plns  (Worthington Biochemical)


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  • 91
    Name:
    Superoxide Dismutase
    Description:
    Chromatographically purified essentially as described by McCord and Fridovich J Biol Chem 244 6049 1969 A dialyzed lyophilized powder
    Catalog Number:
    ls003540
    Price:
    39
    Size:
    2 mg
    Source:
    Bovine Erythrocytes
    Cas Number:
    9054.89.1
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    Structured Review

    Worthington Biochemical plns
    Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining <t>PLNs</t> or pooled axillary and inguinal lymph nodes, respectively. 100 ng of DNA was amplified by PCR using <t>HSV-1</t> or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control (+). A no DNA control was included to rule out contamination (−). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 + T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after infection. Histograms represent 5–10,000 live events.
    Chromatographically purified essentially as described by McCord and Fridovich J Biol Chem 244 6049 1969 A dialyzed lyophilized powder
    https://www.bioz.com/result/plns/product/Worthington Biochemical
    Average 91 stars, based on 1310 article reviews
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    plns - by Bioz Stars, 2020-05
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    Images

    1) Product Images from "Rapid Cytotoxic T Lymphocyte Activation Occurs in the Draining Lymph Nodes After Cutaneous Herpes Simplex Virus Infection as a Result of Early Antigen Presentation and Not the Presence of Virus"

    Article Title: Rapid Cytotoxic T Lymphocyte Activation Occurs in the Draining Lymph Nodes After Cutaneous Herpes Simplex Virus Infection as a Result of Early Antigen Presentation and Not the Presence of Virus

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20012023

    Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining PLNs or pooled axillary and inguinal lymph nodes, respectively. 100 ng of DNA was amplified by PCR using HSV-1 or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control (+). A no DNA control was included to rule out contamination (−). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 + T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after infection. Histograms represent 5–10,000 live events.
    Figure Legend Snippet: Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining PLNs or pooled axillary and inguinal lymph nodes, respectively. 100 ng of DNA was amplified by PCR using HSV-1 or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control (+). A no DNA control was included to rule out contamination (−). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 + T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after infection. Histograms represent 5–10,000 live events.

    Techniques Used: Activation Assay, Mouse Assay, Infection, Isolation, Amplification, Polymerase Chain Reaction, Positive Control, Labeling, Injection

    Activation of CD8 + T cells in the PLNs correlates with the appearance of specific antigen presentation. (A) Mice adoptively transferred with CFSE-labeled gBT-I.1 cells were killed at various times (2–8 h) after footpad infection with HSV-1 and CD8 + CFSE + cells analyzed for the expression of CD69. (B) PLNs from HSV-1–infected C57BL/6 mice were treated with collagenase to form a cell suspension. Graded amounts of these cells were placed into culture with HSV-2.3.2E2 lacZ -inducible hybridoma cells overnight. An X-Gal assay was then performed to stain responding hybridoma cells which were counted microscopically. Numbers presented represent the total number of lacZ + cells per separate PLN at various times (2–48 h) after infection, and error bars represent SD ( n = 8–12).
    Figure Legend Snippet: Activation of CD8 + T cells in the PLNs correlates with the appearance of specific antigen presentation. (A) Mice adoptively transferred with CFSE-labeled gBT-I.1 cells were killed at various times (2–8 h) after footpad infection with HSV-1 and CD8 + CFSE + cells analyzed for the expression of CD69. (B) PLNs from HSV-1–infected C57BL/6 mice were treated with collagenase to form a cell suspension. Graded amounts of these cells were placed into culture with HSV-2.3.2E2 lacZ -inducible hybridoma cells overnight. An X-Gal assay was then performed to stain responding hybridoma cells which were counted microscopically. Numbers presented represent the total number of lacZ + cells per separate PLN at various times (2–48 h) after infection, and error bars represent SD ( n = 8–12).

    Techniques Used: Activation Assay, Mouse Assay, Labeling, Infection, Expressing, Staining

