the BER-resistant abasic site in the template DNA and its suppression by NER. ( A ) Scheme of the reporter for detection of ribonucleotide misincorporation opposite to AP-lesion in the template DNA. Substitution of 613U in mRNA to any other ribonucleotide results in reversion to a fluorescent EGFP. ( B ) Flow cytometry assay for detection of the mRNA single nucleotide substitutions induced by the specified AP lesions (THF, S-THF) in the MRC-5 (group of panels on the left) and XP-A (group of panels on the right) cell lines. Fluorescence scatter plots show full data for individual samples from a representative experiment. The derived EGFP fluorescence distribution plots show overlaid data for EGFP construct without modification (green colour) and EGFP Q205* constructs without modification (blue) or with the indicated lesion (amber). The nature of the nucleotide/modification in the template DNA strand is indicated above the plots. Note the right shift of S-THF plots compared to dA.

    Article Snippet: At 24 h post transfection, cells were fixed for quantitative determination of EGFP expression by flow cytometry using FACSCalibur™ and the CellQuest™ Pro software (Beckton Dickinson GmbH, Heidelberg, Germany), as described in detail previously ( ).

    Techniques: Mutagenesis, Flow Cytometry, Cytometry, Fluorescence, Derivative Assay, Construct, Modification

    Time course and cell type specificity of CDV infections of PBMCs and lymphoid organs. ( A ) Fluorescence-activated cell sorter analysis of PBMCs isolated from an animal infected with 5804P-eGFP/H at 14 d.p.i. ( Lower ), and a noninfected control animal ( Upper ). Cells were stained with Alexa Fluor 647-labeled antibodies against cellular markers ( y axis, see Mateials and Methods ). eGFP expression was plotted on the x axis. ( B ) Percentages of CDV-infected T cells (CD3 positive), B cells (CD79α-positive), and MMs (CD14-positive). Groups of four animals were infected, and PBMCs were collected 0 (D0), 7 (D7), or 14 (D14) d.p.i. The mean values are indicated, with SD shown parenthetically. Total cells include eGFP-positive (eGFP+) cells. ( C ) CDV-infected cells in the lymph nodes (LN), spleen (SP), or thymus (TY) of animals killed 7 (D7) or 14 (D14) d.p.i., or a control animal (D0). In the lines labeled %eGFP+, the percentages of GFP-expressing (CDV-infected) cells within one cell type are indicated.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Tropism illuminated: Lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system

    doi: 10.1073/pnas.0403597101

    Figure Lengend Snippet: Time course and cell type specificity of CDV infections of PBMCs and lymphoid organs. ( A ) Fluorescence-activated cell sorter analysis of PBMCs isolated from an animal infected with 5804P-eGFP/H at 14 d.p.i. ( Lower ), and a noninfected control animal ( Upper ). Cells were stained with Alexa Fluor 647-labeled antibodies against cellular markers ( y axis, see Mateials and Methods ). eGFP expression was plotted on the x axis. ( B ) Percentages of CDV-infected T cells (CD3 positive), B cells (CD79α-positive), and MMs (CD14-positive). Groups of four animals were infected, and PBMCs were collected 0 (D0), 7 (D7), or 14 (D14) d.p.i. The mean values are indicated, with SD shown parenthetically. Total cells include eGFP-positive (eGFP+) cells. ( C ) CDV-infected cells in the lymph nodes (LN), spleen (SP), or thymus (TY) of animals killed 7 (D7) or 14 (D14) d.p.i., or a control animal (D0). In the lines labeled %eGFP+, the percentages of GFP-expressing (CDV-infected) cells within one cell type are indicated.

    Article Snippet: For FACS (Becton Dickinson) analysis, Ficoll gradient centrifugation-purified peripheral blood mononuclear cells (PBMCs), or single-cell suspensions obtained from thymus, spleen, or lymph node homogenates after hypotonic lysis of erythrocytes, were fixed overnight in 1% paraformaldehyde before incubation with Alexa 647-labeled monoclonal antibodies against CD3 (sc-20047, Santa Cruz Biotechnology), CD79α (DAKO), or CD14 (Veterinary Medical Research and Development, Pullman, WA), or a corresponding isotype control for 1 h. Alexa 647 and eGFP expression of each sample was assessed by using a FACScan instrument (Becton Dickinson).

    Techniques: Fluorescence, Isolation, Infection, Staining, Labeling, Expressing

    (A) Schematic representation of PyV hybrid origin constructs used in flow cytometry analysis. (B to D) Time course of long-term EGFP (B) or short-term d1EGFP (C and D) expression in the presence or absence of Geneticin selection for various cell lines.

