Structured Review

Becton Dickinson anti cd81
Artificial reconstitution of a protein corona on HPL EVs. TSEC-EVs (5.4 × 10 8 ) were labeled with anti-CD9, CD63 and <t>CD81</t> tetramix-AF-647 with or without prior albumin-AF-488 corona formation and loaded onto EV profiler chips (ONI). High resolution pictures with overview inserts are shown. ( a ) Negative control albumin-AF-488 solution in the absence of EVs did not bind to the chip; one single positive presumably albumin aggregate is shown in magnification. ( b ) Control tetramix-AF-647-labeled TSEC-EVs without albumin label bound to the chip showing red signal, with negligible signal in the green channel. ( c ) Albumin-AF-488 pre-labeled and tetramix-AF-647 stained TSEC-EV samples produced easily detectable double-positive signals. ( d ) Quantification of AF-647 signals and ( e ) quantification of AF-488 signals on chips loaded as indicated with albumin solution or TSEC-EVs without or with previous fluorescent albumin corona formation. ( f ) In chips loaded with albumin-AF-488-prelabeled TSEC-EVs, represented in ( c ), 6.75 ± 2.18% double-positive, 70.13 ± 11.62% tetramix-647 positive and 23.13 ± 10.70% AF-488 positive events were detected. One-way ANOVA, Tukey, *** p ≤ 0.001, * p ≤ 0.05. Each symbol (circles, squares and triangles) represents a separate independent experiment ( n = 3). Scale bars: 10 µm (low resolution inserts) and 0.5 µm (main panels a – c ).
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1) Product Images from "Synergy of Human Platelet-Derived Extracellular Vesicles with Secretome Proteins Promotes Regenerative Functions"

Article Title: Synergy of Human Platelet-Derived Extracellular Vesicles with Secretome Proteins Promotes Regenerative Functions

Journal: Biomedicines

doi: 10.3390/biomedicines10020238

Artificial reconstitution of a protein corona on HPL EVs. TSEC-EVs (5.4 × 10 8 ) were labeled with anti-CD9, CD63 and CD81 tetramix-AF-647 with or without prior albumin-AF-488 corona formation and loaded onto EV profiler chips (ONI). High resolution pictures with overview inserts are shown. ( a ) Negative control albumin-AF-488 solution in the absence of EVs did not bind to the chip; one single positive presumably albumin aggregate is shown in magnification. ( b ) Control tetramix-AF-647-labeled TSEC-EVs without albumin label bound to the chip showing red signal, with negligible signal in the green channel. ( c ) Albumin-AF-488 pre-labeled and tetramix-AF-647 stained TSEC-EV samples produced easily detectable double-positive signals. ( d ) Quantification of AF-647 signals and ( e ) quantification of AF-488 signals on chips loaded as indicated with albumin solution or TSEC-EVs without or with previous fluorescent albumin corona formation. ( f ) In chips loaded with albumin-AF-488-prelabeled TSEC-EVs, represented in ( c ), 6.75 ± 2.18% double-positive, 70.13 ± 11.62% tetramix-647 positive and 23.13 ± 10.70% AF-488 positive events were detected. One-way ANOVA, Tukey, *** p ≤ 0.001, * p ≤ 0.05. Each symbol (circles, squares and triangles) represents a separate independent experiment ( n = 3). Scale bars: 10 µm (low resolution inserts) and 0.5 µm (main panels a – c ).
Figure Legend Snippet: Artificial reconstitution of a protein corona on HPL EVs. TSEC-EVs (5.4 × 10 8 ) were labeled with anti-CD9, CD63 and CD81 tetramix-AF-647 with or without prior albumin-AF-488 corona formation and loaded onto EV profiler chips (ONI). High resolution pictures with overview inserts are shown. ( a ) Negative control albumin-AF-488 solution in the absence of EVs did not bind to the chip; one single positive presumably albumin aggregate is shown in magnification. ( b ) Control tetramix-AF-647-labeled TSEC-EVs without albumin label bound to the chip showing red signal, with negligible signal in the green channel. ( c ) Albumin-AF-488 pre-labeled and tetramix-AF-647 stained TSEC-EV samples produced easily detectable double-positive signals. ( d ) Quantification of AF-647 signals and ( e ) quantification of AF-488 signals on chips loaded as indicated with albumin solution or TSEC-EVs without or with previous fluorescent albumin corona formation. ( f ) In chips loaded with albumin-AF-488-prelabeled TSEC-EVs, represented in ( c ), 6.75 ± 2.18% double-positive, 70.13 ± 11.62% tetramix-647 positive and 23.13 ± 10.70% AF-488 positive events were detected. One-way ANOVA, Tukey, *** p ≤ 0.001, * p ≤ 0.05. Each symbol (circles, squares and triangles) represents a separate independent experiment ( n = 3). Scale bars: 10 µm (low resolution inserts) and 0.5 µm (main panels a – c ).

Techniques Used: Labeling, Negative Control, Chromatin Immunoprecipitation, Staining, Produced

2) Product Images from "Imaging of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels"

Article Title: Imaging of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S159404

The SR-SIM analysis of hBM-MSCs with intracellular structures visible inside the cells positively stained with lypophilic dyes PKH26 ( A – C ) or tagged with superparamagnetic iron nanoparticles conjugated with rhodamine (Molday ION) ( D and E ) (red). Notes: Coexpression of tetraspanins (exosome markers), such as CD9 ( A and D ), CD63 ( B and E ), and CD81 ( C and F ) (green), was demonstrated. Cell nuclei were stained with Hoechst (blue). Scale bar =50 μm. Abbreviations: hBM-MSCs, human bone marrow mesenchymal stem cells; SR-SIM, super-resolution structured illumination microscopy.
Figure Legend Snippet: The SR-SIM analysis of hBM-MSCs with intracellular structures visible inside the cells positively stained with lypophilic dyes PKH26 ( A – C ) or tagged with superparamagnetic iron nanoparticles conjugated with rhodamine (Molday ION) ( D and E ) (red). Notes: Coexpression of tetraspanins (exosome markers), such as CD9 ( A and D ), CD63 ( B and E ), and CD81 ( C and F ) (green), was demonstrated. Cell nuclei were stained with Hoechst (blue). Scale bar =50 μm. Abbreviations: hBM-MSCs, human bone marrow mesenchymal stem cells; SR-SIM, super-resolution structured illumination microscopy.

Techniques Used: Staining, Microscopy

The SR-SIM analysis of hBM-MSCs, 24 hours after their co-culture with EVs previously stained with different dyes. Notes: EVs labeled with PKH26 ( A – C ) or tagged with Molday ION ( D – F ) (red) taken up by hBM-MSCs are visible inside the cells. Coexpression of tetraspanins: CD9 ( A and D ), CD63 ( B and E ), and CD81 ( C and F ) (green) were demonstrated. Cell nuclei were stained with Hoechst (blue). Scale bar =20 μm. Abbreviations: EVs, extracellular vesicles; hBM-MSCs, human bone marrow mesenchymal stem cells; SR-SIM, super-resolution structured illumination microscopy.
Figure Legend Snippet: The SR-SIM analysis of hBM-MSCs, 24 hours after their co-culture with EVs previously stained with different dyes. Notes: EVs labeled with PKH26 ( A – C ) or tagged with Molday ION ( D – F ) (red) taken up by hBM-MSCs are visible inside the cells. Coexpression of tetraspanins: CD9 ( A and D ), CD63 ( B and E ), and CD81 ( C and F ) (green) were demonstrated. Cell nuclei were stained with Hoechst (blue). Scale bar =20 μm. Abbreviations: EVs, extracellular vesicles; hBM-MSCs, human bone marrow mesenchymal stem cells; SR-SIM, super-resolution structured illumination microscopy.

Techniques Used: Co-Culture Assay, Staining, Labeling, Microscopy

3) Product Images from "Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures"

Article Title: Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures

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

doi: 10.1073/pnas.0915130107

Primary human hepatocytes in MPCCs form polarized cell layers, express HCV entry factors, and support HCV glycoprotein-mediated entry. Bright field images of primary hepatocytes in MPCCs ( A ) and in monocultures ( B ). Wide-field fluorescence images of fixed MPCCs stained for the canilicular marker MRP2 ( C ), and the basolateral marker CD26 ( D ). Nuclear (blue) and antigen-specific staining (green) for CD81 ( E ), SCARB1 (near edge of hepatocyte island) ( F ), CLDN1 (red) ( G ), OCLN ( H ) in MPCCs. ( I ) Merged image of primary hepatocytes stained for MRP2 (green), ZO1 (red), and nuclei (blue). ( J ) 3D rendering of boxed area in I . ( K ) Infection of MPCCs with retroviral pseudoparticles bearing HCV glycoproteins (HCVpp), vesicular stomatitis virus glycoprotein (VSVGpp), or no glycoproteins (Env - pp) and containing an EGFP reporter gene. Representative images are shown for all experiments. ( L ) Anti-CD81 antibody blocks entry of HCVpp (dark bars), but not VSVGpp (white bars). Concentrations of antibody (μg/mL) are noted. Mean and SD are shown. Scale bars: 100 μm (a, b, k), 50 μm (c, d), 20 μm (e-i).
Figure Legend Snippet: Primary human hepatocytes in MPCCs form polarized cell layers, express HCV entry factors, and support HCV glycoprotein-mediated entry. Bright field images of primary hepatocytes in MPCCs ( A ) and in monocultures ( B ). Wide-field fluorescence images of fixed MPCCs stained for the canilicular marker MRP2 ( C ), and the basolateral marker CD26 ( D ). Nuclear (blue) and antigen-specific staining (green) for CD81 ( E ), SCARB1 (near edge of hepatocyte island) ( F ), CLDN1 (red) ( G ), OCLN ( H ) in MPCCs. ( I ) Merged image of primary hepatocytes stained for MRP2 (green), ZO1 (red), and nuclei (blue). ( J ) 3D rendering of boxed area in I . ( K ) Infection of MPCCs with retroviral pseudoparticles bearing HCV glycoproteins (HCVpp), vesicular stomatitis virus glycoprotein (VSVGpp), or no glycoproteins (Env - pp) and containing an EGFP reporter gene. Representative images are shown for all experiments. ( L ) Anti-CD81 antibody blocks entry of HCVpp (dark bars), but not VSVGpp (white bars). Concentrations of antibody (μg/mL) are noted. Mean and SD are shown. Scale bars: 100 μm (a, b, k), 50 μm (c, d), 20 μm (e-i).

Techniques Used: Fluorescence, Staining, Marker, Infection

Utility of primary human hepatocyte MPCCs in antibody and small molecule screening. ( A ) Dose-dependent inhibition of HCVcc replication in MPCCs treated with antibodies against HCV glycoproteins (AP33, 3/11, CBH5, AR3A) or cellular CD81 (JS-81). Antibody concentrations are 0.1 (light gray), 1 (dark gray), and 10 (black) μg/mL ( B ) Dose-dependent inhibition of HCVcc replication in MPCCs treated with IFN-α (up to 0.13 μM) or small molecules (NS3-4A protease inhibitors, BILN2061 and ITMN191, or polymerase inhibitor, 2’CMA). HCVcc-infected MPCCs were pulse-treated for 2 days with compounds and supernatants were collected at days 2 and 4 (shown) postinhibitor treatment. ( C ), followed by treatment of cultures with small molecules for 2 days. In all experiments, HCVcc replication was monitored by luciferase secretion into the supernatants. Mean and standard error of the mean are shown.
Figure Legend Snippet: Utility of primary human hepatocyte MPCCs in antibody and small molecule screening. ( A ) Dose-dependent inhibition of HCVcc replication in MPCCs treated with antibodies against HCV glycoproteins (AP33, 3/11, CBH5, AR3A) or cellular CD81 (JS-81). Antibody concentrations are 0.1 (light gray), 1 (dark gray), and 10 (black) μg/mL ( B ) Dose-dependent inhibition of HCVcc replication in MPCCs treated with IFN-α (up to 0.13 μM) or small molecules (NS3-4A protease inhibitors, BILN2061 and ITMN191, or polymerase inhibitor, 2’CMA). HCVcc-infected MPCCs were pulse-treated for 2 days with compounds and supernatants were collected at days 2 and 4 (shown) postinhibitor treatment. ( C ), followed by treatment of cultures with small molecules for 2 days. In all experiments, HCVcc replication was monitored by luciferase secretion into the supernatants. Mean and standard error of the mean are shown.

Techniques Used: Inhibition, Infection, Luciferase

4) Product Images from "HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection"

Article Title: HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2016.03.004

HIV pEV contains viral accessory proteins and inflammatory effectors. (a) Western blot analysis of individual sucrose gradient fractions from viremic and non-viremic patients. Purified pEV from samples analyzed in Fig. 1 d and viremic patients (n = 4, pools of 24 ml each) were blotted for indicated factors. Red arrows in the boxes depict the positive correlation of Nef with ADAM17 and pro-TNF cleavage. precrs.: precursor. Note: CD63 and CD81 western blot panel in Fig. 1 d and panel (a) in this figure are identical (same samples). Lysates of 293 T cells, transfected with p24 Gag (+ Gag) and/or Nef/Nef-cofactors (Lys.), served as controls (Cont.). (b) Protein array analyzing the content of cytokines, chemokines and soluble factors (CCF) in pEV from healthy controls, viremic patients with low CD4 counts (120–250/μl) and non-viremic HIV patients with high CD4 counts ( > 750/μl) (30 ml pooled plasma, 5 patients). See full CCF list analyzed in supplementary information.
Figure Legend Snippet: HIV pEV contains viral accessory proteins and inflammatory effectors. (a) Western blot analysis of individual sucrose gradient fractions from viremic and non-viremic patients. Purified pEV from samples analyzed in Fig. 1 d and viremic patients (n = 4, pools of 24 ml each) were blotted for indicated factors. Red arrows in the boxes depict the positive correlation of Nef with ADAM17 and pro-TNF cleavage. precrs.: precursor. Note: CD63 and CD81 western blot panel in Fig. 1 d and panel (a) in this figure are identical (same samples). Lysates of 293 T cells, transfected with p24 Gag (+ Gag) and/or Nef/Nef-cofactors (Lys.), served as controls (Cont.). (b) Protein array analyzing the content of cytokines, chemokines and soluble factors (CCF) in pEV from healthy controls, viremic patients with low CD4 counts (120–250/μl) and non-viremic HIV patients with high CD4 counts ( > 750/μl) (30 ml pooled plasma, 5 patients). See full CCF list analyzed in supplementary information.

Techniques Used: Western Blot, Purification, Transfection, Protein Array

5) Product Images from "Indoor dust extracellular vesicles promote cancer lung metastasis by inducing tumour necrosis factor-α"

Article Title: Indoor dust extracellular vesicles promote cancer lung metastasis by inducing tumour necrosis factor-α

Journal: Journal of Extracellular Vesicles

doi: 10.1080/20013078.2020.1766821

Characterization of indoor dust EVs. (a) Transmission electron microscopy images of indoor dust EVs. Scale bar = 200 nm. (b) The size distribution of indoor dust EVs analysed by dynamic light scattering. n = 5. (c, d) ELISA to detect the content of lipid A (a Gram-negative bacterial constituent) (c), and LTA (a Gram-positive bacterial constituent) (d) from indoor dust EVs (blue). E. coli EVs (black) and S. aureus EVs (red) were used as positive controls for Gram-negative and Gram-positive bacterial origins, respectively. E. coli EVs and S. aureus EVs were also employed as negative controls for Gram-positive and Gram-negative bacterial EVs, respectively. RLU, relative luminescence unit. n = 3. (e) Indoor dust EVs (15 µg in total protein amounts) were analysed by Western blotting, using anti-CD63, anti-CD81, anti-CD9 and anti-TSG101. SW480 EVs (5 µg in total protein amounts) were used as controls for human origins.
Figure Legend Snippet: Characterization of indoor dust EVs. (a) Transmission electron microscopy images of indoor dust EVs. Scale bar = 200 nm. (b) The size distribution of indoor dust EVs analysed by dynamic light scattering. n = 5. (c, d) ELISA to detect the content of lipid A (a Gram-negative bacterial constituent) (c), and LTA (a Gram-positive bacterial constituent) (d) from indoor dust EVs (blue). E. coli EVs (black) and S. aureus EVs (red) were used as positive controls for Gram-negative and Gram-positive bacterial origins, respectively. E. coli EVs and S. aureus EVs were also employed as negative controls for Gram-positive and Gram-negative bacterial EVs, respectively. RLU, relative luminescence unit. n = 3. (e) Indoor dust EVs (15 µg in total protein amounts) were analysed by Western blotting, using anti-CD63, anti-CD81, anti-CD9 and anti-TSG101. SW480 EVs (5 µg in total protein amounts) were used as controls for human origins.

Techniques Used: Transmission Assay, Electron Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot

6) Product Images from "Potential Role for CD63 in CCR5-Mediated Human Immunodeficiency Virus Type 1 Infection of Macrophages"

Article Title: Potential Role for CD63 in CCR5-Mediated Human Immunodeficiency Virus Type 1 Infection of Macrophages

Journal: Journal of Virology

doi: 10.1128/JVI.77.6.3624-3633.2003

Anti-CD63 treatment of primary macrophages inhibits infection by R5 HIV-1 isolates. Cells were preincubated with anti-CD63 (mouse IgG1 isotype, 10 μg/ml) or control antibodies (mouse IgG1, 10 μg/ml) and then infected with the blood-derived R5 isolate HIV-1-BL4 (A) or BL6 (B). For the R5 virus HIV-1-SX (C), anti-CD63, anti-CD82, or anti-CD4 MAb at concentrations ranging from 0 to 3 μg/ml was used. Virus production was measured in culture supernatants at 7 days postinfection by p24 ELISA. In some experiments, cross-linking (XL) of mouse anti-CD63 with a secondary rat anti-mouse MAb was used. Anti-CD4 (Leu3a, 0.5 μg/ml) was used as a positive control, and mouse IgG1, CD9, CD81, and CD82 (all tetraspanin proteins) were used as negative controls (10 μg/ml). Data shown are representative of results with seven different donors and seven different R5 virus isolates.
Figure Legend Snippet: Anti-CD63 treatment of primary macrophages inhibits infection by R5 HIV-1 isolates. Cells were preincubated with anti-CD63 (mouse IgG1 isotype, 10 μg/ml) or control antibodies (mouse IgG1, 10 μg/ml) and then infected with the blood-derived R5 isolate HIV-1-BL4 (A) or BL6 (B). For the R5 virus HIV-1-SX (C), anti-CD63, anti-CD82, or anti-CD4 MAb at concentrations ranging from 0 to 3 μg/ml was used. Virus production was measured in culture supernatants at 7 days postinfection by p24 ELISA. In some experiments, cross-linking (XL) of mouse anti-CD63 with a secondary rat anti-mouse MAb was used. Anti-CD4 (Leu3a, 0.5 μg/ml) was used as a positive control, and mouse IgG1, CD9, CD81, and CD82 (all tetraspanin proteins) were used as negative controls (10 μg/ml). Data shown are representative of results with seven different donors and seven different R5 virus isolates.

Techniques Used: Infection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Positive Control

7) Product Images from "Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin"

Article Title: Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

Journal: Journal of Cell Science

doi: 10.1242/jcs.045658

Phosphorylation of ezrin pY353 is Syk-dependent and requires the C-terminal domain of CD81. (A) Flow cytometry analysis of U937-CD81 and U937-CD81-Syk KD. Analysis of Syk (upper panel) and ezrin (middle panel) expression was performed on permeabilized
Figure Legend Snippet: Phosphorylation of ezrin pY353 is Syk-dependent and requires the C-terminal domain of CD81. (A) Flow cytometry analysis of U937-CD81 and U937-CD81-Syk KD. Analysis of Syk (upper panel) and ezrin (middle panel) expression was performed on permeabilized

Techniques Used: Flow Cytometry, Cytometry, Expressing

The transient dephosphorylation of ezrin Thr567 requires the C-terminal domain of CD81 and is Syk-independent. (A) Untreated or R406-treated OCI-LY8 cells were stimulated for the indicated times with the indicated anti-CD81 mAbs. (B) U937-CD81 (left
Figure Legend Snippet: The transient dephosphorylation of ezrin Thr567 requires the C-terminal domain of CD81 and is Syk-independent. (A) Untreated or R406-treated OCI-LY8 cells were stimulated for the indicated times with the indicated anti-CD81 mAbs. (B) U937-CD81 (left

Techniques Used: De-Phosphorylation Assay

CD81 stimulation induces its co-capping with ezrin and F-actin. OCI-LY8 cells were incubated for 10 minutes at 37°C with a FITC-conjugated αCD81 mAb alone (CD81 stimulation), or in the presence of the Syk inhibitor R406 (CD81 stimulation+R406).
Figure Legend Snippet: CD81 stimulation induces its co-capping with ezrin and F-actin. OCI-LY8 cells were incubated for 10 minutes at 37°C with a FITC-conjugated αCD81 mAb alone (CD81 stimulation), or in the presence of the Syk inhibitor R406 (CD81 stimulation+R406).