    Concurrent in vivo proliferation and CTL activity by gB-specific CD8 + T cells in the PLNs after cutaneous infection with HSV-1. (A) CFSE-labeled lymph node cells from gBT-I.1 mice were transferred into C57BL/6 mice before infection with HSV-1. PLN cells were isolated at various times after infection (24–72 h) and dilution of the CFSE fluorescence analyzed by gating on live CD8 + T cells. (B) Cellularity within the draining lymph nodes over a 48-h period was determined using cell suspensions obtained from the PLNs of mice after foot-pad HSV-1 infection. (C) Mice that had (black bars) or had not (white bars) received 10 6 gBT-I.1 cells 24 h earlier were infected with HSV-1 in the footpad and left for various times as shown before intravenous transfer of CFSE-labeled syngeneic target cells. gB-peptide–pulsed splenocytes were labeled with a high concentration of CFSE (CFSE hi ) while unpulsed control targets were labeled with a low concentration of CFSE (CFSE lo ). 4 h after target cell transfer, mice were killed and PLN cells analyzed for relative elimination of the CFSE hi versus CFSE lo populations. Percent specific lysis was calculated as described in reference 5. Error bars represent SD.
    Figure Legend Snippet: Concurrent in vivo proliferation and CTL activity by gB-specific CD8 + T cells in the PLNs after cutaneous infection with HSV-1. (A) CFSE-labeled lymph node cells from gBT-I.1 mice were transferred into C57BL/6 mice before infection with HSV-1. PLN cells were isolated at various times after infection (24–72 h) and dilution of the CFSE fluorescence analyzed by gating on live CD8 + T cells. (B) Cellularity within the draining lymph nodes over a 48-h period was determined using cell suspensions obtained from the PLNs of mice after foot-pad HSV-1 infection. (C) Mice that had (black bars) or had not (white bars) received 10 6 gBT-I.1 cells 24 h earlier were infected with HSV-1 in the footpad and left for various times as shown before intravenous transfer of CFSE-labeled syngeneic target cells. gB-peptide–pulsed splenocytes were labeled with a high concentration of CFSE (CFSE hi ) while unpulsed control targets were labeled with a low concentration of CFSE (CFSE lo ). 4 h after target cell transfer, mice were killed and PLN cells analyzed for relative elimination of the CFSE hi versus CFSE lo populations. Percent specific lysis was calculated as described in reference 5. Error bars represent SD.

    Techniques Used: In Vivo, CTL Assay, Activity Assay, Infection, Labeling, Mouse Assay, Isolation, Fluorescence, Concentration Assay, Lysis

    De novo synthesis of viral peptides is required to elicit a gB-specific T cell response. Mice receiving CFSE-labeled gBT-I.1 CD8 + T cells were infected with wild-type HSV-1 KOS or the gB mutant strains KΔ318 or KΔ5C. 42 h after infection, CD8 + T cells from draining PLNs were analyzed for dilution of the CFSE stain caused by cell division. Histograms represent 5–10,000 live events.
    Figure Legend Snippet: De novo synthesis of viral peptides is required to elicit a gB-specific T cell response. Mice receiving CFSE-labeled gBT-I.1 CD8 + T cells were infected with wild-type HSV-1 KOS or the gB mutant strains KΔ318 or KΔ5C. 42 h after infection, CD8 + T cells from draining PLNs were analyzed for dilution of the CFSE stain caused by cell division. Histograms represent 5–10,000 live events.

    Techniques Used: Mouse Assay, Labeling, Infection, Mutagenesis, Staining

    2) Product Images from "Rapid Cytotoxic T Lymphocyte Activation Occurs in the Draining Lymph Nodes After Cutaneous Herpes Simplex Virus Infection as a Result of Early Antigen Presentation and Not the Presence of Virus"

    Article Title: Rapid Cytotoxic T Lymphocyte Activation Occurs in the Draining Lymph Nodes After Cutaneous Herpes Simplex Virus Infection as a Result of Early Antigen Presentation and Not the Presence of Virus

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20012023

    Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining PLNs or pooled axillary and inguinal lymph nodes, respectively. 100 ng of DNA was amplified by PCR using HSV-1 or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control (+). A no DNA control was included to rule out contamination (−). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 + T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after infection. Histograms represent 5–10,000 live events.
    Figure Legend Snippet: Activation of the gB-specific T cells does not require the presence of viral DNA in the draining lymph nodes. Mice were killed at various times (2–48 h; N, naive) after footpad infection (A), or after flank scarification (B) and DNA isolated from the draining PLNs or pooled axillary and inguinal lymph nodes, respectively. 100 ng of DNA was amplified by PCR using HSV-1 or insulin-specific primers. DNA from the footpad of a mouse infected 24 h earlier was used as a positive control (+). A no DNA control was included to rule out contamination (−). (C) Mice receiving CFSE-labeled gBT-I.1 cells were infected with HSV-1 by footpad injection or flank scarification, and CD8 + T cells from the PLNs or pooled axillary and inguinal lymph nodes, respectively, were analyzed for the presence of proliferating cells via CFSE intensity at 48 h after infection. Histograms represent 5–10,000 live events.

    Techniques Used: Activation Assay, Mouse Assay, Infection, Isolation, Amplification, Polymerase Chain Reaction, Positive Control, Labeling, Injection