    Journal:

    Article Title: Episomal Maintenance of Plasmids with Hybrid Origins in Mouse Cells

    doi: 10.1128/JVI.79.24.15277-15288.2005

    Figure Lengend Snippet: (A) Schematic representation of PyV hybrid origin constructs used in flow cytometry analysis. (B to D) Time course of long-term EGFP (B) or short-term d1EGFP (C and D) expression in the presence or absence of Geneticin selection for various cell lines.

    Article Snippet: EGFP expression was analyzed by flow cytometry using a Becton Dickinson FACSCalibur flow cytometer with associated CellQuest software.

    Techniques: Construct, Flow Cytometry, Cytometry, Expressing, Selection

    ( a ) hCMV-MIE sequence (left) and list of mutants (right). Positions where C to G mutations were introduced are marked with an asterisk. Transcription factor binding sites are underlined. CpG sites are highlighted in grey. hCMV-MIE variants are coded with a three letter identifier referring to the nucleotides at positions −508, −179 and −41. DNA positions are numbered relative to the transcription start site. (b) Transient expression of SEAP driven by hCMV-MIE mutants. Expression levels were normalised to unmutated hCMV-MIE (CCC). Error bars represent SD of eight biological replicates. Two independent experiments were performed. Potential effects of the hCMV-MIE mutations within the same experiment were tested with Tukey HSD (α = 0.05). Significant differences between the promoter mutants and CCC within the same experiment are marked with the respective p-value. (c) eGFP expression of permanently transfected CHO cell pools 34 days (upper panel) and 87 days (lower panel) after transfection. eGFP expression was quantified by FACS and the geometrical mean of the fluorescence intensity (gMFI) was plotted for each cell pool. The identity of the promoter variants and the number (N) of independent cell pools is indicated below the x-axis. The upper and the lower ends of the boxes represent the first and the third quartile of each group. The ends of the whiskers represent the lowest and highest values still within the 1.5fold interquartile range. The grey line indicates the overall mean of the gMFI values. (d) eGFP expression of permanently transfected CHO cell pools 42 days (left panel) and 69 days (right panel) after transfection; all further labelling is identical to ( c ). (e) Percentage methylation levels of hCMV-MIE variants at each CpG cytosine 42 days (upper panels) and 69 days (lower panels) after transfection. Methylation levels are plotted against DNA positions numbered 5′ to 3′. The positions of C-508, C-179, and C-41 are highlighted.

    Journal: Scientific Reports

    Article Title: CMV promoter mutants with a reduced propensity to productivity loss in CHO cells

    doi: 10.1038/srep16952

    Figure Lengend Snippet: ( a ) hCMV-MIE sequence (left) and list of mutants (right). Positions where C to G mutations were introduced are marked with an asterisk. Transcription factor binding sites are underlined. CpG sites are highlighted in grey. hCMV-MIE variants are coded with a three letter identifier referring to the nucleotides at positions −508, −179 and −41. DNA positions are numbered relative to the transcription start site. (b) Transient expression of SEAP driven by hCMV-MIE mutants. Expression levels were normalised to unmutated hCMV-MIE (CCC). Error bars represent SD of eight biological replicates. Two independent experiments were performed. Potential effects of the hCMV-MIE mutations within the same experiment were tested with Tukey HSD (α = 0.05). Significant differences between the promoter mutants and CCC within the same experiment are marked with the respective p-value. (c) eGFP expression of permanently transfected CHO cell pools 34 days (upper panel) and 87 days (lower panel) after transfection. eGFP expression was quantified by FACS and the geometrical mean of the fluorescence intensity (gMFI) was plotted for each cell pool. The identity of the promoter variants and the number (N) of independent cell pools is indicated below the x-axis. The upper and the lower ends of the boxes represent the first and the third quartile of each group. The ends of the whiskers represent the lowest and highest values still within the 1.5fold interquartile range. The grey line indicates the overall mean of the gMFI values. (d) eGFP expression of permanently transfected CHO cell pools 42 days (left panel) and 69 days (right panel) after transfection; all further labelling is identical to ( c ). (e) Percentage methylation levels of hCMV-MIE variants at each CpG cytosine 42 days (upper panels) and 69 days (lower panels) after transfection. Methylation levels are plotted against DNA positions numbered 5′ to 3′. The positions of C-508, C-179, and C-41 are highlighted.

    Article Snippet: Analysis of eGFP and antibody expression eGFP expression in CHO-K1 was examined before passaging the cells with a BD FACS Canto II flow cytometer (BD, Heidelberg, Germany).

    Techniques: Sequencing, Binding Assay, Expressing, Countercurrent Chromatography, Transfection, FACS, Fluorescence, Methylation