Techniques Used: Incubation

Engagement of CD81 induces tyrosine phosphorylation of ezrin. (A) OCI-LY8 cell lysates and immunoprecipitated tyrosine-phosphorylated proteins (αPY IP) following stimulation with αCD81 or IC mAbs were separated by SDS-PAGE and western
Figure Legend Snippet: Engagement of CD81 induces tyrosine phosphorylation of ezrin. (A) OCI-LY8 cell lysates and immunoprecipitated tyrosine-phosphorylated proteins (αPY IP) following stimulation with αCD81 or IC mAbs were separated by SDS-PAGE and western

Techniques Used: Immunoprecipitation, SDS Page, Western Blot

A unique tyrosine-phosphorylated protein is induced in response to engagement of CD81. OCI-LY8 cells were incubated with (A) 1 μg/ml of 5A6, an anti-CD81 (αCD81) or isotype control (IC) mAbs for the indicated periods. (B) Cells were
Figure Legend Snippet: A unique tyrosine-phosphorylated protein is induced in response to engagement of CD81. OCI-LY8 cells were incubated with (A) 1 μg/ml of 5A6, an anti-CD81 (αCD81) or isotype control (IC) mAbs for the indicated periods. (B) Cells were

Techniques Used: Incubation

8) Product Images from "Serum exosomal hsa-miR-135b-5p serves as a potential diagnostic biomarker in steroid-induced osteonecrosis of femoral head"

Article Title: Serum exosomal hsa-miR-135b-5p serves as a potential diagnostic biomarker in steroid-induced osteonecrosis of femoral head

Journal: American Journal of Translational Research

doi:

Characteristics of isolated exosomes from serum samples. A. Morphology of serum-derived exosome was visualized by TEM, indicating the diameter of isolated exosome in 50-100 nm. B. Size distribution of serum-derived exosome was analyzed using NTA, which were most abundant in 75-117 nm. C. The exosome-specific proteins CD63 and CD81 were detected in the serum exosomes by flow cytometry analysis.
Figure Legend Snippet: Characteristics of isolated exosomes from serum samples. A. Morphology of serum-derived exosome was visualized by TEM, indicating the diameter of isolated exosome in 50-100 nm. B. Size distribution of serum-derived exosome was analyzed using NTA, which were most abundant in 75-117 nm. C. The exosome-specific proteins CD63 and CD81 were detected in the serum exosomes by flow cytometry analysis.

Techniques Used: Isolation, Derivative Assay, Transmission Electron Microscopy, Flow Cytometry

9) Product Images from "Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein"

Article Title: Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20011124

CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (10 7 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (10 7 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).
Figure Legend Snippet: CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (10 7 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (10 7 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).

Techniques Used: Purification, Cell Culture, Incubation, Immunoprecipitation, SDS Page

CD81 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ + T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR + T (stippled bar), CD16 + T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR + T; Th1; CD16 + T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16 + T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).
Figure Legend Snippet: CD81 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ + T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR + T (stippled bar), CD16 + T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR + T; Th1; CD16 + T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16 + T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).

Techniques Used: Clone Assay, Ligation, FACS, Inhibition, Expressing, Activation Assay

Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 10 5 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3 [H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).
Figure Legend Snippet: Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 10 5 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3 [H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).

Techniques Used: Activation Assay, Purification, Cell Culture, Flow Cytometry, Expressing, Marker, Activity Assay, Inhibition

CD81 engagement blocks the functions of resting NK cells. PBMCs freshly purified from healthy donors were cultured in complete medium on plastic plates coated with no antibody (A and B), 1 μg/ml of CD16 mAb alone (C and D), or 1 μg/ml of CD16 mAb plus 10 μg/ml of CD81 mAb (E and F). After 4 h of Brefeldin-A treatment, cells were stained for intracellular IFN-γ production (A, C, and E) and for the surface expression of the activation marker CD25. The plots in (A–F) represent the CD3 - CD56 + subpopulation as defined by the staining in H. In G, CD16 stimulation is specific for NK cells as the CD3 + (T cell) PBMC subpopulation did not produce any IFN-γ, as assayed by intracellular staining.
Figure Legend Snippet: CD81 engagement blocks the functions of resting NK cells. PBMCs freshly purified from healthy donors were cultured in complete medium on plastic plates coated with no antibody (A and B), 1 μg/ml of CD16 mAb alone (C and D), or 1 μg/ml of CD16 mAb plus 10 μg/ml of CD81 mAb (E and F). After 4 h of Brefeldin-A treatment, cells were stained for intracellular IFN-γ production (A, C, and E) and for the surface expression of the activation marker CD25. The plots in (A–F) represent the CD3 - CD56 + subpopulation as defined by the staining in H. In G, CD16 stimulation is specific for NK cells as the CD3 + (T cell) PBMC subpopulation did not produce any IFN-γ, as assayed by intracellular staining.

Techniques Used: Purification, Cell Culture, Staining, Expressing, Activation Assay, Marker

10) Product Images from "Infection of Human Liver Myofibroblasts by Hepatitis C Virus: A Direct Mechanism of Liver Fibrosis in Hepatitis C"

Article Title: Infection of Human Liver Myofibroblasts by Hepatitis C Virus: A Direct Mechanism of Liver Fibrosis in Hepatitis C

Journal: PLoS ONE

doi: 10.1371/journal.pone.0134141

Expression of HCV entry receptors in human liver myofibroblasts (HLMF). HLMF were subjected to the analysis of HCV entry receptors (CD81, LDLR, CLDN-1, OCLN and SR-BI) using four techniques. (A) Quantitative RT–PCR. Histograms represent mRNA levels relative to Huh7.5 in PHH, HLMF, HepG2 and the T98G cell line (means ± SD from 7 cell preparations). (B) Flow cytometry (CD81 and LDLR). The histograms of mean fluorescence showed that Huh7.5, PHH and HLMF expressed CD81 and LDLR, whereas HepG2 expressed LDLR but not CD81. Appropriate IgG controls were used as negative controls. (C) Immunofluorescence. HLMF, Huh7.5, PHH, HepG2 and the T98G line were stained for HCV receptors and imaged with a Leica DMR inverted microscope using LAS image analysis (Original magnification X40). The Flow cytometry and IF results are representative of seven HLMF preparations. (D) CLDN-1, OCLN and SR-BI detection using Western blot analysis. Huh7.5 and PHH were used as positive controls and T98G as a negative control for all receptors. The blots are representative of three HLMF preparations.
Figure Legend Snippet: Expression of HCV entry receptors in human liver myofibroblasts (HLMF). HLMF were subjected to the analysis of HCV entry receptors (CD81, LDLR, CLDN-1, OCLN and SR-BI) using four techniques. (A) Quantitative RT–PCR. Histograms represent mRNA levels relative to Huh7.5 in PHH, HLMF, HepG2 and the T98G cell line (means ± SD from 7 cell preparations). (B) Flow cytometry (CD81 and LDLR). The histograms of mean fluorescence showed that Huh7.5, PHH and HLMF expressed CD81 and LDLR, whereas HepG2 expressed LDLR but not CD81. Appropriate IgG controls were used as negative controls. (C) Immunofluorescence. HLMF, Huh7.5, PHH, HepG2 and the T98G line were stained for HCV receptors and imaged with a Leica DMR inverted microscope using LAS image analysis (Original magnification X40). The Flow cytometry and IF results are representative of seven HLMF preparations. (D) CLDN-1, OCLN and SR-BI detection using Western blot analysis. Huh7.5 and PHH were used as positive controls and T98G as a negative control for all receptors. The blots are representative of three HLMF preparations.

Techniques Used: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Fluorescence, Immunofluorescence, Staining, Inverted Microscopy, Western Blot, Negative Control

Infection status of HLMF inoculated with HCVcc. HLMF were inoculated with JFH1-HCVcc 24 hours after plating. Parameters of HCV infection were monitored at the indicated days after inoculation, and in non-infected HLMF or 3T3 and 293T cells inoculated with JFH1-HCVcc, as controls. Strand-specific HCV RNA was measured by RT-PCR: (A) In lysed cells, (B) In filtered culture supernatants. Histograms represent the copies of strand-specific HCV RNA per μg of total cellular RNA or per ml of supernatant (means ± SD, Ten cell preparations). (C) Intracellular levels of HCV NS3 and core proteins were analyzed by Western blot after 72h post infection in comparison with Huh7.5 or 3T3 cells inoculated with JFH1-HCVcc and with non-infected cells. (D) Levels of HCV core protein in filtered culture supernatants were measured by chemiluminescent microparticle immunoassay (means ± SD, two cell preparations). The blots and histograms are representative of seven HLMF preparations. (E) Immunofluorescence on HLMF with co-staining (CD90 and HCV core protein) and on Huh7.5 with co-staining (CLDN-1 and HCV core protein). (F) Inhibition of HCVcc infection by anti-CD81 antibody or IFN-lpha in HLMF. HLMF were incubated with (a-c) an anti-CD81 neutralizing monoclonal antibody or an isotype-matched control antibody, added 1 h before HCVcc inoculation, or with (d-f) IFN-lpha or a vehicle after HCVcc inoculation. The concentrations tested are indicated. HCV infection was evaluated three days after the inoculation by RT-PCR analysis of strand-specific HCV RNA (a, b, d, e) in lysed cells, and (c and f) in filtered culture supernatants. Histograms represent the copies of strand-specific HCV RNA per μg of total cellular RNA or per ml of supernatant, from triplicate independent experiments.
Figure Legend Snippet: Infection status of HLMF inoculated with HCVcc. HLMF were inoculated with JFH1-HCVcc 24 hours after plating. Parameters of HCV infection were monitored at the indicated days after inoculation, and in non-infected HLMF or 3T3 and 293T cells inoculated with JFH1-HCVcc, as controls. Strand-specific HCV RNA was measured by RT-PCR: (A) In lysed cells, (B) In filtered culture supernatants. Histograms represent the copies of strand-specific HCV RNA per μg of total cellular RNA or per ml of supernatant (means ± SD, Ten cell preparations). (C) Intracellular levels of HCV NS3 and core proteins were analyzed by Western blot after 72h post infection in comparison with Huh7.5 or 3T3 cells inoculated with JFH1-HCVcc and with non-infected cells. (D) Levels of HCV core protein in filtered culture supernatants were measured by chemiluminescent microparticle immunoassay (means ± SD, two cell preparations). The blots and histograms are representative of seven HLMF preparations. (E) Immunofluorescence on HLMF with co-staining (CD90 and HCV core protein) and on Huh7.5 with co-staining (CLDN-1 and HCV core protein). (F) Inhibition of HCVcc infection by anti-CD81 antibody or IFN-lpha in HLMF. HLMF were incubated with (a-c) an anti-CD81 neutralizing monoclonal antibody or an isotype-matched control antibody, added 1 h before HCVcc inoculation, or with (d-f) IFN-lpha or a vehicle after HCVcc inoculation. The concentrations tested are indicated. HCV infection was evaluated three days after the inoculation by RT-PCR analysis of strand-specific HCV RNA (a, b, d, e) in lysed cells, and (c and f) in filtered culture supernatants. Histograms represent the copies of strand-specific HCV RNA per μg of total cellular RNA or per ml of supernatant, from triplicate independent experiments.

Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Inhibition, Incubation

Permissiveness of HLMF to HCVpp. Huh7.5, PHH and HLMF were infected with HCVpp (JFH-1a or H77-a), VSVGpp or CTRL-PUC (pUC19 no envelops). (A) The Huh7.5, PHH and HLMF were infected with HCVpp of genotype (1a) or (2a) carrying a lacZ marker gene encoding a nuclear-targeted ß-galactosidase. The expression of LacZ in cultured cells was performed with X-Gal staining after 72h (the nuclei of positive cells are stained blue) and shown by optical microscopy (magnification x20). (B) Histograms represent the infectious titers of HCVpp per ml (TU/ml) (means ± SD, two cell preparations). (C) Effect of anti-CD81 on HCVpp entry. The different types of cells were preincubated with anti-CD81 or irrelevant isotype control for 1h at 37°C, followed with inoculation of HCVpp for 72h and the expression of LacZ was measured by X-Gal staining. Data represent a percent of neutralization of infectious titers HCVpp.
Figure Legend Snippet: Permissiveness of HLMF to HCVpp. Huh7.5, PHH and HLMF were infected with HCVpp (JFH-1a or H77-a), VSVGpp or CTRL-PUC (pUC19 no envelops). (A) The Huh7.5, PHH and HLMF were infected with HCVpp of genotype (1a) or (2a) carrying a lacZ marker gene encoding a nuclear-targeted ß-galactosidase. The expression of LacZ in cultured cells was performed with X-Gal staining after 72h (the nuclei of positive cells are stained blue) and shown by optical microscopy (magnification x20). (B) Histograms represent the infectious titers of HCVpp per ml (TU/ml) (means ± SD, two cell preparations). (C) Effect of anti-CD81 on HCVpp entry. The different types of cells were preincubated with anti-CD81 or irrelevant isotype control for 1h at 37°C, followed with inoculation of HCVpp for 72h and the expression of LacZ was measured by X-Gal staining. Data represent a percent of neutralization of infectious titers HCVpp.

Techniques Used: Infection, Marker, Expressing, Cell Culture, Staining, Microscopy, Neutralization

11) Product Images from "Tumor Necrosis Factor Inhibits Spread of Hepatitis C Virus Among Liver Cells, Independent from Interferons"

Article Title: Tumor Necrosis Factor Inhibits Spread of Hepatitis C Virus Among Liver Cells, Independent from Interferons

Journal: Gastroenterology

doi: 10.1053/j.gastro.2017.04.021

TNFα has little effect on HCV replication or entry A, B, HCV replicons. Huh-7 cells were treated overnight with TNFα (20 ng/ml), IFNβ (5 U/ml), or media as indicated, electroporated with 5 μg of replicon or NS5B mutant (GNN) RNA, and culture continued in the presence or absence of TNFα/IFNβ as indicated. A, Subgenomic JFH1 replicon. Secreted luciferase was measured at 24-hour intervals. B, Full-length HCV replicons (no reporter). NS5A + cells were measured by flow cytometry at days 5 and 10. C, HCV pseudoparticle entry. Huh-7 cells (4 wells/point) were treated overnight with TNFα or media control. Anti-CD81 was added as indicated 2 hours before addition of pseudoparticles bearing envelope proteins from HCV (H77 or Con1) or control RD114, as indicated. After 6 hours, cultures were washed 3× and culture continued in complete medium. Secreted luciferase was measured 72 hours after infection.
Figure Legend Snippet: TNFα has little effect on HCV replication or entry A, B, HCV replicons. Huh-7 cells were treated overnight with TNFα (20 ng/ml), IFNβ (5 U/ml), or media as indicated, electroporated with 5 μg of replicon or NS5B mutant (GNN) RNA, and culture continued in the presence or absence of TNFα/IFNβ as indicated. A, Subgenomic JFH1 replicon. Secreted luciferase was measured at 24-hour intervals. B, Full-length HCV replicons (no reporter). NS5A + cells were measured by flow cytometry at days 5 and 10. C, HCV pseudoparticle entry. Huh-7 cells (4 wells/point) were treated overnight with TNFα or media control. Anti-CD81 was added as indicated 2 hours before addition of pseudoparticles bearing envelope proteins from HCV (H77 or Con1) or control RD114, as indicated. After 6 hours, cultures were washed 3× and culture continued in complete medium. Secreted luciferase was measured 72 hours after infection.

Techniques Used: Mutagenesis, Luciferase, Flow Cytometry, Cytometry, Infection

12) Product Images from "NG2 as an Identity and Quality Marker of Mesenchymal Stem Cell Extracellular Vesicles"

Article Title: NG2 as an Identity and Quality Marker of Mesenchymal Stem Cell Extracellular Vesicles

Journal: Cells

doi: 10.3390/cells8121524

( a ) Representative histograms of particle size distribution by nanoparticle tracking analysis. Mean and standard deviation of n = 5 repeated measures are represented. ( b ) Representative flow cytometry histograms showing isotype (grey) and LL-cbMSC (green) or bmMSC (orange) CFSE fluorescent signals. ( c ) Representative scanning electron microscopy images of extracellular vesicle (EV) budding by MSC. Scale bar is 10 µm for all images. ( d ) Representative transmission electron microscopy images of isolated EV. Scale bar is 500 µm for all images. ( e ) Representative flow cytometry density plots showing fluorescent signal as detected by APC-A channel for different antigens and controls; IgG1 isotype is CD63 and CD9 control; REA control is CD81 control. The vertical bar marks positivity for APC-A signal. ( f ) Histograms showing APC-A signal normalized to the average of mean fluorescence intensity of CD63, CD81, and CD9. Mean and standard deviation are represented, n = 3 for each experimental group; a.u., arbitrary units. ( g , h ) Western blots showing expression of the proteins of interest in EV and the respective parental cells for LL-cbMSC ( g ) and bmMSC ( h ).
Figure Legend Snippet: ( a ) Representative histograms of particle size distribution by nanoparticle tracking analysis. Mean and standard deviation of n = 5 repeated measures are represented. ( b ) Representative flow cytometry histograms showing isotype (grey) and LL-cbMSC (green) or bmMSC (orange) CFSE fluorescent signals. ( c ) Representative scanning electron microscopy images of extracellular vesicle (EV) budding by MSC. Scale bar is 10 µm for all images. ( d ) Representative transmission electron microscopy images of isolated EV. Scale bar is 500 µm for all images. ( e ) Representative flow cytometry density plots showing fluorescent signal as detected by APC-A channel for different antigens and controls; IgG1 isotype is CD63 and CD9 control; REA control is CD81 control. The vertical bar marks positivity for APC-A signal. ( f ) Histograms showing APC-A signal normalized to the average of mean fluorescence intensity of CD63, CD81, and CD9. Mean and standard deviation are represented, n = 3 for each experimental group; a.u., arbitrary units. ( g , h ) Western blots showing expression of the proteins of interest in EV and the respective parental cells for LL-cbMSC ( g ) and bmMSC ( h ).

Techniques Used: Standard Deviation, Flow Cytometry, Cytometry, Electron Microscopy, Transmission Assay, Isolation, Fluorescence, Western Blot, Expressing

( a ) Representative flow cytometry density plots showing fluorescent signal as detected by APC-A channel for different antigens and controls. The vertical bar marks positivity for APC-A signal. ( b ) Histograms showing APC-A signal normalized to the average of mean fluorescence intensity of CD63, CD81, and CD9. Mean and standard deviation are represented, n = 3 for each experimental group; a.u., arbitrary units. ( c , d ) Representative flow cytometry density plots showing fluorescent signal as detected by APC-A channel for CD105 ( c ), NG2 ( d ) compared to isotype IgG1 controls. The vertical bar marks positivity for APC-A signal. ( e ) Histograms showing APC-A signal normalized to the average of mean fluorescence intensity of CD63, CD81, and CD9. Mean and standard deviation are represented, n = 3 for each experimental group. ( f ) Western blot showing the expression of the protein of interest in extracellular vesicle samples.
Figure Legend Snippet: ( a ) Representative flow cytometry density plots showing fluorescent signal as detected by APC-A channel for different antigens and controls. The vertical bar marks positivity for APC-A signal. ( b ) Histograms showing APC-A signal normalized to the average of mean fluorescence intensity of CD63, CD81, and CD9. Mean and standard deviation are represented, n = 3 for each experimental group; a.u., arbitrary units. ( c , d ) Representative flow cytometry density plots showing fluorescent signal as detected by APC-A channel for CD105 ( c ), NG2 ( d ) compared to isotype IgG1 controls. The vertical bar marks positivity for APC-A signal. ( e ) Histograms showing APC-A signal normalized to the average of mean fluorescence intensity of CD63, CD81, and CD9. Mean and standard deviation are represented, n = 3 for each experimental group. ( f ) Western blot showing the expression of the protein of interest in extracellular vesicle samples.

Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Standard Deviation, Western Blot, Expressing

13) Product Images from "(4R,6S)-2-Dihydromenisdaurilide is a Butenolide that Efficiently Inhibits Hepatitis C Virus Entry"

Article Title: (4R,6S)-2-Dihydromenisdaurilide is a Butenolide that Efficiently Inhibits Hepatitis C Virus Entry

Journal: Scientific Reports

doi: 10.1038/srep29969

Confirmation of DHMD’s antiviral effect against HCV particle attachment using ELISA-based virus binding analysis. ( a ) Schematics of the ELISA-based virus binding assay. ( b ) Huh-7.5 cells were treated with DHMD (50 μM) and HCVcc (MOI = 0.01) at 4 °C for 3 h, then washed with PBS before fixation and analysis for cell surface-bound virus particles by ELISA using anti-HCV E2 antibody. Dashed line indicates baseline signals. DMSO = 0.5%; anti-CD81 = 10 μg/ml; S29 cell = CD81-deficient Huh-7 derivative. Data shown are means ± SEM (* P
Figure Legend Snippet: Confirmation of DHMD’s antiviral effect against HCV particle attachment using ELISA-based virus binding analysis. ( a ) Schematics of the ELISA-based virus binding assay. ( b ) Huh-7.5 cells were treated with DHMD (50 μM) and HCVcc (MOI = 0.01) at 4 °C for 3 h, then washed with PBS before fixation and analysis for cell surface-bound virus particles by ELISA using anti-HCV E2 antibody. Dashed line indicates baseline signals. DMSO = 0.5%; anti-CD81 = 10 μg/ml; S29 cell = CD81-deficient Huh-7 derivative. Data shown are means ± SEM (* P

Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

Confocal microscopy analysis of DHMD’s inhibitory effect against HCV adsorption. ( a ) Huh-7.5 cells seeded in chamber wells were co-incubated with HCVcc (MOI = 0.5) and DHMD (50 μM) or the controls at 4 °C for 3 h, then washed with PBS before shifting to 37 °C for 3 h. At the end of the incubation, cells were washed and fixed for immunostaining using anti-HCV core antibody and visualized with a confocal microscope. Nuclei were stained with the mounting medium used, which contained DAPI. DMSO = 0.5%; anti-CD81 = 10 μg/ml; magnification = 60×; scale bar = 50 μm. Data shown are means ± SEM (* P
Figure Legend Snippet: Confocal microscopy analysis of DHMD’s inhibitory effect against HCV adsorption. ( a ) Huh-7.5 cells seeded in chamber wells were co-incubated with HCVcc (MOI = 0.5) and DHMD (50 μM) or the controls at 4 °C for 3 h, then washed with PBS before shifting to 37 °C for 3 h. At the end of the incubation, cells were washed and fixed for immunostaining using anti-HCV core antibody and visualized with a confocal microscope. Nuclei were stained with the mounting medium used, which contained DAPI. DMSO = 0.5%; anti-CD81 = 10 μg/ml; magnification = 60×; scale bar = 50 μm. Data shown are means ± SEM (* P

Techniques Used: Confocal Microscopy, Adsorption, Incubation, Immunostaining, Microscopy, Staining

14) Product Images from "Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation"

Article Title: Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20090766

Cross-neutralization of escape variants infecting the liver graft by antienvelope and antireceptor mAbs. (A) Neutralization of HCVpps from viral isolates by cross-neutralizing anti-E2 mAb AP33. HCVpps derived from different isolates were incubated with 10 µg/ml anti-E2 AP33 or isotype monoclonal control IgG, and neutralization of viral entry in Huh7 cells was determined as described in Fig. 4 (entry in the presence of isotype monoclonal control IgG = 100%). Neutralization was calculated as described in Materials and methods. Means ± SD from three independent experiments (performed in triplicate) are shown. (B and C) Neutralization of HCV isolates having escaped patients’ neutralizing responses by anti-E2 and anti-CD81 mAbs in primary human hepatocytes. HCVpps derived from viral isolates selected after LT (P01VL, P02VI, P03VC, P04VE, P05VF, and P06VI) were incubated with serial dilutions of anti-E2 AP33 (B) or control IgG from mouse (Ctrl) as described in Fig. 4 and then added to primary human hepatocytes. For analysis of neutralization using anti-CD81, primary human hepatocytes were preincubated with anti-CD81 or isotype control IgG for 1 h at 37° before incubation with patient-derived HCVpps (C). Means from one representative experiment (performed in triplicate) out of two independent experiments are shown. 50% neutralization of HCVpp entry is indicated by a dashed line.
Figure Legend Snippet: Cross-neutralization of escape variants infecting the liver graft by antienvelope and antireceptor mAbs. (A) Neutralization of HCVpps from viral isolates by cross-neutralizing anti-E2 mAb AP33. HCVpps derived from different isolates were incubated with 10 µg/ml anti-E2 AP33 or isotype monoclonal control IgG, and neutralization of viral entry in Huh7 cells was determined as described in Fig. 4 (entry in the presence of isotype monoclonal control IgG = 100%). Neutralization was calculated as described in Materials and methods. Means ± SD from three independent experiments (performed in triplicate) are shown. (B and C) Neutralization of HCV isolates having escaped patients’ neutralizing responses by anti-E2 and anti-CD81 mAbs in primary human hepatocytes. HCVpps derived from viral isolates selected after LT (P01VL, P02VI, P03VC, P04VE, P05VF, and P06VI) were incubated with serial dilutions of anti-E2 AP33 (B) or control IgG from mouse (Ctrl) as described in Fig. 4 and then added to primary human hepatocytes. For analysis of neutralization using anti-CD81, primary human hepatocytes were preincubated with anti-CD81 or isotype control IgG for 1 h at 37° before incubation with patient-derived HCVpps (C). Means from one representative experiment (performed in triplicate) out of two independent experiments are shown. 50% neutralization of HCVpp entry is indicated by a dashed line.

Techniques Used: Neutralization, Derivative Assay, Incubation

Mutations in envelope region E2 425–483 mediate enhanced viral entry and escape from neutralizing antibodies. To map envelope regions mediating enhanced entry and viral escape, we exchanged four regions spanning C-terminal E1 and N-terminal E2, E1, E2- HVR1, and E2-HVR2 of the envelope glycoproteins of selected variant VL and nonselected variant VC of patient P01 (see Figs. 2–4 ). These regions include aa 221–483, aa 221–357, aa 358–424, and aa 425–483, respectively. (A) Deduced amino acid sequences of the exchanged region between P01VC (black) and P01VL (blue). Amino acid changes are indicated in red bold letters. (B) Construction of recombinant chimeric HCVpps P01VCVL 221–483 and P01VLVC 221–483 , P01VCVL-E1 and P01VLVC-E1, P01VCVL-HVR1 and P01VLVC-HVR1, and P01VCVL-HVR2 and P01VLVC-HVR2 by exchanging E1E2 envelope domain aa 221–483, aa 221–357, aa 358–424, and aa 425–483, respectively, of nonselected variant VC (patient 01) depicted in white and escape isolate VL (patient 01) depicted in blue (see Figs. 2–4 ). HVR1 and HVR2 are shown in orange, and CD81 binding domains (CD81 BD) are shown in green. Positions of E2 epitopes I and II are indicated ( Zhang et al., 2007 , 2009 ). The number of mutations within each region is shown. (C) Viral entry of HCVpps containing chimeric envelope proteins in Huh7 cells. HCVpps were incubated with Huh7 cells, and infection was analyzed as described in Fig. 3 . Results are expressed in relative light units (RLU) plotted in a logarithmic scale. The threshold for a detectable infection is 3 × 10 3 RLU and was determined as described in Fig. 3 . (D) Neutralization of HCVpps by autologous pretransplant serum was performed as described in Fig. 4 . End point dilution titers are indicated for each variant. Dashed lines indicate the threshold for a positive neutralization titer corresponding to 1:40. Calculation of neutralization and determination of threshold titers are described in Materials and methods. Chimeric HCVpps are depicted in dashed blue. Statistically significant differences (repeated measures ANOVA) in HCVpp entry or neutralization between VC and VL wild-type and mutant variants are indicated by asterisks (**, P
Figure Legend Snippet: Mutations in envelope region E2 425–483 mediate enhanced viral entry and escape from neutralizing antibodies. To map envelope regions mediating enhanced entry and viral escape, we exchanged four regions spanning C-terminal E1 and N-terminal E2, E1, E2- HVR1, and E2-HVR2 of the envelope glycoproteins of selected variant VL and nonselected variant VC of patient P01 (see Figs. 2–4 ). These regions include aa 221–483, aa 221–357, aa 358–424, and aa 425–483, respectively. (A) Deduced amino acid sequences of the exchanged region between P01VC (black) and P01VL (blue). Amino acid changes are indicated in red bold letters. (B) Construction of recombinant chimeric HCVpps P01VCVL 221–483 and P01VLVC 221–483 , P01VCVL-E1 and P01VLVC-E1, P01VCVL-HVR1 and P01VLVC-HVR1, and P01VCVL-HVR2 and P01VLVC-HVR2 by exchanging E1E2 envelope domain aa 221–483, aa 221–357, aa 358–424, and aa 425–483, respectively, of nonselected variant VC (patient 01) depicted in white and escape isolate VL (patient 01) depicted in blue (see Figs. 2–4 ). HVR1 and HVR2 are shown in orange, and CD81 binding domains (CD81 BD) are shown in green. Positions of E2 epitopes I and II are indicated ( Zhang et al., 2007 , 2009 ). The number of mutations within each region is shown. (C) Viral entry of HCVpps containing chimeric envelope proteins in Huh7 cells. HCVpps were incubated with Huh7 cells, and infection was analyzed as described in Fig. 3 . Results are expressed in relative light units (RLU) plotted in a logarithmic scale. The threshold for a detectable infection is 3 × 10 3 RLU and was determined as described in Fig. 3 . (D) Neutralization of HCVpps by autologous pretransplant serum was performed as described in Fig. 4 . End point dilution titers are indicated for each variant. Dashed lines indicate the threshold for a positive neutralization titer corresponding to 1:40. Calculation of neutralization and determination of threshold titers are described in Materials and methods. Chimeric HCVpps are depicted in dashed blue. Statistically significant differences (repeated measures ANOVA) in HCVpp entry or neutralization between VC and VL wild-type and mutant variants are indicated by asterisks (**, P

Techniques Used: Variant Assay, Recombinant, Binding Assay, Incubation, Infection, Neutralization, Mutagenesis

15) Product Images from "Hepatitis C Virus (HCV)-Induced Immunoglobulin Hypermutation Reduces the Affinity and Neutralizing Activities of Antibodies against HCV Envelope Protein "

Article Title: Hepatitis C Virus (HCV)-Induced Immunoglobulin Hypermutation Reduces the Affinity and Neutralizing Activities of Antibodies against HCV Envelope Protein

Journal: Journal of Virology

doi: 10.1128/JVI.02582-07

(A) EBV-transformed human B cells fused to a mouse-human cell line (hybridoma) are listed with regard to protein targets, heavy-chain subtype, and neutralizing activity for the HCV 2b strain. (B) Infectivity of the hybridoma clones by SB strain of HCV. Total RNA was extracted from cells 7, 14, 28, 42, or 56 days postinfection. R04 is a hybridoma producing antibody against cytomegaloviral proteins. CBH4 and -17, hybridoma producing non-neutralizing anti-E2 antibody; CBH2 and -5, hybridomas producing neutralizing anti-E2 antibody. HCV RNA in the cells was quantified by real-time RT-PCR at various days after infection. The minimum detection limit was 80 copies. (C) Immunofluorescence of NS3 protein. (D) Semiquantitative RT-PCR for AID, CD81, and β-actin. The cDNA was serially diluted fivefold twice and used for PCR.
Figure Legend Snippet: (A) EBV-transformed human B cells fused to a mouse-human cell line (hybridoma) are listed with regard to protein targets, heavy-chain subtype, and neutralizing activity for the HCV 2b strain. (B) Infectivity of the hybridoma clones by SB strain of HCV. Total RNA was extracted from cells 7, 14, 28, 42, or 56 days postinfection. R04 is a hybridoma producing antibody against cytomegaloviral proteins. CBH4 and -17, hybridoma producing non-neutralizing anti-E2 antibody; CBH2 and -5, hybridomas producing neutralizing anti-E2 antibody. HCV RNA in the cells was quantified by real-time RT-PCR at various days after infection. The minimum detection limit was 80 copies. (C) Immunofluorescence of NS3 protein. (D) Semiquantitative RT-PCR for AID, CD81, and β-actin. The cDNA was serially diluted fivefold twice and used for PCR.

Techniques Used: Transformation Assay, Activity Assay, Infection, Clone Assay, Quantitative RT-PCR, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

16) Product Images from "Cholesterol sensing by CD81 is important for hepatitis C virus entry"

Article Title: Cholesterol sensing by CD81 is important for hepatitis C virus entry

Journal: bioRxiv

doi: 10.1101/542837

Conformational switch mutants modulate HCV entry. We mutated residues D196 and K201 to prevent stabilizing interactions across the EC2-TMD4 hinge. A. We performed five independent MD simulations of WT and D196A K201A CD81 in the presence of cholesterol. Images provide overlaid snapshots from representative simulations. Helix E, TMD4 and cholesterol are color coded by time. For clarity the remaining structure is shown in grey for the t=0ns snapshot only. Structures were orientated using TMD4 as a reference B. The change in angle between Helix E and TMD4, by comparison to the CD81 crystal structure, was measured over time for each D196A K201A simulation; compare to Fig 2B . C. The cumulative time spent in the open conformation for either WT or D196A K201A CD81. D. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT CD81, N18A E219A (cholesterol binding mutant), D196A K201A (open mutant) or K116A D117A (closed mutant). HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2, 4 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). There was no significant difference between N18A E219A and D196A K201A. Error bars indicate standard error of the mean.
Figure Legend Snippet: Conformational switch mutants modulate HCV entry. We mutated residues D196 and K201 to prevent stabilizing interactions across the EC2-TMD4 hinge. A. We performed five independent MD simulations of WT and D196A K201A CD81 in the presence of cholesterol. Images provide overlaid snapshots from representative simulations. Helix E, TMD4 and cholesterol are color coded by time. For clarity the remaining structure is shown in grey for the t=0ns snapshot only. Structures were orientated using TMD4 as a reference B. The change in angle between Helix E and TMD4, by comparison to the CD81 crystal structure, was measured over time for each D196A K201A simulation; compare to Fig 2B . C. The cumulative time spent in the open conformation for either WT or D196A K201A CD81. D. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT CD81, N18A E219A (cholesterol binding mutant), D196A K201A (open mutant) or K116A D117A (closed mutant). HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2, 4 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). There was no significant difference between N18A E219A and D196A K201A. Error bars indicate standard error of the mean.

Techniques Used: Transduction, Binding Assay, Mutagenesis, Infection, Expressing, Clone Assay

Cell surface functionality of CD81 mutants. Huh-7 CD81 KO cells were co-transduced with lentivectors encoding human CD19 and CD81 or empty vector. A. Representative flow cytometry histograms, all samples received CD19 lentivector plus the indicated CD81/control vector. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays CD81-dependent trafficking of CD19 to the cell surface (ii). B. CD81 expression on CHO cells confers binding on soluble HCV E2. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays sE2 binding to transduced CHO cells (ii). C. Quantification of sE2 binding expressed relative to WT CD81. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.
Figure Legend Snippet: Cell surface functionality of CD81 mutants. Huh-7 CD81 KO cells were co-transduced with lentivectors encoding human CD19 and CD81 or empty vector. A. Representative flow cytometry histograms, all samples received CD19 lentivector plus the indicated CD81/control vector. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays CD81-dependent trafficking of CD19 to the cell surface (ii). B. CD81 expression on CHO cells confers binding on soluble HCV E2. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays sE2 binding to transduced CHO cells (ii). C. Quantification of sE2 binding expressed relative to WT CD81. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

Techniques Used: Transduction, Plasmid Preparation, Flow Cytometry, Expressing, Binding Assay

Cholesterol sensing is important for authentic HCV infection. Huh-7 CD81 KO cells were transduced with lentivectors expressing the stated CD81 mutants and were then challenged with J6/JFH HCVcc. A. Representative micrographs of HCVcc infection in transduced cells; DAPI nuclei shown in blue, viral antigen NS5A displayed in orange, scale bar = 100 μ m. B. Quantification of infection, compiled from four independent experiments, data is expressed relative to infection in cells expressing WT CD81. C. Huh-7 Lunet N cells stably expressing the stated CD81 mutants were challenged with a panel of diverse HCVcc bearing the glycoproteins of genotypes 1, 2, 3, 4 and 5. Infection was quantified via a virally encoded luciferase reporter and is expressed relative to WT CD81. Data from three representative clones and a summary plot of all HCVcc are shown. Asterisks indicate statistical significance from WT (n=3, one-way ANOVA, Prism). Error bars indicate standard error of the mean.
Figure Legend Snippet: Cholesterol sensing is important for authentic HCV infection. Huh-7 CD81 KO cells were transduced with lentivectors expressing the stated CD81 mutants and were then challenged with J6/JFH HCVcc. A. Representative micrographs of HCVcc infection in transduced cells; DAPI nuclei shown in blue, viral antigen NS5A displayed in orange, scale bar = 100 μ m. B. Quantification of infection, compiled from four independent experiments, data is expressed relative to infection in cells expressing WT CD81. C. Huh-7 Lunet N cells stably expressing the stated CD81 mutants were challenged with a panel of diverse HCVcc bearing the glycoproteins of genotypes 1, 2, 3, 4 and 5. Infection was quantified via a virally encoded luciferase reporter and is expressed relative to WT CD81. Data from three representative clones and a summary plot of all HCVcc are shown. Asterisks indicate statistical significance from WT (n=3, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

Techniques Used: Infection, Transduction, Expressing, Stable Transfection, Luciferase, Clone Assay

Conformational switch mutants exhibit altered protein interaction networks. A. Volcano plot visualizing differences from co-IPs of Huh-7 Lunet N CD81 WT versus Lunet N control cells (n=4 biological replicates for each cell line). LFQ intensity differences (log2) are plotted against the t-test p value (−logP). Significant interactors were defined by a permutation-based FDR using S0=1 as described [ 94 ]. Reference proteins (CD81, SCARB1, CLDN1, EGFR, TFRC, CAPN5 ITGB and CD151) are highlighted, color coded as in B. B. Mean LFQ intensity differences (log2) of interactors in CD81 co-IP (Huh-7 Lunet N CD81 WT and mutants versus Lunet N control cells). Error bars indicate standard error of the mean (n=4) C. Venn diagrams showing the overlap of significantly enriched proteins found in CD81 co-IPs from WT in grey, N18A E219A (Chl) in orange, D196A K201A (O) in purple and K116A D117A (C) in green. Values below each title indicate significant interactors for each CD81 variant, values in the center of each Venn diagram indicate overlapping interactors.
Figure Legend Snippet: Conformational switch mutants exhibit altered protein interaction networks. A. Volcano plot visualizing differences from co-IPs of Huh-7 Lunet N CD81 WT versus Lunet N control cells (n=4 biological replicates for each cell line). LFQ intensity differences (log2) are plotted against the t-test p value (−logP). Significant interactors were defined by a permutation-based FDR using S0=1 as described [ 94 ]. Reference proteins (CD81, SCARB1, CLDN1, EGFR, TFRC, CAPN5 ITGB and CD151) are highlighted, color coded as in B. B. Mean LFQ intensity differences (log2) of interactors in CD81 co-IP (Huh-7 Lunet N CD81 WT and mutants versus Lunet N control cells). Error bars indicate standard error of the mean (n=4) C. Venn diagrams showing the overlap of significantly enriched proteins found in CD81 co-IPs from WT in grey, N18A E219A (Chl) in orange, D196A K201A (O) in purple and K116A D117A (C) in green. Values below each title indicate significant interactors for each CD81 variant, values in the center of each Venn diagram indicate overlapping interactors.