    Activation of CD8 + T cells in the PLNs correlates with the appearance of specific antigen presentation. (A) Mice adoptively transferred with CFSE-labeled gBT-I.1 cells were killed at various times (2–8 h) after footpad infection with HSV-1 and CD8 + CFSE + cells analyzed for the expression of CD69. (B) PLNs from HSV-1–infected C57BL/6 mice were treated with collagenase to form a cell suspension. Graded amounts of these cells were placed into culture with HSV-2.3.2E2 lacZ -inducible hybridoma cells overnight. An X-Gal assay was then performed to stain responding hybridoma cells which were counted microscopically. Numbers presented represent the total number of lacZ + cells per separate PLN at various times (2–48 h) after infection, and error bars represent SD ( n = 8–12).
    Figure Legend Snippet: Activation of CD8 + T cells in the PLNs correlates with the appearance of specific antigen presentation. (A) Mice adoptively transferred with CFSE-labeled gBT-I.1 cells were killed at various times (2–8 h) after footpad infection with HSV-1 and CD8 + CFSE + cells analyzed for the expression of CD69. (B) PLNs from HSV-1–infected C57BL/6 mice were treated with collagenase to form a cell suspension. Graded amounts of these cells were placed into culture with HSV-2.3.2E2 lacZ -inducible hybridoma cells overnight. An X-Gal assay was then performed to stain responding hybridoma cells which were counted microscopically. Numbers presented represent the total number of lacZ + cells per separate PLN at various times (2–48 h) after infection, and error bars represent SD ( n = 8–12).

    Techniques Used: Activation Assay, Mouse Assay, Labeling, Infection, Expressing, Staining

    Concurrent in vivo proliferation and CTL activity by gB-specific CD8 + T cells in the PLNs after cutaneous infection with HSV-1. (A) CFSE-labeled lymph node cells from gBT-I.1 mice were transferred into C57BL/6 mice before infection with HSV-1. PLN cells were isolated at various times after infection (24–72 h) and dilution of the CFSE fluorescence analyzed by gating on live CD8 + T cells. (B) Cellularity within the draining lymph nodes over a 48-h period was determined using cell suspensions obtained from the PLNs of mice after foot-pad HSV-1 infection. (C) Mice that had (black bars) or had not (white bars) received 10 6 gBT-I.1 cells 24 h earlier were infected with HSV-1 in the footpad and left for various times as shown before intravenous transfer of CFSE-labeled syngeneic target cells. gB-peptide–pulsed splenocytes were labeled with a high concentration of CFSE (CFSE hi ) while unpulsed control targets were labeled with a low concentration of CFSE (CFSE lo ). 4 h after target cell transfer, mice were killed and PLN cells analyzed for relative elimination of the CFSE hi versus CFSE lo populations. Percent specific lysis was calculated as described in reference 5. Error bars represent SD.
    Figure Legend Snippet: Concurrent in vivo proliferation and CTL activity by gB-specific CD8 + T cells in the PLNs after cutaneous infection with HSV-1. (A) CFSE-labeled lymph node cells from gBT-I.1 mice were transferred into C57BL/6 mice before infection with HSV-1. PLN cells were isolated at various times after infection (24–72 h) and dilution of the CFSE fluorescence analyzed by gating on live CD8 + T cells. (B) Cellularity within the draining lymph nodes over a 48-h period was determined using cell suspensions obtained from the PLNs of mice after foot-pad HSV-1 infection. (C) Mice that had (black bars) or had not (white bars) received 10 6 gBT-I.1 cells 24 h earlier were infected with HSV-1 in the footpad and left for various times as shown before intravenous transfer of CFSE-labeled syngeneic target cells. gB-peptide–pulsed splenocytes were labeled with a high concentration of CFSE (CFSE hi ) while unpulsed control targets were labeled with a low concentration of CFSE (CFSE lo ). 4 h after target cell transfer, mice were killed and PLN cells analyzed for relative elimination of the CFSE hi versus CFSE lo populations. Percent specific lysis was calculated as described in reference 5. Error bars represent SD.

    Techniques Used: In Vivo, CTL Assay, Activity Assay, Infection, Labeling, Mouse Assay, Isolation, Fluorescence, Concentration Assay, Lysis

    De novo synthesis of viral peptides is required to elicit a gB-specific T cell response. Mice receiving CFSE-labeled gBT-I.1 CD8 + T cells were infected with wild-type HSV-1 KOS or the gB mutant strains KΔ318 or KΔ5C. 42 h after infection, CD8 + T cells from draining PLNs were analyzed for dilution of the CFSE stain caused by cell division. Histograms represent 5–10,000 live events.
    Figure Legend Snippet: De novo synthesis of viral peptides is required to elicit a gB-specific T cell response. Mice receiving CFSE-labeled gBT-I.1 CD8 + T cells were infected with wild-type HSV-1 KOS or the gB mutant strains KΔ318 or KΔ5C. 42 h after infection, CD8 + T cells from draining PLNs were analyzed for dilution of the CFSE stain caused by cell division. Histograms represent 5–10,000 live events.

    Techniques Used: Mouse Assay, Labeling, Infection, Mutagenesis, Staining

    3) Product Images from "Sodium-myoinositol cotransporter-1, SMIT1, mediates the production of reactive oxygen species induced by hyperglycemia in the heart"

    Article Title: Sodium-myoinositol cotransporter-1, SMIT1, mediates the production of reactive oxygen species induced by hyperglycemia in the heart

    Journal: Scientific Reports

    doi: 10.1038/srep41166

    Detection of SGLT isoforms in mouse heart and cardiomyocytes. ( A ) SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from hearts (n = 4) and isolated cardiomyocytes (cardio. n = 4) of mice. Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5 and brain for SGLT6 and SMIT1. mRNA copy number per μg of RNA of SGLT1 ( B ) and SMIT1 ( C ) were measured in mice hearts (n = 4) and cardiomyocytes (n = 4) and compared to a positive control (n = 3). Data are means ± SEM. Statistical analysis was by one-way ANOVA. *Indicates values statistically different from corresponding control tissue, p ≤ 0.05. ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA between hearts (n = 4) and cardiomyocytes (n = 4). Data were normalized to ribosomal protein L32 (RPL32) and expressed as Log10 copy numbers/μg RNA. ( E ) SMIT1 protein expression in murine heart compared to murine brain. eEF-2 detection is used as loading control. Full-length blots are presented in Supplementary Figure 5 .
    Figure Legend Snippet: Detection of SGLT isoforms in mouse heart and cardiomyocytes. ( A ) SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from hearts (n = 4) and isolated cardiomyocytes (cardio. n = 4) of mice. Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5 and brain for SGLT6 and SMIT1. mRNA copy number per μg of RNA of SGLT1 ( B ) and SMIT1 ( C ) were measured in mice hearts (n = 4) and cardiomyocytes (n = 4) and compared to a positive control (n = 3). Data are means ± SEM. Statistical analysis was by one-way ANOVA. *Indicates values statistically different from corresponding control tissue, p ≤ 0.05. ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA between hearts (n = 4) and cardiomyocytes (n = 4). Data were normalized to ribosomal protein L32 (RPL32) and expressed as Log10 copy numbers/μg RNA. ( E ) SMIT1 protein expression in murine heart compared to murine brain. eEF-2 detection is used as loading control. Full-length blots are presented in Supplementary Figure 5 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Staining, Isolation, Mouse Assay, Positive Control, Expressing

    Impact of SGLT1 deletion on HG-induced NOX2 activation and ROS production. ( A ) LVEDV, ( B ) EF and ( C ) LV mass were measured by echocardiography of SGLT1 WT under usual diet (n = 10), of SGLT1 WT submitted to glucose and galactose free diet (glu/gal free diet, n = 10) and of SGLT1 KO (n = 10) mice. Echocardiographic data in M-mode and 2D parasternal long axis are presented in Supplementary Table 4 . ( D ) Detection of SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from the hearts of SGLT1 KO mice (n = 3) compared to WT mice (n = 3). Positive controls were intestine for SGLT1, kidneys for SGLT2, SGLT3, SGLT4 and SGLT5, brain for SGLT6 and SMIT1. ( E ) Quantification of HG-induced p47phox translocation close to cav3 in SGLT1 WT mice (with and without glu/gal free diet) compared to SGLT1 KO mice. Adult mouse cardiomyocytes were isolated from SGLT1 WT (n = 7), SGLT1 WT submitted to glu/gal free diet (n = 7) and SGLT1 KO (n = 7) hearts. PLA was performed 90 min after stimulation with HG and compared to LG. White lines correspond to 20 μm. ( F ) ROS production induced by 3 h of incubation with HG in cardiomyocytes isolated from SGLT1 WT (n = 6), SGLT1 WT submitted to glu/gal free diet (n = 6) and SGLT1 KO (n = 6) mice. Data are means ± SEM. Statistical analysis was by two-way ANOVA. $ Indicates values statistically different from LG, p ≤ 0.05.
    Figure Legend Snippet: Impact of SGLT1 deletion on HG-induced NOX2 activation and ROS production. ( A ) LVEDV, ( B ) EF and ( C ) LV mass were measured by echocardiography of SGLT1 WT under usual diet (n = 10), of SGLT1 WT submitted to glucose and galactose free diet (glu/gal free diet, n = 10) and of SGLT1 KO (n = 10) mice. Echocardiographic data in M-mode and 2D parasternal long axis are presented in Supplementary Table 4 . ( D ) Detection of SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from the hearts of SGLT1 KO mice (n = 3) compared to WT mice (n = 3). Positive controls were intestine for SGLT1, kidneys for SGLT2, SGLT3, SGLT4 and SGLT5, brain for SGLT6 and SMIT1. ( E ) Quantification of HG-induced p47phox translocation close to cav3 in SGLT1 WT mice (with and without glu/gal free diet) compared to SGLT1 KO mice. Adult mouse cardiomyocytes were isolated from SGLT1 WT (n = 7), SGLT1 WT submitted to glu/gal free diet (n = 7) and SGLT1 KO (n = 7) hearts. PLA was performed 90 min after stimulation with HG and compared to LG. White lines correspond to 20 μm. ( F ) ROS production induced by 3 h of incubation with HG in cardiomyocytes isolated from SGLT1 WT (n = 6), SGLT1 WT submitted to glu/gal free diet (n = 6) and SGLT1 KO (n = 6) mice. Data are means ± SEM. Statistical analysis was by two-way ANOVA. $ Indicates values statistically different from LG, p ≤ 0.05.