Techniques Used: Co-Immunoprecipitation Assay, Variant Assay

Conformational switching of CD81 in the absence of cholesterol. We performed five independent 500ns MD simulations of WT CD81 with and without cholesterol. A. Snapshots summarising representative simulations from either condition. The Δ° measurement reflects the change in the angle between helix E of the EC2 and TMD4 (as annotated), by comparison to the CD81 crystal structure. For each snapshot the region from which the measurement was taken is color-coded by time. Cholesterol is shown in red. Structures were orientated using TMD4 as a reference. Examples of the orientation of D196 and K201 are shown as insets. B. The angle between helix E and TMD4 was measured over time for each simulation, 25° was chosen as a threshold to indicate conformational switching. C. The cumulative time spent in the open conformation was calculated across all simulations for either experimental condition. D. The distance between D196 K201 was measured over time for each simulation, the dashed line indicates the distance under which electrostatic interactions and hydrogen bonding occurs (10Å). E. The average distance between D196 and K201 with and without cholesterol, data points represent the mean value for each simulation, asterisk indicates statistical significance (n=5 simulations, unpaired T-test, Prism).
Figure Legend Snippet: Conformational switching of CD81 in the absence of cholesterol. We performed five independent 500ns MD simulations of WT CD81 with and without cholesterol. A. Snapshots summarising representative simulations from either condition. The Δ° measurement reflects the change in the angle between helix E of the EC2 and TMD4 (as annotated), by comparison to the CD81 crystal structure. For each snapshot the region from which the measurement was taken is color-coded by time. Cholesterol is shown in red. Structures were orientated using TMD4 as a reference. Examples of the orientation of D196 and K201 are shown as insets. B. The angle between helix E and TMD4 was measured over time for each simulation, 25° was chosen as a threshold to indicate conformational switching. C. The cumulative time spent in the open conformation was calculated across all simulations for either experimental condition. D. The distance between D196 K201 was measured over time for each simulation, the dashed line indicates the distance under which electrostatic interactions and hydrogen bonding occurs (10Å). E. The average distance between D196 and K201 with and without cholesterol, data points represent the mean value for each simulation, asterisk indicates statistical significance (n=5 simulations, unpaired T-test, Prism).

Techniques Used:

Mutations in the cholesterol binding pocket of CD81 modulate HCV entry. A. Cholesterol (red) is coordinated in the intramembrane cavity of CD81 by hydrogen bonds with inward facing residues N18 and E219. We made various mutations at these sites to disrupt this interaction. B. The cholesterol molecule sits in the centre of an intramembrane binding pocket. In the V68W M72W A108W V212W mutant this space is occupied by tryptophan residues (blue residues). C. The cell surface expression levels of each mutant CD81 was assessed by flow cytometry. D. Huh-7 CD81 KO cells were transduced with lentivector encoding WT CD81 or empty vector control. The cells were surface labelled with anti-CD81 mAb and lysed in Brij-98 detergent buffer. CD81-mAb complexes were pulled-down with protein G beads and associated free cholesterol was measured. Our positive control demonstrates the accuracy of the assay. The dashed line indicates the limit of detection E. We assessed cholesterol association with WT and mutant CD81. Data is expressed relative to WT CD81, asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). A representative western blot demonstrating equivalent levels of CD81 in the whole cell lysate (WCL) and pull-down (IP) F. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT and mutant CD81. HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.
Figure Legend Snippet: Mutations in the cholesterol binding pocket of CD81 modulate HCV entry. A. Cholesterol (red) is coordinated in the intramembrane cavity of CD81 by hydrogen bonds with inward facing residues N18 and E219. We made various mutations at these sites to disrupt this interaction. B. The cholesterol molecule sits in the centre of an intramembrane binding pocket. In the V68W M72W A108W V212W mutant this space is occupied by tryptophan residues (blue residues). C. The cell surface expression levels of each mutant CD81 was assessed by flow cytometry. D. Huh-7 CD81 KO cells were transduced with lentivector encoding WT CD81 or empty vector control. The cells were surface labelled with anti-CD81 mAb and lysed in Brij-98 detergent buffer. CD81-mAb complexes were pulled-down with protein G beads and associated free cholesterol was measured. Our positive control demonstrates the accuracy of the assay. The dashed line indicates the limit of detection E. We assessed cholesterol association with WT and mutant CD81. Data is expressed relative to WT CD81, asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). A representative western blot demonstrating equivalent levels of CD81 in the whole cell lysate (WCL) and pull-down (IP) F. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT and mutant CD81. HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

Techniques Used: Binding Assay, Mutagenesis, Expressing, Flow Cytometry, Transduction, Plasmid Preparation, Positive Control, Western Blot, Infection, Clone Assay

17) Product Images from "Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture"

Article Title: Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000910

Virus neutralization by anti-CD81 and anti-HCV antibodies. (A) Huh7.5 cells were pre-incubated with anti-CD81 before infection with filter-clarified supernatants from LB-piVe cells or (B) J6/JFH-1-infected cells. M2, isotype-control antibody. J6/JFH-1 was titrated by an end-point dilution assay using indirect immunofluorescence. Wells were scored positive if at least 1 positive cell was detected. (C) LB-piVe was neutralized by incubation with human anti-HCIGIV [35] or (D) anti-E2 monoclonal antibodies [36] . LBpiVe virus titers in A, C and D were determined by a CPE-based TCID 50 assay. HCV titers were calculated using the method of Reed and Muench [31] . Error bars, ±SD.
Figure Legend Snippet: Virus neutralization by anti-CD81 and anti-HCV antibodies. (A) Huh7.5 cells were pre-incubated with anti-CD81 before infection with filter-clarified supernatants from LB-piVe cells or (B) J6/JFH-1-infected cells. M2, isotype-control antibody. J6/JFH-1 was titrated by an end-point dilution assay using indirect immunofluorescence. Wells were scored positive if at least 1 positive cell was detected. (C) LB-piVe was neutralized by incubation with human anti-HCIGIV [35] or (D) anti-E2 monoclonal antibodies [36] . LBpiVe virus titers in A, C and D were determined by a CPE-based TCID 50 assay. HCV titers were calculated using the method of Reed and Muench [31] . Error bars, ±SD.

Techniques Used: Neutralization, Incubation, Infection, End-point Dilution Assay, Immunofluorescence

18) Product Images from "Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein"

Article Title: Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20011124

CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (10 7 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (10 7 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).
Figure Legend Snippet: CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (10 7 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (10 7 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).

Techniques Used: Purification, Cell Culture, Incubation, Immunoprecipitation, SDS Page

CD81 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ + T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR + T (stippled bar), CD16 + T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR + T; Th1; CD16 + T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16 + T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).
Figure Legend Snippet: CD81 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ + T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR + T (stippled bar), CD16 + T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR + T; Th1; CD16 + T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16 + T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).

Techniques Used: Clone Assay, Ligation, FACS, Inhibition, Expressing, Activation Assay

Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 10 5 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3 [H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).
Figure Legend Snippet: Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 10 5 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3 [H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).

Techniques Used: Activation Assay, Purification, Cell Culture, Flow Cytometry, Expressing, Marker, Activity Assay, Inhibition

CD81 engagement blocks the functions of resting NK cells. PBMCs freshly purified from healthy donors were cultured in complete medium on plastic plates coated with no antibody (A and B), 1 μg/ml of CD16 mAb alone (C and D), or 1 μg/ml of CD16 mAb plus 10 μg/ml of CD81 mAb (E and F). After 4 h of Brefeldin-A treatment, cells were stained for intracellular IFN-γ production (A, C, and E) and for the surface expression of the activation marker CD25. The plots in (A–F) represent the CD3 - CD56 + subpopulation as defined by the staining in H. In G, CD16 stimulation is specific for NK cells as the CD3 + (T cell) PBMC subpopulation did not produce any IFN-γ, as assayed by intracellular staining.
Figure Legend Snippet: CD81 engagement blocks the functions of resting NK cells. PBMCs freshly purified from healthy donors were cultured in complete medium on plastic plates coated with no antibody (A and B), 1 μg/ml of CD16 mAb alone (C and D), or 1 μg/ml of CD16 mAb plus 10 μg/ml of CD81 mAb (E and F). After 4 h of Brefeldin-A treatment, cells were stained for intracellular IFN-γ production (A, C, and E) and for the surface expression of the activation marker CD25. The plots in (A–F) represent the CD3 - CD56 + subpopulation as defined by the staining in H. In G, CD16 stimulation is specific for NK cells as the CD3 + (T cell) PBMC subpopulation did not produce any IFN-γ, as assayed by intracellular staining.

Techniques Used: Purification, Cell Culture, Staining, Expressing, Activation Assay, Marker

19) Product Images from "Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes"

Article Title: Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

Journal: Journal of Extracellular Vesicles

doi: 10.3402/jev.v2i0.20677

Detection and characterization of extracellular vesicles (EVs) by flow cytometry. The CD9, CD63 and CD81 expression on HMC-1 and TF-1 cells (A) and their expression on different vesicles, using anti-CD63-coated beads, are shown. (B) Cells and vesicles were immunostained against the tetraspanin (open curve) CD9 (in black), CD63 (in blue) and CD81 (in red) and compared with their appropriate isotype control (filled curve). The graphs are representative of n=3.
Figure Legend Snippet: Detection and characterization of extracellular vesicles (EVs) by flow cytometry. The CD9, CD63 and CD81 expression on HMC-1 and TF-1 cells (A) and their expression on different vesicles, using anti-CD63-coated beads, are shown. (B) Cells and vesicles were immunostained against the tetraspanin (open curve) CD9 (in black), CD63 (in blue) and CD81 (in red) and compared with their appropriate isotype control (filled curve). The graphs are representative of n=3.

Techniques Used: Flow Cytometry, Cytometry, Expressing

20) Product Images from "Semipermeable Cellulose Beads Allow Selective and Continuous Release of Small Extracellular Vesicles (sEV) From Encapsulated Cells"

Article Title: Semipermeable Cellulose Beads Allow Selective and Continuous Release of Small Extracellular Vesicles (sEV) From Encapsulated Cells

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2020.00679

Expression of classic sEV markers in sEV-cap and sEV-2D. (A) Flow cytometry characterization of sEV-Cap and sEV isolated from 2D-cultured MenSCs shows that are positive for CD63, CD9, and CD81, characteristic markers for sEV. (B) Mean fluorescence index from the isotype controls and the markers analyzed.
Figure Legend Snippet: Expression of classic sEV markers in sEV-cap and sEV-2D. (A) Flow cytometry characterization of sEV-Cap and sEV isolated from 2D-cultured MenSCs shows that are positive for CD63, CD9, and CD81, characteristic markers for sEV. (B) Mean fluorescence index from the isotype controls and the markers analyzed.

Techniques Used: Expressing, Flow Cytometry, Isolation, Cell Culture, Fluorescence

21) Product Images from "Human Immunodeficiency Virus Type 1 and Influenza Virus Exit via Different Membrane Microdomains ▿Human Immunodeficiency Virus Type 1 and Influenza Virus Exit via Different Membrane Microdomains ▿ †"

Article Title: Human Immunodeficiency Virus Type 1 and Influenza Virus Exit via Different Membrane Microdomains ▿Human Immunodeficiency Virus Type 1 and Influenza Virus Exit via Different Membrane Microdomains ▿ †

Journal:

doi: 10.1128/JVI.01255-07

The anti-CD9 antibody K41 inhibits the release of HIV-1 but not of influenza virus. HeLa cells expressing HIV-1 were radiolabeled and treated or not with 1.5 μg/ml of anti-CD63, anti-CD81, anti-CD82, anti-CD9 K41, or isotype control antibody (A),
Figure Legend Snippet: The anti-CD9 antibody K41 inhibits the release of HIV-1 but not of influenza virus. HeLa cells expressing HIV-1 were radiolabeled and treated or not with 1.5 μg/ml of anti-CD63, anti-CD81, anti-CD82, anti-CD9 K41, or isotype control antibody (A),

Techniques Used: Expressing

22) Product Images from "Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization"

Article Title: Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization

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

doi: 10.1073/pnas.0711044105

Blocking of CD81 inhibits ED43/JFH1 infection. Huh7.5 cells were preincubated with anti-CD81 antibody or anti-HIV-p24 isotype-matched control antibody before addition of ≈100 TCID 50 ED43/JFH1-γ third-passage virus. The count of FFUs per well after an incubation period of 2 days is indicated. Each data point represents triplicate experiments. Error bars indicate standard errors of the mean. nd, not determined.
Figure Legend Snippet: Blocking of CD81 inhibits ED43/JFH1 infection. Huh7.5 cells were preincubated with anti-CD81 antibody or anti-HIV-p24 isotype-matched control antibody before addition of ≈100 TCID 50 ED43/JFH1-γ third-passage virus. The count of FFUs per well after an incubation period of 2 days is indicated. Each data point represents triplicate experiments. Error bars indicate standard errors of the mean. nd, not determined.

Techniques Used: Blocking Assay, Infection, Incubation

23) Product Images from "Cholesterol sensing by CD81 is important for hepatitis C virus entry"

Article Title: Cholesterol sensing by CD81 is important for hepatitis C virus entry

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA120.014761

Cell-surface functionality of CD81 mutants. Huh-7 CD81 KO cells were co-transduced with lentivectors encoding human CD19 and CD81 or empty vector. A , representative flow cytometry histograms. All samples received CD19 lentivector plus the indicated CD81/control vector. Panel i demonstrates CD81 surface expression, and panel ii displays CD81-dependent trafficking of CD19 to the cell surface. B , CD81 expression on CHO cells confers binding on soluble HCV E2. Panel i demonstrates CD81 surface expression, and panel ii displays sE2 binding to transduced CHO cells. C , quantification of sE2 binding expressed relative to WT CD81. An asterisk indicates statistical significance from WT ( n = 3, one-way ANOVA, Prism). Error bars indicate standard deviation of the mean.
Figure Legend Snippet: Cell-surface functionality of CD81 mutants. Huh-7 CD81 KO cells were co-transduced with lentivectors encoding human CD19 and CD81 or empty vector. A , representative flow cytometry histograms. All samples received CD19 lentivector plus the indicated CD81/control vector. Panel i demonstrates CD81 surface expression, and panel ii displays CD81-dependent trafficking of CD19 to the cell surface. B , CD81 expression on CHO cells confers binding on soluble HCV E2. Panel i demonstrates CD81 surface expression, and panel ii displays sE2 binding to transduced CHO cells. C , quantification of sE2 binding expressed relative to WT CD81. An asterisk indicates statistical significance from WT ( n = 3, one-way ANOVA, Prism). Error bars indicate standard deviation of the mean.

Techniques Used: Transduction, Plasmid Preparation, Flow Cytometry, Expressing, Binding Assay, Standard Deviation

Cholesterol sensing is important for authentic HCV infection. Huh-7 CD81 KO cells were transduced with lentivectors expressing the stated CD81 mutants and were then challenged with J6/JFH HCVcc. Equal cell-surface expression of WT and mutant CD81 was confirmed by flow cytometry (representative data are provided in Fig. 4 ). A , representative micrographs of HCVcc infection in transduced cells. The 4′,6-diamino-2-phenylindole nuclei shown in blue , and viral antigen NS5A is displayed in orange . Scale bar , 100 μm. B , quantification of infection. The data are expressed relative to infection in cells expressing WT CD81, and an asterisk indicates statistical significance from WT ( n = 4, one-way ANOVA, Prism). C , Huh-7 Lunet N cells stably expressing the stated CD81 mutants were challenged with a panel of diverse HCVcc bearing the glycoproteins of genotypes 1, 2, 3, 4, and 5. Infection was quantified via a virally encoded luciferase reporter and is shown, relative to WT CD81, for three representative clones. An asterisks indicates statistical significance from WT ( n = 3, one-way ANOVA, Prism). D , summary data displaying mean relative infection, as in C , for 12 HCVcc chimeras. An asterisk indicates statistical significance from WT ( n = 12 one-way ANOVA, Prism). In all plots, error bars indicate standard deviation of the mean.
Figure Legend Snippet: Cholesterol sensing is important for authentic HCV infection. Huh-7 CD81 KO cells were transduced with lentivectors expressing the stated CD81 mutants and were then challenged with J6/JFH HCVcc. Equal cell-surface expression of WT and mutant CD81 was confirmed by flow cytometry (representative data are provided in Fig. 4 ). A , representative micrographs of HCVcc infection in transduced cells. The 4′,6-diamino-2-phenylindole nuclei shown in blue , and viral antigen NS5A is displayed in orange . Scale bar , 100 μm. B , quantification of infection. The data are expressed relative to infection in cells expressing WT CD81, and an asterisk indicates statistical significance from WT ( n = 4, one-way ANOVA, Prism). C , Huh-7 Lunet N cells stably expressing the stated CD81 mutants were challenged with a panel of diverse HCVcc bearing the glycoproteins of genotypes 1, 2, 3, 4, and 5. Infection was quantified via a virally encoded luciferase reporter and is shown, relative to WT CD81, for three representative clones. An asterisks indicates statistical significance from WT ( n = 3, one-way ANOVA, Prism). D , summary data displaying mean relative infection, as in C , for 12 HCVcc chimeras. An asterisk indicates statistical significance from WT ( n = 12 one-way ANOVA, Prism). In all plots, error bars indicate standard deviation of the mean.

Techniques Used: Infection, Transduction, Expressing, Mutagenesis, Flow Cytometry, Stable Transfection, Luciferase, Clone Assay, Standard Deviation

Conformational switch mutants modulate HCV entry. We mutated residues Asp 196 and Lys 201 to prevent stabilizing interactions across the EC2–TMD4 hinge. A , we performed five independent MD simulations of WT and D196A/K201A CD81 in the presence of cholesterol. Images provide overlaid snapshots from representative simulations. Helix E, TMD4, and cholesterol are color-coded by time. For clarity the remaining structure is shown in gray for the t = 0 ns snapshot only. Structures were orientated using TMD4 as a reference. B , the change in angle between helix E and TMD4, by comparison with the CD81 crystal structure, was measured over time for each D196A/K201A simulation (compare with Fig. 2 B ). The cumulative time spent in the open conformation across all simulations was 400 ns for WT and 1050 ns for D196A/K201A. C , the average distance between residues 196 and 201 for WT and D196A/K201A in the presence of cholesterol. The dashed line indicates the distance under which electrostatic interactions and hydrogen bonding can occur (10 Å). The data points represent the mean value for each simulation, and an asterisk indicates statistical significance ( n = 5 simulations, unpaired t test, Prism). D , Huh-7 CD81 KO cells were transduced with lentivectors encoding WT CD81, N18A/E219A (cholesterol-binding mutant), D196A/K201A (open mutant), or K116A/D117A (closed mutant), equal cell-surface expression was confirmed by flow cytometry (representative data are provided in Fig. 4 ). HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2, 4, and 5). HCVpp infection, from three representative clones, is shown relative to cells expressing WT CD81. An asterisk indicates statistical significance from WT ( n = 4, one-way ANOVA, Prism). There was no significant difference between N18A/E219A and D196A/K201A. E , summary data displaying mean relative infection, as in D , for eight HCVpp clones. An asterisk indicates statistical significance from WT ( n = 8, one-way ANOVA, Prism). In all plots error bars indicate standard deviation of the mean.
Figure Legend Snippet: Conformational switch mutants modulate HCV entry. We mutated residues Asp 196 and Lys 201 to prevent stabilizing interactions across the EC2–TMD4 hinge. A , we performed five independent MD simulations of WT and D196A/K201A CD81 in the presence of cholesterol. Images provide overlaid snapshots from representative simulations. Helix E, TMD4, and cholesterol are color-coded by time. For clarity the remaining structure is shown in gray for the t = 0 ns snapshot only. Structures were orientated using TMD4 as a reference. B , the change in angle between helix E and TMD4, by comparison with the CD81 crystal structure, was measured over time for each D196A/K201A simulation (compare with Fig. 2 B ). The cumulative time spent in the open conformation across all simulations was 400 ns for WT and 1050 ns for D196A/K201A. C , the average distance between residues 196 and 201 for WT and D196A/K201A in the presence of cholesterol. The dashed line indicates the distance under which electrostatic interactions and hydrogen bonding can occur (10 Å). The data points represent the mean value for each simulation, and an asterisk indicates statistical significance ( n = 5 simulations, unpaired t test, Prism). D , Huh-7 CD81 KO cells were transduced with lentivectors encoding WT CD81, N18A/E219A (cholesterol-binding mutant), D196A/K201A (open mutant), or K116A/D117A (closed mutant), equal cell-surface expression was confirmed by flow cytometry (representative data are provided in Fig. 4 ). HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2, 4, and 5). HCVpp infection, from three representative clones, is shown relative to cells expressing WT CD81. An asterisk indicates statistical significance from WT ( n = 4, one-way ANOVA, Prism). There was no significant difference between N18A/E219A and D196A/K201A. E , summary data displaying mean relative infection, as in D , for eight HCVpp clones. An asterisk indicates statistical significance from WT ( n = 8, one-way ANOVA, Prism). In all plots error bars indicate standard deviation of the mean.