    Techniques Used: Activation Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Translocation Assay, Isolation, Proximity Ligation Assay, Incubation

    Impact of SMIT1 overexpression on NOX2 activation and ROS production. Adult rat cardiomyocytes were infected with adenoviruses (24 h, 200 MOI) expressing SMIT1 (Ad-SMIT1) or β-galactosidase (Ad-Ctl). ( A ) SMIT1 mRNA level measured by qRT-PCR (n = 3). Data were normalized to HPRT1 and expressed as relative expression vs Ad-Ctl. ( B ) SMIT1 protein expression in plasma membrane fractions obtained after cellular fractionation. Full-length blots are presented in Supplementary Figure 6 . ( C ) Quantification of picomoles myo-[3 H]inositol uptake per min and mg of proteins (n = 4). ( D ) p47phox translocation close to cav3 (n = 6) and ( E ) ROS production (n = 7) in response to increased glucose concentration (5–10 and 21 mM of glucose). ( F ) Gp91dstat and scrambled peptide were added 15 min prior to glucose (5 or 10 mM glucose). ROS production was quantified 2 h after change in glucose concentration (n = 3). Data are means ± SEM. Statistical analysis was by ( A–C ) Student’s t-test or ( D,E,F ) two-way ANOVA. $ Indicates values statistically different from LG, p ≤ 0.05. *Indicates values statistically different from ( A–E ) Ad-Ctl, and ( F ) scr, p ≤ 0.05.
    Figure Legend Snippet: Impact of SMIT1 overexpression on NOX2 activation and ROS production. Adult rat cardiomyocytes were infected with adenoviruses (24 h, 200 MOI) expressing SMIT1 (Ad-SMIT1) or β-galactosidase (Ad-Ctl). ( A ) SMIT1 mRNA level measured by qRT-PCR (n = 3). Data were normalized to HPRT1 and expressed as relative expression vs Ad-Ctl. ( B ) SMIT1 protein expression in plasma membrane fractions obtained after cellular fractionation. Full-length blots are presented in Supplementary Figure 6 . ( C ) Quantification of picomoles myo-[3 H]inositol uptake per min and mg of proteins (n = 4). ( D ) p47phox translocation close to cav3 (n = 6) and ( E ) ROS production (n = 7) in response to increased glucose concentration (5–10 and 21 mM of glucose). ( F ) Gp91dstat and scrambled peptide were added 15 min prior to glucose (5 or 10 mM glucose). ROS production was quantified 2 h after change in glucose concentration (n = 3). Data are means ± SEM. Statistical analysis was by ( A–C ) Student’s t-test or ( D,E,F ) two-way ANOVA. $ Indicates values statistically different from LG, p ≤ 0.05. *Indicates values statistically different from ( A–E ) Ad-Ctl, and ( F ) scr, p ≤ 0.05.

    Techniques Used: Over Expression, Activation Assay, Infection, Expressing, CTL Assay, Quantitative RT-PCR, Cell Fractionation, Translocation Assay, Concentration Assay

    Detection of SGLT isoforms in rat heart and cardiomyocytes. ( A ) SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from hearts (n = 4) and isolated cardiomyocytes (cardio. n = 4) of rats. Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5 and brain for SGLT6 and SMIT1. mRNA copy number per μg of RNA of SGLT1 ( B ) and SMIT1 ( C ) were measured in rat hearts (n = 4) and cardiomyocytes (n = 4) and compared to a positive control (n = 3). Data are means ± SEM. Statistical analysis was by one-way ANOVA. *Indicates values statistically different from corresponding control tissue, p ≤ 0.05. ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA between hearts (n = 4) and cardiomyocytes (n = 4). Data were normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT1) and expressed as Log10 copy numbers/μg RNA. ( E ) SMIT1 protein expression in rat heart compared to rat brain and in isolated rat cardiomyocytes in culture compared to total heart extract. eEF-2 detection is used as loading control. Full-length blots are presented in Supplementary Figure 4 .
    Figure Legend Snippet: Detection of SGLT isoforms in rat heart and cardiomyocytes. ( A ) SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5, SGLT6 and SMIT1 detection by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from hearts (n = 4) and isolated cardiomyocytes (cardio. n = 4) of rats. Positive controls were intestine for SGLT1, kidney for SGLT2, SGLT3, SGLT4 and SGLT5 and brain for SGLT6 and SMIT1. mRNA copy number per μg of RNA of SGLT1 ( B ) and SMIT1 ( C ) were measured in rat hearts (n = 4) and cardiomyocytes (n = 4) and compared to a positive control (n = 3). Data are means ± SEM. Statistical analysis was by one-way ANOVA. *Indicates values statistically different from corresponding control tissue, p ≤ 0.05. ( D ) Comparison of SGLT1 and SMIT1 mRNA copy numbers/μg of RNA between hearts (n = 4) and cardiomyocytes (n = 4). Data were normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT1) and expressed as Log10 copy numbers/μg RNA. ( E ) SMIT1 protein expression in rat heart compared to rat brain and in isolated rat cardiomyocytes in culture compared to total heart extract. eEF-2 detection is used as loading control. Full-length blots are presented in Supplementary Figure 4 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Staining, Isolation, Positive Control, Expressing