Techniques Used: Transduction, Binding Assay, Mutagenesis, Expressing, Flow Cytometry, Infection, Clone Assay, Standard Deviation

Conformational switch mutants exhibit altered protein interaction networks. A , volcano plot visualizing differences from co-IPs of Huh-7 Lunet N CD81 WT versus Lunet N control cells ( n = 4 biological replicates for each cell line). LFQ intensity differences (log2) are plotted against the t test p value (−logP). Significant interactors were defined by a permutation-based FDR using S0 = 1 as described ( 93 ). Reference proteins (CD81, SCARB1, CLDN1, EGFR, TFRC, CAPN5 ITGB, and CD151) are highlighted and color-coded as in B . B , mean LFQ intensity differences (log2) of interactors in CD81 co-IP (Huh-7 Lunet N CD81 WT and mutants versus Lunet N control cells). Error bars indicate standard deviation of the mean ( n = 4). C , Venn diagrams showing the overlap of significantly enriched proteins found in CD81 co-IPs from WT in gray , N18A/E219A ( Chl ) in orange , D196A/K201A ( O ) in purple , and K116A/D117A ( C ) in green . The values below each title indicate significant interactors for each CD81 variant, and the values in the center of each Venn diagram indicate overlapping interactors.
Figure Legend Snippet: Conformational switch mutants exhibit altered protein interaction networks. A , volcano plot visualizing differences from co-IPs of Huh-7 Lunet N CD81 WT versus Lunet N control cells ( n = 4 biological replicates for each cell line). LFQ intensity differences (log2) are plotted against the t test p value (−logP). Significant interactors were defined by a permutation-based FDR using S0 = 1 as described ( 93 ). Reference proteins (CD81, SCARB1, CLDN1, EGFR, TFRC, CAPN5 ITGB, and CD151) are highlighted and color-coded as in B . B , mean LFQ intensity differences (log2) of interactors in CD81 co-IP (Huh-7 Lunet N CD81 WT and mutants versus Lunet N control cells). Error bars indicate standard deviation of the mean ( n = 4). C , Venn diagrams showing the overlap of significantly enriched proteins found in CD81 co-IPs from WT in gray , N18A/E219A ( Chl ) in orange , D196A/K201A ( O ) in purple , and K116A/D117A ( C ) in green . The values below each title indicate significant interactors for each CD81 variant, and the values in the center of each Venn diagram indicate overlapping interactors.

Techniques Used: Co-Immunoprecipitation Assay, Standard Deviation, Variant Assay

24) Product Images from "Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner"

Article Title: Hepatitis C Virus Stimulates Murine CD8α-Like Dendritic Cells to Produce Type I Interferon in a TRIF-Dependent Manner

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005736

Type I IFN production by Flt3-L DC cultures is dependent on HCV RNA replication and independent of cell-to-cell contact. (A) Huh7.5 cells were mock transfected or transfected with SGR, Jc1 or Jc1ΔGDD (ΔGDD) RNA, co-cultivated with Flt3-L derived DC cultures and the amount of IFN-α in the supernatant was determined (n = 3). (B) Mock or HCV RNA transfected hepatoma cells were treated with 0.5 μg/mL RNAse or 1 unit DNAse before Flt3-L DC were added in a coculture. After 18 h, IFN-α was detected in the cell-free supernatants (n = 3). Flt3-L derived DC were seeded and stimulated with Jc1 (C) or 5 μL concentrated SN from Mock or HCV SGR transfected cells (D) (n = 3). (E) Extracellular vesicles were isolated from concentrated SN from Mock, pUCΔGDD (ΔGDD) or HCV SGR transfected cells. 5 μL of isolated vesicles were used to stimulate Flt3-L DC for 18 h and IFN-α was quantified in the cell-free supernatant by ELISA (n = 6). (F) Protein content of isolated extracellular vesicles was analyzed using antibodies against polypeptides typically enriched in exosomes (Hsp70, AnxII, CD81, CD63 and actin). Dashed line indicates the lowest value of the standard of the respective ELISA assay, n.d. not detected. (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; Mann-Whitney test and 2-way ANOVA, means + SD; n.s. not significant).
Figure Legend Snippet: Type I IFN production by Flt3-L DC cultures is dependent on HCV RNA replication and independent of cell-to-cell contact. (A) Huh7.5 cells were mock transfected or transfected with SGR, Jc1 or Jc1ΔGDD (ΔGDD) RNA, co-cultivated with Flt3-L derived DC cultures and the amount of IFN-α in the supernatant was determined (n = 3). (B) Mock or HCV RNA transfected hepatoma cells were treated with 0.5 μg/mL RNAse or 1 unit DNAse before Flt3-L DC were added in a coculture. After 18 h, IFN-α was detected in the cell-free supernatants (n = 3). Flt3-L derived DC were seeded and stimulated with Jc1 (C) or 5 μL concentrated SN from Mock or HCV SGR transfected cells (D) (n = 3). (E) Extracellular vesicles were isolated from concentrated SN from Mock, pUCΔGDD (ΔGDD) or HCV SGR transfected cells. 5 μL of isolated vesicles were used to stimulate Flt3-L DC for 18 h and IFN-α was quantified in the cell-free supernatant by ELISA (n = 6). (F) Protein content of isolated extracellular vesicles was analyzed using antibodies against polypeptides typically enriched in exosomes (Hsp70, AnxII, CD81, CD63 and actin). Dashed line indicates the lowest value of the standard of the respective ELISA assay, n.d. not detected. (****, p≤ 0.0001, ***, p≤ 0.001; **, P≤0.01; *, P≤0.05; Mann-Whitney test and 2-way ANOVA, means + SD; n.s. not significant).

Techniques Used: Transfection, Derivative Assay, Isolation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

25) Product Images from "Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1"

Article Title: Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200508165

CD9, CD63, CD81, and CD82 cocluster in surface TEMs. To identify surface TEMs containing three of the tetraspanins analyzed here, HeLa cells were surface stained as described in Materials and methods. Insets show sixfold-magnified views of the boxed region in the cell. The percentages of TEMs containing one (30 ± 9), two (24 ± 7), or three (46 ± 14) of the tetraspanins visualized in these triple stainings were assessed as described in Materials and methods ( n > 300). Bar, 10 μm.
Figure Legend Snippet: CD9, CD63, CD81, and CD82 cocluster in surface TEMs. To identify surface TEMs containing three of the tetraspanins analyzed here, HeLa cells were surface stained as described in Materials and methods. Insets show sixfold-magnified views of the boxed region in the cell. The percentages of TEMs containing one (30 ± 9), two (24 ± 7), or three (46 ± 14) of the tetraspanins visualized in these triple stainings were assessed as described in Materials and methods ( n > 300). Bar, 10 μm.

Techniques Used: Staining

Quantitative analysis of surface TEM composition. Cells were triple stained with anti-tetraspanin antibodies as described in the legend for Fig. 2 , and the relative presence in surface TEMs of CD9, CD63, CD81, and CD82 was analyzed as described in Materials and methods. (A) A representative micrograph reveals that the relative contribution of the tetraspanins to the build-up of individual TEMs varies. (B) Statistical analyses confirming the stochastic nature of the tetraspanin contributions to TEM formation. Each bar represents a single TEM that showed staining for three of the above tetraspanins. The relative contribution of each tetraspanin was determined as described in Materials and methods. The bars were sorted in ascending order according to CD63 signal. Bar, 0.5 μm.
Figure Legend Snippet: Quantitative analysis of surface TEM composition. Cells were triple stained with anti-tetraspanin antibodies as described in the legend for Fig. 2 , and the relative presence in surface TEMs of CD9, CD63, CD81, and CD82 was analyzed as described in Materials and methods. (A) A representative micrograph reveals that the relative contribution of the tetraspanins to the build-up of individual TEMs varies. (B) Statistical analyses confirming the stochastic nature of the tetraspanin contributions to TEM formation. Each bar represents a single TEM that showed staining for three of the above tetraspanins. The relative contribution of each tetraspanin was determined as described in Materials and methods. The bars were sorted in ascending order according to CD63 signal. Bar, 0.5 μm.

Techniques Used: Transmission Electron Microscopy, Staining

26) Product Images from "miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR‐186‐5p as a potential antiretroviral factor in macrophages"

Article Title: miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR‐186‐5p as a potential antiretroviral factor in macrophages

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12952

EV composition during the menstrual cycle. (A) Nanoparticle concentrations of CVL UC pellets monitored weekly over 5 weeks for two SIV‐negative (“control”) and four SIV‐infected rhesus macaques (Mean ± SD). Red arrows indicate time of ovulation for two control animals, which were absent for SIV‐infected animals. (B) Transmission electron micrographs of CVL pellets from the 10 000 g pellet (left) and 110 000 g pellet (right) confirm presence of bacteria and EVs/EV‐like particles, with several respective diameters indicated. Scale bar = 500 nm. (C) Western blot analysis suggests enrichment of EV markers CD63, CD81, and TSG101 in 110k pellet fraction of CVL from uninfected animals ( n = 2). Vaginal tissue homogenate and DC (LK23) 110k pellet controls were also positive for CD63 and CD81. Nuclear marker nucleoporin was detected in tissue homogenate but not in putative EV samples. Dashed lines were added to indicate the grouping of the ladder and the samples on the TSG101 and nucleoporin blots. (D) SP‐IRIS confirmation of EV markers on CVL and DC EVs. Shown are averages of tetraspanin‐positive particles bound to anti‐CD63 and anti‐CD81 antibodies and detected by label‐free imaging (mean ± SD).
Figure Legend Snippet: EV composition during the menstrual cycle. (A) Nanoparticle concentrations of CVL UC pellets monitored weekly over 5 weeks for two SIV‐negative (“control”) and four SIV‐infected rhesus macaques (Mean ± SD). Red arrows indicate time of ovulation for two control animals, which were absent for SIV‐infected animals. (B) Transmission electron micrographs of CVL pellets from the 10 000 g pellet (left) and 110 000 g pellet (right) confirm presence of bacteria and EVs/EV‐like particles, with several respective diameters indicated. Scale bar = 500 nm. (C) Western blot analysis suggests enrichment of EV markers CD63, CD81, and TSG101 in 110k pellet fraction of CVL from uninfected animals ( n = 2). Vaginal tissue homogenate and DC (LK23) 110k pellet controls were also positive for CD63 and CD81. Nuclear marker nucleoporin was detected in tissue homogenate but not in putative EV samples. Dashed lines were added to indicate the grouping of the ladder and the samples on the TSG101 and nucleoporin blots. (D) SP‐IRIS confirmation of EV markers on CVL and DC EVs. Shown are averages of tetraspanin‐positive particles bound to anti‐CD63 and anti‐CD81 antibodies and detected by label‐free imaging (mean ± SD).

Techniques Used: Infection, Transmission Assay, Western Blot, Marker, Imaging

27) Product Images from "A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I"

Article Title: A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I

Journal: Journal of Virology

doi: 10.1128/JVI.01011-16

Subset of growth-enhanced mutants are more sensitive to CD81-LEL and NMAb inhibition. Parental or mutant viruses were preincubated with a dilution series of CD81-LEL (A) or anti-E2 antibody H77.39 (B) or HC84.26 (C) for 1 h at 37°C and then added
Figure Legend Snippet: Subset of growth-enhanced mutants are more sensitive to CD81-LEL and NMAb inhibition. Parental or mutant viruses were preincubated with a dilution series of CD81-LEL (A) or anti-E2 antibody H77.39 (B) or HC84.26 (C) for 1 h at 37°C and then added

Techniques Used: Inhibition, Mutagenesis

Dependence on CD81 and CLDN1 for growth-adapted mutants is similar to that of the parental virus. Huh7.5 cells were preincubated with a dilution series of anti-CD81 (A) or anti-CLDN1 (B) for 1 h at 37°C and then infected with parental and growth-adapted
Figure Legend Snippet: Dependence on CD81 and CLDN1 for growth-adapted mutants is similar to that of the parental virus. Huh7.5 cells were preincubated with a dilution series of anti-CD81 (A) or anti-CLDN1 (B) for 1 h at 37°C and then infected with parental and growth-adapted

Techniques Used: Infection

28) Product Images from "Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability"

Article Title: Zeta Potential of Extracellular Vesicles: Toward Understanding the Attributes that Determine Colloidal Stability

Journal: ACS Omega

doi: 10.1021/acsomega.0c01582

Western blot analysis with EV-specific markers: EVs isolated from the conditioned medium of human choriocarcinoma (JAr) cells were probed/immunoblotted with CD9, CD63, CD81, and HSP70.
Figure Legend Snippet: Western blot analysis with EV-specific markers: EVs isolated from the conditioned medium of human choriocarcinoma (JAr) cells were probed/immunoblotted with CD9, CD63, CD81, and HSP70.

Techniques Used: Western Blot, Isolation

Physiology of exosomes: fusion of the multivesicular bodies (MVBs) with the cell membrane releases the exosomes decorated with markers such as CD9, CD63, CD81, and HSP70 and composed of intraluminal vesicles (ILVs) derived from the endoplasmic reticulum (ER) as part of the secretory and/or endocytic pathways. Exosomes carry nucleic acids, cytokines, and proteins (α-synuclein, superoxide dismutase/SOD1, PrP) and achieve intercellular communication via a range of mechanisms, including uptake by the receptor cell by clathrin-mediated pathway or pinocytosis, while the toxic materials (e.g., β-amyloid) in the exosomes are cleared by the microglia and macrophages. Adapted as a freely available open access material under Creative Commons Attribution License (CC BY) from Soria et al., 2017. 7
Figure Legend Snippet: Physiology of exosomes: fusion of the multivesicular bodies (MVBs) with the cell membrane releases the exosomes decorated with markers such as CD9, CD63, CD81, and HSP70 and composed of intraluminal vesicles (ILVs) derived from the endoplasmic reticulum (ER) as part of the secretory and/or endocytic pathways. Exosomes carry nucleic acids, cytokines, and proteins (α-synuclein, superoxide dismutase/SOD1, PrP) and achieve intercellular communication via a range of mechanisms, including uptake by the receptor cell by clathrin-mediated pathway or pinocytosis, while the toxic materials (e.g., β-amyloid) in the exosomes are cleared by the microglia and macrophages. Adapted as a freely available open access material under Creative Commons Attribution License (CC BY) from Soria et al., 2017. 7

Techniques Used: Derivative Assay

29) Product Images from "A Novel Inhibitor IDPP Interferes with Entry and Egress of HCV by Targeting Glycoprotein E1 in a Genotype-Specific Manner"

Article Title: A Novel Inhibitor IDPP Interferes with Entry and Egress of HCV by Targeting Glycoprotein E1 in a Genotype-Specific Manner

Journal: Scientific Reports

doi: 10.1038/srep44676

IDPP interferes with HCV cell-to-cell spread. ( A ) Viral spread assay was performed as described in concept image. After JFH1 (GFP-tagged) infected Huh-7.5 cells (producer cells, P) were co-cultured with naïve TagRFP-NLS-IPS cells (target cells, T), cell-to-cell spread of virus was assessed by counting of red nuclei (spread cells, S) (upper panel). P cells were co-cultured with T cells in the presence of IDPP or α-CD81 mAb (1 μg/mL), and S cells were assessed at 72 h post treatment. ( B ) S cells were counted and data are shown as the mean ± SD of four independent experiments. *** p
Figure Legend Snippet: IDPP interferes with HCV cell-to-cell spread. ( A ) Viral spread assay was performed as described in concept image. After JFH1 (GFP-tagged) infected Huh-7.5 cells (producer cells, P) were co-cultured with naïve TagRFP-NLS-IPS cells (target cells, T), cell-to-cell spread of virus was assessed by counting of red nuclei (spread cells, S) (upper panel). P cells were co-cultured with T cells in the presence of IDPP or α-CD81 mAb (1 μg/mL), and S cells were assessed at 72 h post treatment. ( B ) S cells were counted and data are shown as the mean ± SD of four independent experiments. *** p

Techniques Used: Infection, Cell Culture

30) Product Images from "Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry"

Article Title: Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry

Journal: Journal of Virology

doi: 10.1128/JVI.00684-12

Intracellular (A) and extracellular (B) infectivity titers of transfected S29 cells at 48 h posttransfection. In vitro transcripts of 1b-E2 H77C/JFH1 V787A Q1247L recombinants with and without E2 compensatory mutations were transfected into S29 hepatoma cells lacking CD81. H77C/JFH1 V787A Q1247L and JFH1 ΔE1/E2 were included as positive and negative controls, respectively. All measurements are presented as mean values of triplicates, with error bars displaying standard errors of the means (SEM). The lower limit of quantification was 10 0.7 FFU/well (A) or 10 1.3 FFU/ml (B). Samples with titers under the detection limit are indicated by an asterisk.
Figure Legend Snippet: Intracellular (A) and extracellular (B) infectivity titers of transfected S29 cells at 48 h posttransfection. In vitro transcripts of 1b-E2 H77C/JFH1 V787A Q1247L recombinants with and without E2 compensatory mutations were transfected into S29 hepatoma cells lacking CD81. H77C/JFH1 V787A Q1247L and JFH1 ΔE1/E2 were included as positive and negative controls, respectively. All measurements are presented as mean values of triplicates, with error bars displaying standard errors of the means (SEM). The lower limit of quantification was 10 0.7 FFU/well (A) or 10 1.3 FFU/ml (B). Samples with titers under the detection limit are indicated by an asterisk.

Techniques Used: Infection, Transfection, In Vitro

Dose-dependent inhibition of DH5-E2 with or without S707L binding to the CD81 receptor using CD81 specific antibody. In vitro -generated RNA transcripts of DH5-E2 and DH5-E2 S707L were transfected into S29 cells for production of HCVcc. At 48 h posttransfection, particle-containing supernatant was harvested and DH5-E2 with or without S707L inoculum was applied to naïve Huh7.5 cells which had been incubated with anti-CD81 antibody at concentrations ranging from 0.004 to 2.5 μg/ml for 1 h preinfection. Results are presented as the means of 4 replicates at each antibody concentration, with error bars representing SEM. Percent inhibition was calculated relative to infectivity titers of 6 replicates of virus only. Open symbols represent percent inhibition relative to virus only using an isotype-matched negative control at a concentration of 2.5 μl/ml.
Figure Legend Snippet: Dose-dependent inhibition of DH5-E2 with or without S707L binding to the CD81 receptor using CD81 specific antibody. In vitro -generated RNA transcripts of DH5-E2 and DH5-E2 S707L were transfected into S29 cells for production of HCVcc. At 48 h posttransfection, particle-containing supernatant was harvested and DH5-E2 with or without S707L inoculum was applied to naïve Huh7.5 cells which had been incubated with anti-CD81 antibody at concentrations ranging from 0.004 to 2.5 μg/ml for 1 h preinfection. Results are presented as the means of 4 replicates at each antibody concentration, with error bars representing SEM. Percent inhibition was calculated relative to infectivity titers of 6 replicates of virus only. Open symbols represent percent inhibition relative to virus only using an isotype-matched negative control at a concentration of 2.5 μl/ml.