    Impact of SMIT1 deletion on HG-induced NOX2 activation and ROS production. ( A ) LVEDV, ( B ) EF and ( C ) LV mass were measured by echocardiography of SMIT1 WT (n = 10) and KO (n = 10) mice. Echocardiographic data in M-mode and 2D parasternal long axis are presented in Supplementary Table 5 . ( D ) Detection of SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5 and SGLT6 by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from the hearts of SMIT1 KO mice (n = 3) compared to WT mice (n = 3). Positive controls were intestine for SGLT1, kidneys for SGLT2, SGLT3, SGLT4 and SGLT5, brain for SGLT6 and SMIT1. ( E ) Quantification of HG-induced p47phox translocation close to cav3 in SMIT1 WT mice compared to SMIT1 KO mice. Adult mouse cardiomyocytes were isolated from SMIT1 WT (n = 4) or SMIT1 KO (n = 4) hearts. PLA was performed 90 min after stimulation with HG and compared to 5 mM of glucose. White lines correspond to 20 μm. ( F ) ROS production induced by 3 h of incubation with HG in cardiomyocytes isolated from SMIT1 WT (n = 6) or SMIT1 KO (n = 6) mice. ( G ) Cardiac glucose uptake in SMIT1 WT (n = 6) vs KO (n = 6) mice was measured under LG, HG and after insulin (3.10 −9 M insulin 30 min). Data are means ± SEM. Statistical analysis was by two-way ANOVA ( E–F ). $ Indicates values statistically different from LG, p ≤ 0.05.
    Figure Legend Snippet: Impact of SMIT1 deletion on HG-induced NOX2 activation and ROS production. ( A ) LVEDV, ( B ) EF and ( C ) LV mass were measured by echocardiography of SMIT1 WT (n = 10) and KO (n = 10) mice. Echocardiographic data in M-mode and 2D parasternal long axis are presented in Supplementary Table 5 . ( D ) Detection of SGLT1, SGLT2, SGLT3b, SGLT4, SGLT5 and SGLT6 by RT-PCR and ethidium bromide-stained agarose gels on mRNA extracted from the hearts of SMIT1 KO mice (n = 3) compared to WT mice (n = 3). Positive controls were intestine for SGLT1, kidneys for SGLT2, SGLT3, SGLT4 and SGLT5, brain for SGLT6 and SMIT1. ( E ) Quantification of HG-induced p47phox translocation close to cav3 in SMIT1 WT mice compared to SMIT1 KO mice. Adult mouse cardiomyocytes were isolated from SMIT1 WT (n = 4) or SMIT1 KO (n = 4) hearts. PLA was performed 90 min after stimulation with HG and compared to 5 mM of glucose. White lines correspond to 20 μm. ( F ) ROS production induced by 3 h of incubation with HG in cardiomyocytes isolated from SMIT1 WT (n = 6) or SMIT1 KO (n = 6) mice. ( G ) Cardiac glucose uptake in SMIT1 WT (n = 6) vs KO (n = 6) mice was measured under LG, HG and after insulin (3.10 −9 M insulin 30 min). Data are means ± SEM. Statistical analysis was by two-way ANOVA ( E–F ). $ Indicates values statistically different from LG, p ≤ 0.05.

    Techniques Used: Activation Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Translocation Assay, Isolation, Proximity Ligation Assay, Incubation

    4) Product Images from "Using the TAP Component of the Antigen-Processing Machinery as a Molecular Adjuvant"

    Article Title: Using the TAP Component of the Antigen-Processing Machinery as a Molecular Adjuvant