Techniques Used: Inhibition, Binding Assay, In Vitro, Generated, Transfection, Incubation, Concentration Assay, Infection, Negative Control

Influence of compensatory E2 stem region mutations on HCV E1/E2 heterodimerization. Immunoblots of elution fractions of coimmunoprecipitation using CD81-LEL-GST- or GST-tagged coated beads on lysates from transfected 293T cells (A) and of input fractions of 293T cell lysates prior to coimmunoprecipitation using anti-E1 and anti-E2 specific antibodies (B) are shown. An H77C E1/E2 positive control was transfected in duplicate and served as a specificity control, as co-IP was conducted using both CD81-LEL-GST-coated beads (H77 E1/E2) and GST-tagged coated beads only (H77 E1/E2 GST). The ΔE1/E2 construct (phCMV-ires, in which the partial E1/E2 HCV sequence was deleted) was included as an E1/E2 negative control. β-Actin served as loading control.
Figure Legend Snippet: Influence of compensatory E2 stem region mutations on HCV E1/E2 heterodimerization. Immunoblots of elution fractions of coimmunoprecipitation using CD81-LEL-GST- or GST-tagged coated beads on lysates from transfected 293T cells (A) and of input fractions of 293T cell lysates prior to coimmunoprecipitation using anti-E1 and anti-E2 specific antibodies (B) are shown. An H77C E1/E2 positive control was transfected in duplicate and served as a specificity control, as co-IP was conducted using both CD81-LEL-GST-coated beads (H77 E1/E2) and GST-tagged coated beads only (H77 E1/E2 GST). The ΔE1/E2 construct (phCMV-ires, in which the partial E1/E2 HCV sequence was deleted) was included as an E1/E2 negative control. β-Actin served as loading control.

Techniques Used: Western Blot, Transfection, Positive Control, Co-Immunoprecipitation Assay, Construct, Sequencing, Negative Control

31) Product Images from "TLR-4 engagement of dendritic cells confers a BST-2/tetherin-mediated restriction of HIV-1 infection to CD4+ T cells across the virological synapse"

Article Title: TLR-4 engagement of dendritic cells confers a BST-2/tetherin-mediated restriction of HIV-1 infection to CD4+ T cells across the virological synapse

Journal: Retrovirology

doi: 10.1186/1742-4690-10-6

LPS-induced relocalization of BST-2/tetherin in HIV-containing tetraspanin-enriched compartment. (A) Confocal immunofluorescence analysis of HIV-Gag (green), BST-2/tetherin (red) and CD81 (blue) of DC pre-treated (or not) with IFN-α or LPS and left uninfected or challenged with HIV-WT or HIV-ΔVpu for 3 days. Data are representative of four independent experiments. Scale bars correspond to 5 μm. (B) Graphs represent the quantification of BST-2/tetherin and CD81 co-localisation with HIV-Gag from at least 24 cells per condition from 3 independent experiments.
Figure Legend Snippet: LPS-induced relocalization of BST-2/tetherin in HIV-containing tetraspanin-enriched compartment. (A) Confocal immunofluorescence analysis of HIV-Gag (green), BST-2/tetherin (red) and CD81 (blue) of DC pre-treated (or not) with IFN-α or LPS and left uninfected or challenged with HIV-WT or HIV-ΔVpu for 3 days. Data are representative of four independent experiments. Scale bars correspond to 5 μm. (B) Graphs represent the quantification of BST-2/tetherin and CD81 co-localisation with HIV-Gag from at least 24 cells per condition from 3 independent experiments.

Techniques Used: Immunofluorescence

32) Product Images from "An Improved Detection of Circulating Tumor DNA in Extracellular Vesicles-Depleted Plasma"

Article Title: An Improved Detection of Circulating Tumor DNA in Extracellular Vesicles-Depleted Plasma

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2021.691798

Identity analysis of fraction 3 (large microvesicles) and fraction 5 (exosomes). (A) Transmission electron microscopy. The round shape of large microvesicles (LMV) and exosomes by negatively staining the background with phosphotungstic acid. The bar represents 200 nm. (B) Nanosight analysis. Particle sizes of fractions 3 and 5 are different with 405.5 nm and 100.3 nm in the main peak value, respectively. (C, D) Flow cytometry of characteristic protein analysis. Results of CD63 (C) and CD81 (D) positive ratio show 26.4 and 11.3% in fraction 3, and 66.4 and 88.2% in fraction 5, respectively.
Figure Legend Snippet: Identity analysis of fraction 3 (large microvesicles) and fraction 5 (exosomes). (A) Transmission electron microscopy. The round shape of large microvesicles (LMV) and exosomes by negatively staining the background with phosphotungstic acid. The bar represents 200 nm. (B) Nanosight analysis. Particle sizes of fractions 3 and 5 are different with 405.5 nm and 100.3 nm in the main peak value, respectively. (C, D) Flow cytometry of characteristic protein analysis. Results of CD63 (C) and CD81 (D) positive ratio show 26.4 and 11.3% in fraction 3, and 66.4 and 88.2% in fraction 5, respectively.

Techniques Used: Transmission Assay, Electron Microscopy, Staining, Flow Cytometry

33) Product Images from "Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A"

Article Title: Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006214

L665W decreased AR5A binding to H77/JFH1 particles and to the E1/E2 complex in H77/JFH1 infected cells, but did not alter receptor dependency. (A) Immunoprecipitation was carried out using anti-E1/E2 antibodies AR5A, AR4A, or the anti-E2 antibody, AR3A, or an irrelevant IgG as described in Material and Methods. RNA was measured in duplicates by RT-PCR (values given in international units, IU). The results represent the mean of the total amount of RNA in each sample. The error bar represents standard deviation. *HCV RNA titer below assay cut-off. (B) Huh7.5 cells were infected with virus H77/JFH1 or H77/JFH1 L665W . After 48 hours cells were fixed and incubated with primary antibodies against NS5A (9E10) and E1/E2 (AR5A). Nuclei were counter-stained using Hoechst. Antibody binding was visualized using specific secondary antibodies coupled to fluorophores Alexa488 or Alexa594. Images were acquired using a Zeiss Axio Observer Z1. (C-E) Huh7.5 cells were incubated for 1 hour with dilution series of blocking antibodies against either (C) CD81, (D) SR-BI, or (E) LDLr or control antibody (see Material and Methods for specific antibodies). The indicated virus supernatants were added to Huh7.5 cells and incubated for 4 hour prior to wash and addition of fresh medium. Following a total of 48 hour infection the cells were immunostained and the number of FFUs per well were counted. Values are means of four replicates and normalized to 8 replicates of virus only. Three-parameter curve-fitting was used to obtain sigmoidal dose-response curves. Errors bars represent the standard errors of the mean.
Figure Legend Snippet: L665W decreased AR5A binding to H77/JFH1 particles and to the E1/E2 complex in H77/JFH1 infected cells, but did not alter receptor dependency. (A) Immunoprecipitation was carried out using anti-E1/E2 antibodies AR5A, AR4A, or the anti-E2 antibody, AR3A, or an irrelevant IgG as described in Material and Methods. RNA was measured in duplicates by RT-PCR (values given in international units, IU). The results represent the mean of the total amount of RNA in each sample. The error bar represents standard deviation. *HCV RNA titer below assay cut-off. (B) Huh7.5 cells were infected with virus H77/JFH1 or H77/JFH1 L665W . After 48 hours cells were fixed and incubated with primary antibodies against NS5A (9E10) and E1/E2 (AR5A). Nuclei were counter-stained using Hoechst. Antibody binding was visualized using specific secondary antibodies coupled to fluorophores Alexa488 or Alexa594. Images were acquired using a Zeiss Axio Observer Z1. (C-E) Huh7.5 cells were incubated for 1 hour with dilution series of blocking antibodies against either (C) CD81, (D) SR-BI, or (E) LDLr or control antibody (see Material and Methods for specific antibodies). The indicated virus supernatants were added to Huh7.5 cells and incubated for 4 hour prior to wash and addition of fresh medium. Following a total of 48 hour infection the cells were immunostained and the number of FFUs per well were counted. Values are means of four replicates and normalized to 8 replicates of virus only. Three-parameter curve-fitting was used to obtain sigmoidal dose-response curves. Errors bars represent the standard errors of the mean.

Techniques Used: Binding Assay, Infection, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Incubation, Staining, Blocking Assay

34) Product Images from "CD81/CD9 tetraspanins aid plasmacytoid dendritic cells in recognition of HCV-infected cells and induction of IFNα"

Article Title: CD81/CD9 tetraspanins aid plasmacytoid dendritic cells in recognition of HCV-infected cells and induction of IFNα

Journal: Hepatology (Baltimore, Md.)

doi: 10.1002/hep.25827

Blocking CD81 inhibits pDC IFNα induction by HCV replicons or JFH-1 infected cells
Figure Legend Snippet: Blocking CD81 inhibits pDC IFNα induction by HCV replicons or JFH-1 infected cells

Techniques Used: Blocking Assay, Infection

Inhibition of CD81 on hepatoma cells does not affect IFNα production by PBMCss
Figure Legend Snippet: Inhibition of CD81 on hepatoma cells does not affect IFNα production by PBMCss

Techniques Used: Inhibition

CD81 protein, lipid rafts and endocytosis pathways are required for IFNα induction in PBMCs and hepatoma co-cultures
Figure Legend Snippet: CD81 protein, lipid rafts and endocytosis pathways are required for IFNα induction in PBMCs and hepatoma co-cultures

Techniques Used:

35) Product Images from "Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein"

Article Title: Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20011124

CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (10 7 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (10 7 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).
Figure Legend Snippet: CD81 engagement inhibits specific CD16-triggered tyrosine phosphorylation events. Purified, cultured NK cells (10 7 per sample) were incubated with the indicated antibodies for 1 min and tyrosine phosphorylated proteins were immunoprecipitated from cell lysates, resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with antiphosphotyrosine mAb (A). NK cells (10 7 per sample) were stimulated with the indicated cross-linked mAb's for 1 or 3 min, as indicated in the figure (B and C). Total cell lysates were resolved by SDS PAGE and immunoblotted (B) first with a rabbit polyclonal antiphospho-p44/42 MAPK (erk-2) antibody (top) and then reprobed with a rabbit polyclonal p44/42 MAPK (erk-2) antibody (bottom). Under the same conditions cell lysates were subjected to immunoprecipitation with anti-ζ polyclonal antibody (C). Immunoprecipitated proteins were immunoblotted first with antiphosphotyrosine mAb (top) and then with the immunoprecipitating polyclonal antibody (bottom). In these experiments, SDS-PAGE was performed in nonreducing conditions to detect the 32 kD ζ homodimers (ζ2).

Techniques Used: Purification, Cell Culture, Incubation, Immunoprecipitation, SDS Page

CD81 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ + T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR + T (stippled bar), CD16 + T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR + T; Th1; CD16 + T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16 + T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).
Figure Legend Snippet: CD81 cross-linking has opposite effects on NK and T cells. NK (A) and T (B) cell clones from the same healthy donor were stimulated for 24 h and the supernatants were analyzed for the presence of IFN-γ. The NK cell clones (A) were stimulated with the indicated concentrations of anti-CD16 alone (•) or in combination with 10 μg/ml of: anti-CD81 (○) or anti-HCV-E2 + rHCV-E2 (□). The “classical” TCR αβ + T cell clones (B) were stimulated with decreasing concentrations of anti-CD3 alone (•) or in the presence of 10 μg/ml: anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Control antibodies for anti-CD56 (NK cells) or anti-class I (T cells) had no effect and neither did treatment with the anti-HCV-E2 reagent alone (data not shown). In (C) the effects of CD81 ligation on different T and NK cell subsets is summarized. NKT (gray bar), KIR + T (stippled bar), CD16 + T (hatched bar), Th1 (striped bar), Th2 (white bar, and NK cell (black bar) clones were obtained from the same healthy donor by single cell sorting. The scheme represents the effect of CD81 cross-linking on these different cell types when activated by the appropriate stimulus (anti-CD16 mAb for NK cells, anti-CD3 mAb for the other T cell types). Cytokine production (IFN-γ: KIR + T; Th1; CD16 + T, NK or IL-4: Th2 cell clones), or proliferation (NKT) were used as readouts for CD81-mediated costimulation or inhibition. Results are presented as percentage change compared with treatment with 0.3 μg/ml of anti-CD16 (NK cells) or anti-CD3 (T cells). CD16 + T cells were also analyzed for proliferation, their ability to produce TNF-α and their expression of activation markers after CD81 ligation. In all cases this treatment had no effect (data not shown).

Techniques Used: Clone Assay, Ligation, FACS, Inhibition, Expressing, Activation Assay

Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 10 5 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3 [H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).
Figure Legend Snippet: Cross-linking of CD81 by HCV-E2 or anti-CD81 antibody blocks NK cell activation, cytokine production, and cytotoxic granule release induced by CD16 and IL-2 induced proliferation. Purified, cultured NK cells were stimulated for 24 or 48 h and the supernatants were analyzed for cytokine (TNF-α or IFN-γ) production (A and B). NK cells were stimulated for 24 h and then analyzed by flow cytometry to evaluate the expression level of the activation marker CD25 (C). 10 5 purified NK cells were stimulated for 4 h and supernatants were assayed for BLT-esterase activity which is defined as the percentage of the total BLT-esterase activity obtained from the same number of lysed NK cells (D). For these experiments (A–D) NK cells were cultured in the presence of the indicated concentrations of the anti-CD16 antibody alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti–HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). In E, NK cell proliferation in the presence or absence of rIL-2 was determined by 3 [H]thymidine incorporation. NK cells were cultured at the indicated doses of rIL-2 alone (♦) or in combination with 10 μg/ml of: anti-CD56 (▴); anti-HCV-E2 (▪); anti-CD81 (○) or anti–HCV-E2 + rHCV-E2 (□). Experiments to determine the optimal concentrations of anti-CD81 or anti–HCV-E2 + rHCV-E2 required for NK cell inhibition, demonstrated that the negative effect was detectable over a broad range of concentrations (2.5–20 μg/ml), with 10 μg/ml giving the most potent and consistent inhibition compared with controls (data not shown).

Techniques Used: Activation Assay, Purification, Cell Culture, Flow Cytometry, Cytometry, Expressing, Marker, Activity Assay, Inhibition

CD81 engagement blocks the functions of resting NK cells. PBMCs freshly purified from healthy donors were cultured in complete medium on plastic plates coated with no antibody (A and B), 1 μg/ml of CD16 mAb alone (C and D), or 1 μg/ml of CD16 mAb plus 10 μg/ml of CD81 mAb (E and F). After 4 h of Brefeldin-A treatment, cells were stained for intracellular IFN-γ production (A, C, and E) and for the surface expression of the activation marker CD25. The plots in (A–F) represent the CD3 - CD56 + subpopulation as defined by the staining in H. In G, CD16 stimulation is specific for NK cells as the CD3 + (T cell) PBMC subpopulation did not produce any IFN-γ, as assayed by intracellular staining.
Figure Legend Snippet: CD81 engagement blocks the functions of resting NK cells. PBMCs freshly purified from healthy donors were cultured in complete medium on plastic plates coated with no antibody (A and B), 1 μg/ml of CD16 mAb alone (C and D), or 1 μg/ml of CD16 mAb plus 10 μg/ml of CD81 mAb (E and F). After 4 h of Brefeldin-A treatment, cells were stained for intracellular IFN-γ production (A, C, and E) and for the surface expression of the activation marker CD25. The plots in (A–F) represent the CD3 - CD56 + subpopulation as defined by the staining in H. In G, CD16 stimulation is specific for NK cells as the CD3 + (T cell) PBMC subpopulation did not produce any IFN-γ, as assayed by intracellular staining.

Techniques Used: Purification, Cell Culture, Staining, Expressing, Activation Assay, Marker

36) Product Images from "Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro"

Article Title: Flow Cytometry Based Detection and Isolation of Plasmodium falciparum Liver Stages In Vitro

Journal: PLoS ONE

doi: 10.1371/journal.pone.0129623

Influence of CD81 blocking by mAb 1D6 on P . falciparum hepatocyte infection. Sporozoites were added at a 3:1 sporozoite-to-hepatocyte ratio. 1D6 or isotype were added to cultures at 10μg/ml prior to infection (-2 to 0 hours), during invasion (0 to 6 hours) or after invasion (6 to 24 hours). Representative flow plots shown for (A) HC-04 48 hours postinfection and (B) number and percentage of GFP-positive events in duplicate. (C) Flow plots for donor 4051 96 hours postinfection and (D) graphs indicated the number and percentage of GFP-positive events in duplicate. Mean +/- SD shown on all graphs.
Figure Legend Snippet: Influence of CD81 blocking by mAb 1D6 on P . falciparum hepatocyte infection. Sporozoites were added at a 3:1 sporozoite-to-hepatocyte ratio. 1D6 or isotype were added to cultures at 10μg/ml prior to infection (-2 to 0 hours), during invasion (0 to 6 hours) or after invasion (6 to 24 hours). Representative flow plots shown for (A) HC-04 48 hours postinfection and (B) number and percentage of GFP-positive events in duplicate. (C) Flow plots for donor 4051 96 hours postinfection and (D) graphs indicated the number and percentage of GFP-positive events in duplicate. Mean +/- SD shown on all graphs.

Techniques Used: Blocking Assay, Infection, Flow Cytometry

Expression of surface CD81 by human hepatocyte cell lines and primary hepatocyte donors. Surface CD81 was stained using specific antibodies or an isotype control followed by flow cytometry for detection. (A) Representative plots for all cells are shown and geometric MFI is indicated for both isotype (black) and anti-CD81 staining (red). (B) Surface staining of a mock and transient transfection of HC-04. Transiently transfected HC-04 were infected and run on flow cytometry 96 hours postinfection; (C) GFP-positive number and (D) geometric MFI shown. Mean +/- SD shown.
Figure Legend Snippet: Expression of surface CD81 by human hepatocyte cell lines and primary hepatocyte donors. Surface CD81 was stained using specific antibodies or an isotype control followed by flow cytometry for detection. (A) Representative plots for all cells are shown and geometric MFI is indicated for both isotype (black) and anti-CD81 staining (red). (B) Surface staining of a mock and transient transfection of HC-04. Transiently transfected HC-04 were infected and run on flow cytometry 96 hours postinfection; (C) GFP-positive number and (D) geometric MFI shown. Mean +/- SD shown.

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry, Transfection, Infection

37) Product Images from "RNA-sequencing and bioinformatics analysis of long noncoding RNAs and mRNAs in the asthenozoospermia"

Article Title: RNA-sequencing and bioinformatics analysis of long noncoding RNAs and mRNAs in the asthenozoospermia

Journal: Bioscience Reports

doi: 10.1042/BSR20194041

The Characterization of seminal plasma exosomes ( A ) Transmission electron microscope image of exosomes in normal. ( B ) Transmission electron microscope image of asthenozoospermia. ( C ) Western blotting demonstrating the expression of CD63, and CD81 in normal and asthenozoospermia group.
Figure Legend Snippet: The Characterization of seminal plasma exosomes ( A ) Transmission electron microscope image of exosomes in normal. ( B ) Transmission electron microscope image of asthenozoospermia. ( C ) Western blotting demonstrating the expression of CD63, and CD81 in normal and asthenozoospermia group.