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0010036

    A Viral-Challenge Experiment Was Used to Measure the Protection Provided by Low-Dose Vaccination with VV-hTAP1,2 (A) Three groups of mice were vaccinated with escalating doses of VV-hTAP1,2 (3e3 PFU, 3e4 PFU, 3e5 PFU) and were challenged 14 d later with a lethal dose of VV-WR (1e5 PFU). Percentage weight change was measured as an indication of death and morbidity. Three doses of low-dose VV were administered. (B) Three Groups of mice were vaccinated with escalating doses of VV-PJS-5 (3e3 PFU, 3e4 PFU, 3e5 PFU), and were challenged 14 d later with a lethal dose of VV-WR (1e5 PFU). These groups served as negative controls for the effect of VV-hTAP1,2 on protection from lethal viral challenge. Mice vaccinated with PBS served as positive controls for lethal viral challenge. Data points represent mean weight changes ± standard error of the mean ( n = 6) recorded daily.
    Figure Legend Snippet: A Viral-Challenge Experiment Was Used to Measure the Protection Provided by Low-Dose Vaccination with VV-hTAP1,2 (A) Three groups of mice were vaccinated with escalating doses of VV-hTAP1,2 (3e3 PFU, 3e4 PFU, 3e5 PFU) and were challenged 14 d later with a lethal dose of VV-WR (1e5 PFU). Percentage weight change was measured as an indication of death and morbidity. Three doses of low-dose VV were administered. (B) Three Groups of mice were vaccinated with escalating doses of VV-PJS-5 (3e3 PFU, 3e4 PFU, 3e5 PFU), and were challenged 14 d later with a lethal dose of VV-WR (1e5 PFU). These groups served as negative controls for the effect of VV-hTAP1,2 on protection from lethal viral challenge. Mice vaccinated with PBS served as positive controls for lethal viral challenge. Data points represent mean weight changes ± standard error of the mean ( n = 6) recorded daily.

    Techniques Used: Mouse Assay

    Antigen-Specific Tetramer Staining Was Used to Determine T-Cell Responses in Coinfections with VV-hTAP1,2 and VSV The percentage of CD8 + splenocytes specific for H-2K b –VSV-NP 52–59 was determined by flow cytometry using double labeling with an anti-CD8 + antibody and a VSV-NP–specific tetramer. The value in the upper-right quadrant of the scatter-plots represents the percentage of CD8 + cells specific for H-2K b –VSV-NP 52–59 for mice infected with a low dose of VSV and VV-hTAP1,2. The mice coinfected with both VSV and VV-PJS-5 or with a low dose of VSV, or uninfected mice, were used as negative controls for VV-hTAP1,2. The mice infected with a high dose of VSV alone were used as a positive control.
    Figure Legend Snippet: Antigen-Specific Tetramer Staining Was Used to Determine T-Cell Responses in Coinfections with VV-hTAP1,2 and VSV The percentage of CD8 + splenocytes specific for H-2K b –VSV-NP 52–59 was determined by flow cytometry using double labeling with an anti-CD8 + antibody and a VSV-NP–specific tetramer. The value in the upper-right quadrant of the scatter-plots represents the percentage of CD8 + cells specific for H-2K b –VSV-NP 52–59 for mice infected with a low dose of VSV and VV-hTAP1,2. The mice coinfected with both VSV and VV-PJS-5 or with a low dose of VSV, or uninfected mice, were used as negative controls for VV-hTAP1,2. The mice infected with a high dose of VSV alone were used as a positive control.

    Techniques Used: Staining, Flow Cytometry, Cytometry, Labeling, Mouse Assay, Infection, Positive Control

    VV-hTAP1,2 Increases Antigen Presentation and Immune Responses to SV and VV in Mice (A) A standard chromium-release assay was used to determine the ability of VV-hTAP1,2 to increase immune responses to SV. RMA cells pulsed with SV-NP peptides were used as targets, and effectors were obtained from the mice coinfected with a low dose of SV and VV-hTAP1,2. The mice coinfected with a low dose of SV and VV-PJS-5 or with a low dose of SV alone were used as negative controls. Effectors from the mice infected with a high dose of SV were used as positive controls for maximal SV-specific CTL activity. (B) A standard chromium-release assay was used to determine the ability of VV-hTAP1,2 to stimulate VV-specific CTL responses. RMA cells infected with VV-PJS-5 were used as targets, and effectors were obtained from the mice vaccinated with a low dose of VV-hTAP1,2. Effectors from the mice vaccinated with an equivalent low dose of VV-PJS-5 were used as negative controls, and effectors from the mice vaccinated with a high dose of VV-PJS-5 were used as positive controls for maximal CTL activity. (C) A standard chromium-release assay was used to measure the ability of human TAP expression to increase antigen presentation in normal mouse splenocytes. Naïve splenocytes, which had been stimulated overnight with LPS (LPS blasts) and infected with VV-hTAP1,2, were used as targets for VV-specific effectors; VV-specific effectors were obtained from mice infected with VV-PJS-5. LPS blasts infected with VV-PJS-5 were used as negative controls. Values represent mean of triplicate measurements ± standard error of the mean.
    Figure Legend Snippet: VV-hTAP1,2 Increases Antigen Presentation and Immune Responses to SV and VV in Mice (A) A standard chromium-release assay was used to determine the ability of VV-hTAP1,2 to increase immune responses to SV. RMA cells pulsed with SV-NP peptides were used as targets, and effectors were obtained from the mice coinfected with a low dose of SV and VV-hTAP1,2. The mice coinfected with a low dose of SV and VV-PJS-5 or with a low dose of SV alone were used as negative controls. Effectors from the mice infected with a high dose of SV were used as positive controls for maximal SV-specific CTL activity. (B) A standard chromium-release assay was used to determine the ability of VV-hTAP1,2 to stimulate VV-specific CTL responses. RMA cells infected with VV-PJS-5 were used as targets, and effectors were obtained from the mice vaccinated with a low dose of VV-hTAP1,2. Effectors from the mice vaccinated with an equivalent low dose of VV-PJS-5 were used as negative controls, and effectors from the mice vaccinated with a high dose of VV-PJS-5 were used as positive controls for maximal CTL activity. (C) A standard chromium-release assay was used to measure the ability of human TAP expression to increase antigen presentation in normal mouse splenocytes. Naïve splenocytes, which had been stimulated overnight with LPS (LPS blasts) and infected with VV-hTAP1,2, were used as targets for VV-specific effectors; VV-specific effectors were obtained from mice infected with VV-PJS-5. LPS blasts infected with VV-PJS-5 were used as negative controls. Values represent mean of triplicate measurements ± standard error of the mean.