Techniques Used: Transmission Assay, Microscopy, Western Blot, Expressing

38) Product Images from "Macropinocytosis and Cytoskeleton Contribute to Dendritic Cell-mediated HIV-1 Transmission to CD4+ T Cells"

Article Title: Macropinocytosis and Cytoskeleton Contribute to Dendritic Cell-mediated HIV-1 Transmission to CD4+ T Cells

Journal: Virology

doi: 10.1016/j.virol.2008.08.028

Cytoskeleton inhibitors alter HIV trafficking in mDCs. (A) CytoD treatment alters HIV trafficking in mDCs. DCs were pretreated with DMSO, CytoD, or DMA and pulsed with HIV-Vpr-GFP (green) (40 ng of p24) in the presence of the appropriate inhibitors. DCs were fixed and then stained for F-actin (red, phalloidin) and nucleus (blue, DAPI). (B) Nocodazole treatment alters HIV trafficking in mDCs. DCs were pulsed with HIV-Vpr-GFP (green) (40 ng of p24) in the presence of DMSO or nocodazole. DCs were fixed and stained for α-tubulin (red) and analyzed by confocal microscopy. Quantitative image analysis of HIV-positive DCs in the presence or absence of ( C ) CytoD, DMA, and (D) nocodazole. DCs were counted based on the experiments described in A and B. (E) Localization of CD81 and HIV in DCs treated with CytoD or nocodazole. DCs were pulsed separately with HIV-Vpr-GFP (green) (40 ng of p24) in the presence of DMSO or the inhibitors and stained for CD81 (red). (F) Pearson’s correlation analysis of HIV colocalization with CD81 in DCs. The analysis was based on the experiment described in E. The dots represent the r 2 values of Pearson coefficient derived from individual DC images. Noc, nocodazole. Scale bars, 5 μm (A and E), and 3 μm (B).
Figure Legend Snippet: Cytoskeleton inhibitors alter HIV trafficking in mDCs. (A) CytoD treatment alters HIV trafficking in mDCs. DCs were pretreated with DMSO, CytoD, or DMA and pulsed with HIV-Vpr-GFP (green) (40 ng of p24) in the presence of the appropriate inhibitors. DCs were fixed and then stained for F-actin (red, phalloidin) and nucleus (blue, DAPI). (B) Nocodazole treatment alters HIV trafficking in mDCs. DCs were pulsed with HIV-Vpr-GFP (green) (40 ng of p24) in the presence of DMSO or nocodazole. DCs were fixed and stained for α-tubulin (red) and analyzed by confocal microscopy. Quantitative image analysis of HIV-positive DCs in the presence or absence of ( C ) CytoD, DMA, and (D) nocodazole. DCs were counted based on the experiments described in A and B. (E) Localization of CD81 and HIV in DCs treated with CytoD or nocodazole. DCs were pulsed separately with HIV-Vpr-GFP (green) (40 ng of p24) in the presence of DMSO or the inhibitors and stained for CD81 (red). (F) Pearson’s correlation analysis of HIV colocalization with CD81 in DCs. The analysis was based on the experiment described in E. The dots represent the r 2 values of Pearson coefficient derived from individual DC images. Noc, nocodazole. Scale bars, 5 μm (A and E), and 3 μm (B).

Techniques Used: Staining, Confocal Microscopy, Derivative Assay

39) Product Images from "Hepatitis C Virus E2-CD81 Interaction Induces Hypermutation of the Immunoglobulin Gene in B Cells †"

Article Title: Hepatitis C Virus E2-CD81 Interaction Induces Hypermutation of the Immunoglobulin Gene in B Cells †

Journal: Journal of Virology

doi: 10.1128/JVI.79.13.8079-8089.2005

E2-CD81 interaction induces TNF-α production by Raji cells. (A) TNF-α production as determined by ELISA at various time points after E2 or antibody binding. (B) TNF-α production after HCV infection. (C) Detection of HCV RNA in infected cells by RT-PCR. M, mock.
Figure Legend Snippet: E2-CD81 interaction induces TNF-α production by Raji cells. (A) TNF-α production as determined by ELISA at various time points after E2 or antibody binding. (B) TNF-α production after HCV infection. (C) Detection of HCV RNA in infected cells by RT-PCR. M, mock.

Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Infection, Reverse Transcription Polymerase Chain Reaction

E2 binding induces DSBs in Raji cells and PBMC, as determined by ligation-mediated PCR (LM-PCR). (A) Cells were treated with E1, E2 (genotypes 1a and 1b), or anti-CD81 antibody, and the cellular DNA was used for LM-PCR for detecting DSBs in general or in V H or p53 specifically (see Materials and Methods). DNA from HCV-infected or uninfected Raji cells was used as a control. HCV RNA was detected with RT-PCR. Control PCR with β -actin served as an internal control. (B) DSBs in cells pretreated with (+) or without (−) an inhibitory anti-CD81 antibody before binding with E1, E2, or a stimulatory anti-CD81 antibody. (C) Comparison of DSBs in E2 (1a)-treated or HCV-infected Raji cells. DNA samples were serially diluted. (D) DSBs in PBMC. The conditions of treatment were the same as in panel A.
Figure Legend Snippet: E2 binding induces DSBs in Raji cells and PBMC, as determined by ligation-mediated PCR (LM-PCR). (A) Cells were treated with E1, E2 (genotypes 1a and 1b), or anti-CD81 antibody, and the cellular DNA was used for LM-PCR for detecting DSBs in general or in V H or p53 specifically (see Materials and Methods). DNA from HCV-infected or uninfected Raji cells was used as a control. HCV RNA was detected with RT-PCR. Control PCR with β -actin served as an internal control. (B) DSBs in cells pretreated with (+) or without (−) an inhibitory anti-CD81 antibody before binding with E1, E2, or a stimulatory anti-CD81 antibody. (C) Comparison of DSBs in E2 (1a)-treated or HCV-infected Raji cells. DNA samples were serially diluted. (D) DSBs in PBMC. The conditions of treatment were the same as in panel A.

Techniques Used: Binding Assay, Ligation, Polymerase Chain Reaction, Infection, Reverse Transcription Polymerase Chain Reaction

CD81 interacts with recombinant HCV E2 protein. (A) Lysates from Sf9 cells expressing HCV E2 or E1 proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. E2 or E1 was detected by immunoblotting with anti-E2 MAb or His probe, respectively. Monomeric and aggregated E2 species are indicated. (B) Binding of E2 to Raji and Hep-CD81 cells in the presence or absence of various antibodies. The percentage of cells binding E2 was measured by FACS. Average values from two replicates are presented. (C) Dose-response curve for E2 binding to Raji cells. Different amounts of E2 from genotypes 1a and 1b were used in the binding experiment as in panel B. (D) Morphological changes of Raji cells at 48 h after treatment with E1, E2, or various antibodies.
Figure Legend Snippet: CD81 interacts with recombinant HCV E2 protein. (A) Lysates from Sf9 cells expressing HCV E2 or E1 proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. E2 or E1 was detected by immunoblotting with anti-E2 MAb or His probe, respectively. Monomeric and aggregated E2 species are indicated. (B) Binding of E2 to Raji and Hep-CD81 cells in the presence or absence of various antibodies. The percentage of cells binding E2 was measured by FACS. Average values from two replicates are presented. (C) Dose-response curve for E2 binding to Raji cells. Different amounts of E2 from genotypes 1a and 1b were used in the binding experiment as in panel B. (D) Morphological changes of Raji cells at 48 h after treatment with E1, E2, or various antibodies.

Techniques Used: Recombinant, Expressing, Polyacrylamide Gel Electrophoresis, Binding Assay, FACS

Effects of silencing of AID and polymerases ι and ζ on DSBs. (A to D) The silencing of AID and polymerase ι expression by siRNA transfection in Raji cells as determined by RT-PCR of RNA (A and C) and immunoblotting of proteins (B and D). Samples were collected 2 days after stimulation by E2. (E and F) The expression of polymerase ζ mRNA in Raji cells transfected with the specific antisense oligodeoxynucleotide (AS) as determined by semiquantitative RT-PCR (E) or real-time quantitative RT-PCR (F). (G) E2-induced DSBs in Raji cells after siRNA or antisense DNA transfection as in panels A to F. (H) DSB formation in HepG2 or Hep-CD81 cells treated with E1, E2 or anti-CD81 antibody. (I) Semiquantitative RT-PCR of AID and β -actin transcripts in HepG2 or Hep-CD81 cells after the various treatments.
Figure Legend Snippet: Effects of silencing of AID and polymerases ι and ζ on DSBs. (A to D) The silencing of AID and polymerase ι expression by siRNA transfection in Raji cells as determined by RT-PCR of RNA (A and C) and immunoblotting of proteins (B and D). Samples were collected 2 days after stimulation by E2. (E and F) The expression of polymerase ζ mRNA in Raji cells transfected with the specific antisense oligodeoxynucleotide (AS) as determined by semiquantitative RT-PCR (E) or real-time quantitative RT-PCR (F). (G) E2-induced DSBs in Raji cells after siRNA or antisense DNA transfection as in panels A to F. (H) DSB formation in HepG2 or Hep-CD81 cells treated with E1, E2 or anti-CD81 antibody. (I) Semiquantitative RT-PCR of AID and β -actin transcripts in HepG2 or Hep-CD81 cells after the various treatments.

Techniques Used: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

40) Product Images from "Hepatitis C Virus Protects Human B Lymphocytes from Fas-Mediated Apoptosis via E2-CD81 Engagement"

Article Title: Hepatitis C Virus Protects Human B Lymphocytes from Fas-Mediated Apoptosis via E2-CD81 Engagement

Journal: PLoS ONE

doi: 10.1371/journal.pone.0018933

E2-CD81 engagement activates phosphorylation of IκBα and increases expression of NF-κB. (A). Proteasome inhibitor MG-132 treated naïve or CD81-silenced Raji cells were added to wild type E2 or E2-W529/A coated plates, at the indicated time points (minutes), the cells were lysed and the lysates were subject to Western blot analysis with anti-phospho-IκBα mAb and anti-total IκBα mAb. (B). Raji cells were cultured in HCV E2 protein coated plates or incubated with HCVcc, three days later, the cells were lysed, and then NF-κB in the lysates were analyzed using immuno-blotting, the ratios were obtained of the densitometric intensity of NF-κB band relative to the loading control GAPDH band. 1, Naïve Raji cells; 2, E2 treated Raji cells; 3, E2 treated CD81-silenced Raji cells; 4, E2-W529/A treated Raji cells; 5, HCVcc treated Raji cells; 6, HCVcc treated CD81-silenced Raji cells.
Figure Legend Snippet: E2-CD81 engagement activates phosphorylation of IκBα and increases expression of NF-κB. (A). Proteasome inhibitor MG-132 treated naïve or CD81-silenced Raji cells were added to wild type E2 or E2-W529/A coated plates, at the indicated time points (minutes), the cells were lysed and the lysates were subject to Western blot analysis with anti-phospho-IκBα mAb and anti-total IκBα mAb. (B). Raji cells were cultured in HCV E2 protein coated plates or incubated with HCVcc, three days later, the cells were lysed, and then NF-κB in the lysates were analyzed using immuno-blotting, the ratios were obtained of the densitometric intensity of NF-κB band relative to the loading control GAPDH band. 1, Naïve Raji cells; 2, E2 treated Raji cells; 3, E2 treated CD81-silenced Raji cells; 4, E2-W529/A treated Raji cells; 5, HCVcc treated Raji cells; 6, HCVcc treated CD81-silenced Raji cells.

Techniques Used: Expressing, Western Blot, Cell Culture, Incubation

E2 blocks Raji cells apoptosis induced by anti-Fas antibody. (A). Raji cells or CD81-silenced Raji cells were placed in 96-well plates coated with or without HCV E2 protein, cell viability was measured by MTS assay at various time courses. Data represent the means ± standard deviations of triplicate determinations. The treatments of the cells were: Raji cells cultured in 96 wells without coating with HCV E2 protein (open triangles), CD81 silenced Raji cells cultured in 96 wells without coating with HCV E2 protein (filled triangles), E2-treated Raji cells (open squares), E2-W529/A-treated Raji cells (filled squares), E2-treated CD81 silenced Raji cells (open diamonds), E2-W529/A-treated CD81 silenced Raji cells (filled diamonds). (B). Raji cells or CD81-silenced Raji cells were cultured in 96-well plates coated with or without HCV E2 protein for 24 h, and then incubated with CH11 at various concentrations for 5 h. Apoptotic cells were measured by Hoechst 33342 staining. Data points represent the means ± standard deviations of triplicate determinations. The treatments of the cells were described above. Student's t test was used to determine the statistical significance. Double asterisks, p
Figure Legend Snippet: E2 blocks Raji cells apoptosis induced by anti-Fas antibody. (A). Raji cells or CD81-silenced Raji cells were placed in 96-well plates coated with or without HCV E2 protein, cell viability was measured by MTS assay at various time courses. Data represent the means ± standard deviations of triplicate determinations. The treatments of the cells were: Raji cells cultured in 96 wells without coating with HCV E2 protein (open triangles), CD81 silenced Raji cells cultured in 96 wells without coating with HCV E2 protein (filled triangles), E2-treated Raji cells (open squares), E2-W529/A-treated Raji cells (filled squares), E2-treated CD81 silenced Raji cells (open diamonds), E2-W529/A-treated CD81 silenced Raji cells (filled diamonds). (B). Raji cells or CD81-silenced Raji cells were cultured in 96-well plates coated with or without HCV E2 protein for 24 h, and then incubated with CH11 at various concentrations for 5 h. Apoptotic cells were measured by Hoechst 33342 staining. Data points represent the means ± standard deviations of triplicate determinations. The treatments of the cells were described above. Student's t test was used to determine the statistical significance. Double asterisks, p

Techniques Used: MTS Assay, Cell Culture, Incubation, Staining

Effect of E2-CD81 engagement on expression of CD80, CD86, CD21 and CD81 on Raji and PHB cells. Raji cells (A) and PHB cells (B) were treated with HCV E2 protein or HCVcc, and the expressions of CD80, CD86, CD21 and CD81 were measured using a FACS-based assay. The mean fluorescence intensity (MFI) relative to untreated cells was calculated. Results are the means + standard deviations of three independent experiments. Asterisk, p
Figure Legend Snippet: Effect of E2-CD81 engagement on expression of CD80, CD86, CD21 and CD81 on Raji and PHB cells. Raji cells (A) and PHB cells (B) were treated with HCV E2 protein or HCVcc, and the expressions of CD80, CD86, CD21 and CD81 were measured using a FACS-based assay. The mean fluorescence intensity (MFI) relative to untreated cells was calculated. Results are the means + standard deviations of three independent experiments. Asterisk, p

Techniques Used: Expressing, FACS, Fluorescence

Expression of HCV receptors on Raji cells. (A). Expression of CD81 on naïve Raji cells, mock lentivirus infected Raji cells and CD81 shRNA lentivirus infected Raji cells were assayed by FACS. The primary antibodies used were anti-CD81 mAb JS81 and mouse isotype IgG1. (B) Expression of SR-BI on Raji cells. The primary antibodies used were mouse anti-SR-BI sera and control mouse sera. (C). Lysates of Raji, Huh7.5 and CHO cells were analyzed for expression of SR-BI, CLDN1 and OCLN by immuno-blotting. The primary antibodies used were mouse anti-human SR-BI, rabbit anti-human CLDN1 and mouse anti-human OCLN.
Figure Legend Snippet: Expression of HCV receptors on Raji cells. (A). Expression of CD81 on naïve Raji cells, mock lentivirus infected Raji cells and CD81 shRNA lentivirus infected Raji cells were assayed by FACS. The primary antibodies used were anti-CD81 mAb JS81 and mouse isotype IgG1. (B) Expression of SR-BI on Raji cells. The primary antibodies used were mouse anti-SR-BI sera and control mouse sera. (C). Lysates of Raji, Huh7.5 and CHO cells were analyzed for expression of SR-BI, CLDN1 and OCLN by immuno-blotting. The primary antibodies used were mouse anti-human SR-BI, rabbit anti-human CLDN1 and mouse anti-human OCLN.

Techniques Used: Expressing, Infection, shRNA, FACS

Effect of E2-CD81 engagement on expression of Bcl-2 family proteins. Raji cells (A) and PHB cells (B) were treated with HCV E2 protein or HCVcc as described above, three days later, cell lysates were prepared and Bcl-2, Bcl-xL and Bax were determined by Western blot analysis, the ratios were obtained of the densitometric intensity of anti-apoptotic or pro-apoptotic protein band relative to the loading control GAPDH. A: 1, Naïve Raji cells; 2, E2 treated Raji cells; 3, E2 treated CD81-silenced Raji cells; 4, E2-W529/A treated Raji cells; 5, HCVcc treated Raji cells; 6, HCVcc treated CD81-silenced Raji cells. B: 1, untreated PHB cells; 2, E2 treated PHB cells; 3, E2-W529/A treated PHB cells; 5, HCVcc treated PHB cells.
Figure Legend Snippet: Effect of E2-CD81 engagement on expression of Bcl-2 family proteins. Raji cells (A) and PHB cells (B) were treated with HCV E2 protein or HCVcc as described above, three days later, cell lysates were prepared and Bcl-2, Bcl-xL and Bax were determined by Western blot analysis, the ratios were obtained of the densitometric intensity of anti-apoptotic or pro-apoptotic protein band relative to the loading control GAPDH. A: 1, Naïve Raji cells; 2, E2 treated Raji cells; 3, E2 treated CD81-silenced Raji cells; 4, E2-W529/A treated Raji cells; 5, HCVcc treated Raji cells; 6, HCVcc treated CD81-silenced Raji cells. B: 1, untreated PHB cells; 2, E2 treated PHB cells; 3, E2-W529/A treated PHB cells; 5, HCVcc treated PHB cells.

Techniques Used: Expressing, Western Blot

The role of CD81 in mediating HCV E2 binding to Raji cells. (A). 293T cells were transfected with HCV E2 expression plasmid, E2-W529/A expression plasmid, or mock plasmid, respectively. The cells were lysed at 72 h post-transfection and expression of E2 protein was analyzed using immuno-blotting. (B). The binding of cell extract containing HCV E2 protein with naïve or CD81 expression plasmid transfected CHO cells was measured using a FACS-based assay. E2 binding was expressed as the percentages of mean fluorescence intensity (MFI) relative to that of wild type E2 to CHO-CD81. Results are the means + standard deviations of three independent experiments. (C). The binding of cell extract containing HCV E2 protein with naïve or CD81-silenced Raji cells was measured using a FACS-based assay. E2 binding was expressed as the percentages of mean fluorescence intensity (MFI) relative to that of wild type E2 to Raji cells. Results are the means + standard deviations of three independent experiments.
Figure Legend Snippet: The role of CD81 in mediating HCV E2 binding to Raji cells. (A). 293T cells were transfected with HCV E2 expression plasmid, E2-W529/A expression plasmid, or mock plasmid, respectively. The cells were lysed at 72 h post-transfection and expression of E2 protein was analyzed using immuno-blotting. (B). The binding of cell extract containing HCV E2 protein with naïve or CD81 expression plasmid transfected CHO cells was measured using a FACS-based assay. E2 binding was expressed as the percentages of mean fluorescence intensity (MFI) relative to that of wild type E2 to CHO-CD81. Results are the means + standard deviations of three independent experiments. (C). The binding of cell extract containing HCV E2 protein with naïve or CD81-silenced Raji cells was measured using a FACS-based assay. E2 binding was expressed as the percentages of mean fluorescence intensity (MFI) relative to that of wild type E2 to Raji cells. Results are the means + standard deviations of three independent experiments.

Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, FACS, Fluorescence

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    Becton Dickinson rat anti cd8
    Preventive but not therapeutic treatment impairs the increase of <t>CD8+</t> T-lymphocyte numbers in PLPmut mice. a Representative immune fluorescence microscopy of CD8+ T-lymphocytes (arrows) in longitudinal optic nerve sections from Wt , untreated mutants ( PLPmut ), preventively treated mutants (150 days, starting from postnatal month 4; PLPmut + teriflunomide preventive), and PLPmut mice after treatment interruption at 75 days after treatment onset (PLPmut + teriflunomide terminated). Mice were investigated at 9 months of age. b Quantification of CD8+ T-lymphocytes in optic nerve sections of Wt and PLPmut mice and in PLPmut mice after 150 days of preventive treatment. The numbers of CD8+ T cells were significantly increased in the untreated PLPmut mice, which was attenuated upon preventive treatment. Mice were investigated at 9 months of age. c Quantification of CD8+ T-lymphocytes in optic nerve sections of PLPmut mice after 75 days of preventive treatment, followed by 75 days without treatment. Treatment termination did not lead to an overshoot or rebound of T-lymphocyte number, but failed to significantly preserve T-lymphocyte reduction. Mice were investigated at 9 months of age. d Immunofluorescent depiction of CD8+ T-lymphocytes (left) and their quantification (right) in optic nerve sections of Wt and PLPmut mice and in PLPmut mice after 150 days of therapeutic treatment (PLPmut + teriflunomide therapeutic) starting at 10 months of age. Therapeutic treatment did not attenuate the increase of CD8+ T-lymphocyte numbers in PLPmut mice. Mice were investigated at 15 months of age. Scale bar 30 μm. One-way ANOVA and Tukey’s post hoc tests. * P
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    CoV-S mediated entry into tetraspanin KO cells. (A) Western blot analysis of 293T and HeLa clonal cell lines. Actin and the tetraspanin CD63 are used as loading controls. (B) Immunofluorescent analysis of HeLa clonal cell lines. Unpermeabilized cells were incubated with primary antibodies against CD9, <t>CD81</t> or CD63 as indicated. 293T WT or CD9KO cells were transfected with the appropriate receptors and CD9 where indicated. These cells were transduced with viruses pseudotyped with S proteins from MERS (C), 229E (D), SARS (E), or MHV (F). Pseudovirus transduction was measured by luciferase assay.
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    Becton Dickinson biotin conjugated anti mouse cd8
    The immunoproteasome is critical for IFN-γ production by CD4 + and <t>CD8</t> + T cells after B. abortus infection. Flow cytometry analysis of C57BL/6, TKO, and IFN-γ −/− splenocytes obtained after 1, 2, and 4 weeks of infection with B. abortus was performed after 4 h of incubation with brefeldin A and concanavalin A. Cells were assessed for CD3 + CD4 + IFN-γ + (A) and CD3 + CD8 + IFN-γ + (B) populations. A total of 100,000 events was obtained and analyzed. +++ indicates death of animals. Data are the means ± standard deviations of results for five mice/group and are representative of data from three independent experiments. *, P
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    Preventive but not therapeutic treatment impairs the increase of CD8+ T-lymphocyte numbers in PLPmut mice. a Representative immune fluorescence microscopy of CD8+ T-lymphocytes (arrows) in longitudinal optic nerve sections from Wt , untreated mutants ( PLPmut ), preventively treated mutants (150 days, starting from postnatal month 4; PLPmut + teriflunomide preventive), and PLPmut mice after treatment interruption at 75 days after treatment onset (PLPmut + teriflunomide terminated). Mice were investigated at 9 months of age. b Quantification of CD8+ T-lymphocytes in optic nerve sections of Wt and PLPmut mice and in PLPmut mice after 150 days of preventive treatment. The numbers of CD8+ T cells were significantly increased in the untreated PLPmut mice, which was attenuated upon preventive treatment. Mice were investigated at 9 months of age. c Quantification of CD8+ T-lymphocytes in optic nerve sections of PLPmut mice after 75 days of preventive treatment, followed by 75 days without treatment. Treatment termination did not lead to an overshoot or rebound of T-lymphocyte number, but failed to significantly preserve T-lymphocyte reduction. Mice were investigated at 9 months of age. d Immunofluorescent depiction of CD8+ T-lymphocytes (left) and their quantification (right) in optic nerve sections of Wt and PLPmut mice and in PLPmut mice after 150 days of therapeutic treatment (PLPmut + teriflunomide therapeutic) starting at 10 months of age. Therapeutic treatment did not attenuate the increase of CD8+ T-lymphocyte numbers in PLPmut mice. Mice were investigated at 15 months of age. Scale bar 30 μm. One-way ANOVA and Tukey’s post hoc tests. * P

    Journal: Journal of Neuroinflammation

    Article Title: Teriflunomide attenuates neuroinflammation-related neural damage in mice carrying human PLP1 mutations

    doi: 10.1186/s12974-018-1228-z

    Figure Lengend Snippet: Preventive but not therapeutic treatment impairs the increase of CD8+ T-lymphocyte numbers in PLPmut mice. a Representative immune fluorescence microscopy of CD8+ T-lymphocytes (arrows) in longitudinal optic nerve sections from Wt , untreated mutants ( PLPmut ), preventively treated mutants (150 days, starting from postnatal month 4; PLPmut + teriflunomide preventive), and PLPmut mice after treatment interruption at 75 days after treatment onset (PLPmut + teriflunomide terminated). Mice were investigated at 9 months of age. b Quantification of CD8+ T-lymphocytes in optic nerve sections of Wt and PLPmut mice and in PLPmut mice after 150 days of preventive treatment. The numbers of CD8+ T cells were significantly increased in the untreated PLPmut mice, which was attenuated upon preventive treatment. Mice were investigated at 9 months of age. c Quantification of CD8+ T-lymphocytes in optic nerve sections of PLPmut mice after 75 days of preventive treatment, followed by 75 days without treatment. Treatment termination did not lead to an overshoot or rebound of T-lymphocyte number, but failed to significantly preserve T-lymphocyte reduction. Mice were investigated at 9 months of age. d Immunofluorescent depiction of CD8+ T-lymphocytes (left) and their quantification (right) in optic nerve sections of Wt and PLPmut mice and in PLPmut mice after 150 days of therapeutic treatment (PLPmut + teriflunomide therapeutic) starting at 10 months of age. Therapeutic treatment did not attenuate the increase of CD8+ T-lymphocyte numbers in PLPmut mice. Mice were investigated at 15 months of age. Scale bar 30 μm. One-way ANOVA and Tukey’s post hoc tests. * P

    Article Snippet: Sections were blocked using 5% bovine serum albumin in PBS and incubated over night at 4 °C with one or an appropriate combination of up to three of the following antibodies: rat anti-CD4 (1:1000, Bio-Rad AbD Serotec), rat anti-CD8 (1:500, Bio-Rad AbD Serotec), rat anti-CD11b (1:100, Bio-Rad AbD Serotec), rat anti-CD169 (1:300, Bio-Rad AbD Serotec), mouse anti-SMI32 (1:1000, BioLegend), rat anti-PD-1 (1:100, AbD Serotec), rabbit anti-Ki67 (1:200, abcam), and rat anti-CD8 biotinylated (1:500, BD Biosciences).

    Techniques: Mouse Assay, Fluorescence, Microscopy

    Therapeutic treatment fosters proliferation of regulatory CD8+ PD-1+ T-lymphocytes in optic nerves of PLPmut mice. a Representative example of the close apposition of a presumed effector T cell (CD8+ PD-1−, arrow) with a regulatory CD8+ PD-1+ T-lymphocyte (arrowhead) in the optic nerve of a 15-month-old therapeutically treated PLPmut mouse. b Quantification of immunocytochemically labeled CD8+ T cells in optic nerves of 15-month-old PLPmut mice revealed a relative increase of CD8+ PD-1+ regulatory T cells upon therapeutic treatment. c , d Quantitative triple-immunocytochemistry combining antibodies against CD8 (red), against PD-1 (green) and against the proliferation marker Ki67 (gray scale) revealed increased proliferation activity in regulatory CD8+ PD-1+ T-lymphocytes, but not in CD8+ PD-1− presumed effector T cells in optic nerves of therapeutically treated 15-month-old PLPmut mice. Scale bars: 10 μm. Kruskal-Wallis test and Bonferroni-Holm correction. * P

    Journal: Journal of Neuroinflammation

    Article Title: Teriflunomide attenuates neuroinflammation-related neural damage in mice carrying human PLP1 mutations

    doi: 10.1186/s12974-018-1228-z

    Figure Lengend Snippet: Therapeutic treatment fosters proliferation of regulatory CD8+ PD-1+ T-lymphocytes in optic nerves of PLPmut mice. a Representative example of the close apposition of a presumed effector T cell (CD8+ PD-1−, arrow) with a regulatory CD8+ PD-1+ T-lymphocyte (arrowhead) in the optic nerve of a 15-month-old therapeutically treated PLPmut mouse. b Quantification of immunocytochemically labeled CD8+ T cells in optic nerves of 15-month-old PLPmut mice revealed a relative increase of CD8+ PD-1+ regulatory T cells upon therapeutic treatment. c , d Quantitative triple-immunocytochemistry combining antibodies against CD8 (red), against PD-1 (green) and against the proliferation marker Ki67 (gray scale) revealed increased proliferation activity in regulatory CD8+ PD-1+ T-lymphocytes, but not in CD8+ PD-1− presumed effector T cells in optic nerves of therapeutically treated 15-month-old PLPmut mice. Scale bars: 10 μm. Kruskal-Wallis test and Bonferroni-Holm correction. * P

    Article Snippet: Sections were blocked using 5% bovine serum albumin in PBS and incubated over night at 4 °C with one or an appropriate combination of up to three of the following antibodies: rat anti-CD4 (1:1000, Bio-Rad AbD Serotec), rat anti-CD8 (1:500, Bio-Rad AbD Serotec), rat anti-CD11b (1:100, Bio-Rad AbD Serotec), rat anti-CD169 (1:300, Bio-Rad AbD Serotec), mouse anti-SMI32 (1:1000, BioLegend), rat anti-PD-1 (1:100, AbD Serotec), rabbit anti-Ki67 (1:200, abcam), and rat anti-CD8 biotinylated (1:500, BD Biosciences).

    Techniques: Mouse Assay, Labeling, Immunocytochemistry, Marker, Activity Assay

    CoV-S mediated entry into tetraspanin KO cells. (A) Western blot analysis of 293T and HeLa clonal cell lines. Actin and the tetraspanin CD63 are used as loading controls. (B) Immunofluorescent analysis of HeLa clonal cell lines. Unpermeabilized cells were incubated with primary antibodies against CD9, CD81 or CD63 as indicated. 293T WT or CD9KO cells were transfected with the appropriate receptors and CD9 where indicated. These cells were transduced with viruses pseudotyped with S proteins from MERS (C), 229E (D), SARS (E), or MHV (F). Pseudovirus transduction was measured by luciferase assay.

    Journal: PLoS Pathogens

    Article Title: The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases

    doi: 10.1371/journal.ppat.1006546

    Figure Lengend Snippet: CoV-S mediated entry into tetraspanin KO cells. (A) Western blot analysis of 293T and HeLa clonal cell lines. Actin and the tetraspanin CD63 are used as loading controls. (B) Immunofluorescent analysis of HeLa clonal cell lines. Unpermeabilized cells were incubated with primary antibodies against CD9, CD81 or CD63 as indicated. 293T WT or CD9KO cells were transfected with the appropriate receptors and CD9 where indicated. These cells were transduced with viruses pseudotyped with S proteins from MERS (C), 229E (D), SARS (E), or MHV (F). Pseudovirus transduction was measured by luciferase assay.

    Article Snippet: AntibodiesMonoclonal mouse antibodies against CD9 (clone M-L13), CD63 (clone H5C6), and CD81 (clone JS-81) were obtained from BD Pharmingen.

    Techniques: Western Blot, Incubation, Transfection, Transduction, Luciferase

    Association of CoV entry factors with CHAPS-resistant membranes in the presence or absence of CD9 or CD81. 293T WT, CD9KO, or CD81KO cells were transfected with the CoV receptors DPP4 (A), APN (B), ACE2 (C), CEACAM (D), or the protease TMPRSS2 (E). KO cells were also complemented with the appropriate tetraspanin. Cell-surface proteins were biotinylated before cells were lysed in cold CHAPS and cleared lysates were subjected to ultracentrifugation. Cell surface proteins were isolated by streptavidin pulldown and analyzed in high density (HD) and low density (LD) fractions by western blot.

    Journal: PLoS Pathogens

    Article Title: The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases

    doi: 10.1371/journal.ppat.1006546

    Figure Lengend Snippet: Association of CoV entry factors with CHAPS-resistant membranes in the presence or absence of CD9 or CD81. 293T WT, CD9KO, or CD81KO cells were transfected with the CoV receptors DPP4 (A), APN (B), ACE2 (C), CEACAM (D), or the protease TMPRSS2 (E). KO cells were also complemented with the appropriate tetraspanin. Cell-surface proteins were biotinylated before cells were lysed in cold CHAPS and cleared lysates were subjected to ultracentrifugation. Cell surface proteins were isolated by streptavidin pulldown and analyzed in high density (HD) and low density (LD) fractions by western blot.

    Article Snippet: AntibodiesMonoclonal mouse antibodies against CD9 (clone M-L13), CD63 (clone H5C6), and CD81 (clone JS-81) were obtained from BD Pharmingen.

    Techniques: Transfection, Isolation, Western Blot

    The immunoproteasome is critical for IFN-γ production by CD4 + and CD8 + T cells after B. abortus infection. Flow cytometry analysis of C57BL/6, TKO, and IFN-γ −/− splenocytes obtained after 1, 2, and 4 weeks of infection with B. abortus was performed after 4 h of incubation with brefeldin A and concanavalin A. Cells were assessed for CD3 + CD4 + IFN-γ + (A) and CD3 + CD8 + IFN-γ + (B) populations. A total of 100,000 events was obtained and analyzed. +++ indicates death of animals. Data are the means ± standard deviations of results for five mice/group and are representative of data from three independent experiments. *, P

    Journal: Infection and Immunity

    Article Title: Immunoproteasome Subunits Are Required for CD8+ T Cell Function and Host Resistance to Brucella abortus Infection in Mice

    doi: 10.1128/IAI.00615-17

    Figure Lengend Snippet: The immunoproteasome is critical for IFN-γ production by CD4 + and CD8 + T cells after B. abortus infection. Flow cytometry analysis of C57BL/6, TKO, and IFN-γ −/− splenocytes obtained after 1, 2, and 4 weeks of infection with B. abortus was performed after 4 h of incubation with brefeldin A and concanavalin A. Cells were assessed for CD3 + CD4 + IFN-γ + (A) and CD3 + CD8 + IFN-γ + (B) populations. A total of 100,000 events was obtained and analyzed. +++ indicates death of animals. Data are the means ± standard deviations of results for five mice/group and are representative of data from three independent experiments. *, P

    Article Snippet: Cells were incubated for 20 min at 4°C with an antibody solution (0.15 M PBS, 0.5% bovine serum albumin, 2 mM NaN3 ) containing phycoerythrin (PE)-Cy7-conjugated anti-mouse CD4 (1:200 dilution) (clone GK1.5; BD Biosciences), biotin-conjugated anti-mouse CD8 (1:200) (clone 53-6.7; BD Biosciences), and biotin-conjugated anti-mouse CD3 (1:200) (clone 500A2; BD Biosciences) surface markers.

    Techniques: Infection, Flow Cytometry, Cytometry, Incubation, Mouse Assay

    Schematic model of the CD8 + T cell response during B. abortus infection in TKO animals. B. abortus proteins secreted into the cytoplasm of dendritic cells are degraded by the immunoproteasome, generating a variety of peptides. These peptides are then transported to the endoplasmic reticulum and bind to the MHC-I molecule. The MHC-I–peptide complex is exported to the cell membrane and recognized by CD8 + T lymphocytes. After this, CD8 + T cells produce granzyme B and IFN-γ in response to infection. The red dotted arrows indicate the partially impaired process in TKO dendritic and CD8 + T cells. P, proteasome; IP, immunoproteasome; ER, endoplasmic reticulum; DC, dendritic cell.

    Journal: Infection and Immunity

    Article Title: Immunoproteasome Subunits Are Required for CD8+ T Cell Function and Host Resistance to Brucella abortus Infection in Mice

    doi: 10.1128/IAI.00615-17

    Figure Lengend Snippet: Schematic model of the CD8 + T cell response during B. abortus infection in TKO animals. B. abortus proteins secreted into the cytoplasm of dendritic cells are degraded by the immunoproteasome, generating a variety of peptides. These peptides are then transported to the endoplasmic reticulum and bind to the MHC-I molecule. The MHC-I–peptide complex is exported to the cell membrane and recognized by CD8 + T lymphocytes. After this, CD8 + T cells produce granzyme B and IFN-γ in response to infection. The red dotted arrows indicate the partially impaired process in TKO dendritic and CD8 + T cells. P, proteasome; IP, immunoproteasome; ER, endoplasmic reticulum; DC, dendritic cell.

    Article Snippet: Cells were incubated for 20 min at 4°C with an antibody solution (0.15 M PBS, 0.5% bovine serum albumin, 2 mM NaN3 ) containing phycoerythrin (PE)-Cy7-conjugated anti-mouse CD4 (1:200 dilution) (clone GK1.5; BD Biosciences), biotin-conjugated anti-mouse CD8 (1:200) (clone 53-6.7; BD Biosciences), and biotin-conjugated anti-mouse CD3 (1:200) (clone 500A2; BD Biosciences) surface markers.

    Techniques: Infection

    TKO CD8 + T cell cytotoxic activity is reduced in B. abortus -infected mice. Bone marrow-derived dendritic cells (5 × 10 5 cells/well) obtained from C57BL/6 and TKO mice were infected with B. abortus (MOI of 100:1) and used as target cells. (A and C) CD8 + (A) or CD4 + (C) T lymphocytes (1 × 10 6 cells/well) obtained by cell sorting from C57BL/6 and TKO splenocytes at the second week of infection were used as effector cells for cytotoxicity assays and were cocultured with dendritic cells in 24-well plates in DMEM. CD8 + T cells were added to target cells at a 1:2 ratio, CD4 + T cells were added to target cells in duplicate at a 1:1 ratio, and 24 h after coculture, the supernatant was collected to quantify LDH release according to the manufacturer's instructions. (B and D) Percentages of CD3 + CD8 + granzyme B-positive (B) and CD3 + CD4 + granzyme B-positive (D) cells measured by flow cytometry in splenocytes from C57BL/6 and TKO mice. Data are the means ± standard deviations of results for five mice/group and are representative of data from three independent experiments. *, P

    Journal: Infection and Immunity

    Article Title: Immunoproteasome Subunits Are Required for CD8+ T Cell Function and Host Resistance to Brucella abortus Infection in Mice

    doi: 10.1128/IAI.00615-17

    Figure Lengend Snippet: TKO CD8 + T cell cytotoxic activity is reduced in B. abortus -infected mice. Bone marrow-derived dendritic cells (5 × 10 5 cells/well) obtained from C57BL/6 and TKO mice were infected with B. abortus (MOI of 100:1) and used as target cells. (A and C) CD8 + (A) or CD4 + (C) T lymphocytes (1 × 10 6 cells/well) obtained by cell sorting from C57BL/6 and TKO splenocytes at the second week of infection were used as effector cells for cytotoxicity assays and were cocultured with dendritic cells in 24-well plates in DMEM. CD8 + T cells were added to target cells at a 1:2 ratio, CD4 + T cells were added to target cells in duplicate at a 1:1 ratio, and 24 h after coculture, the supernatant was collected to quantify LDH release according to the manufacturer's instructions. (B and D) Percentages of CD3 + CD8 + granzyme B-positive (B) and CD3 + CD4 + granzyme B-positive (D) cells measured by flow cytometry in splenocytes from C57BL/6 and TKO mice. Data are the means ± standard deviations of results for five mice/group and are representative of data from three independent experiments. *, P

    Article Snippet: Cells were incubated for 20 min at 4°C with an antibody solution (0.15 M PBS, 0.5% bovine serum albumin, 2 mM NaN3 ) containing phycoerythrin (PE)-Cy7-conjugated anti-mouse CD4 (1:200 dilution) (clone GK1.5; BD Biosciences), biotin-conjugated anti-mouse CD8 (1:200) (clone 53-6.7; BD Biosciences), and biotin-conjugated anti-mouse CD3 (1:200) (clone 500A2; BD Biosciences) surface markers.

    Techniques: Activity Assay, Infection, Mouse Assay, Derivative Assay, FACS, Flow Cytometry, Cytometry