    Techniques Used: Mouse Assay, Release Assay, Infection, CTL Assay, Activity Assay, Expressing

    The Effect of VV-hTAP1,2 and VV-mTAP1 Infection on the Cross-Presentation Activity of OVA/SIINFEKL by Normal Spleen-Derived DCs DCs infected with VV-hTAP1,2 expressed greater (A) H-2K b –SIINFEKL and (B) total H-2K b than DCs infected with VV-PJS-5. DCs infected with VV-mTAP1 also expressed greater (C) H-2K b –SIINFEKL and (D) total H-2K b than DCs infected with VV-PJS-5. DCs infected with VV-PJS-5, but not incubated with OVA, served as negative controls for cross-presentation. The data are representative of the experiment performed in duplicate.
    Figure Legend Snippet: The Effect of VV-hTAP1,2 and VV-mTAP1 Infection on the Cross-Presentation Activity of OVA/SIINFEKL by Normal Spleen-Derived DCs DCs infected with VV-hTAP1,2 expressed greater (A) H-2K b –SIINFEKL and (B) total H-2K b than DCs infected with VV-PJS-5. DCs infected with VV-mTAP1 also expressed greater (C) H-2K b –SIINFEKL and (D) total H-2K b than DCs infected with VV-PJS-5. DCs infected with VV-PJS-5, but not incubated with OVA, served as negative controls for cross-presentation. The data are representative of the experiment performed in duplicate.

    Techniques Used: Infection, Activity Assay, Derivative Assay, Incubation

    Human TAP Expression and Activity Was Determined in Splenocytes 24 h after the Mice Were Infected with VV-hTAP1,2 (A) Human TAP1 protein expression in mouse splenocytes was determined by Western blot. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression. (B) The expression of human TAP1 and human TAP2 was detected by RT-PCR 24 h after the mice were infected with VV-hTAP1,2. The mice infected with VV-PJS-5 were negative for human TAP1 and TAP2. (C) Immunofluorescence visualized with confocal microscopy identified human TAP1 expression in antigen-presenting splenocytes isolated from mice 24 h after infection with VV-hTAP1,2. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression (green fluorescence) (I). Cell-surface markers (red fluorescence) identified cell types. Representative images show the following cell types: (I) B cell from a mouse infected with VV-PJS-5 (negative control); (II) B cell that is positive for human TAP1; (III) macrophage that is positive for human TAP1; and (IV) DC that is positive for human TAP1. (D) ATP-dependent TAP activity was measured in splenocytes taken 24 h after the mice were infected with VV-hTAP1,2 or VV-PJS-5 (negative control). Active transport activity was measured in the presence or absence of ATP by a peptide-transport assay that determined the translocation of radioactive peptides from the cytosol into the ER. Normal uninfected mice, uninfected TAP −/− mice, and mice infected with VV-PJS-5 were used as negative controls when assessing the effect of VV-hTAP1,2 infections on peptide-transport activity. The bars represent the mean value ± standard error of the mean of triplicate measurements. The data are representative of the experiment performed in duplicate.
    Figure Legend Snippet: Human TAP Expression and Activity Was Determined in Splenocytes 24 h after the Mice Were Infected with VV-hTAP1,2 (A) Human TAP1 protein expression in mouse splenocytes was determined by Western blot. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression. (B) The expression of human TAP1 and human TAP2 was detected by RT-PCR 24 h after the mice were infected with VV-hTAP1,2. The mice infected with VV-PJS-5 were negative for human TAP1 and TAP2. (C) Immunofluorescence visualized with confocal microscopy identified human TAP1 expression in antigen-presenting splenocytes isolated from mice 24 h after infection with VV-hTAP1,2. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression (green fluorescence) (I). Cell-surface markers (red fluorescence) identified cell types. Representative images show the following cell types: (I) B cell from a mouse infected with VV-PJS-5 (negative control); (II) B cell that is positive for human TAP1; (III) macrophage that is positive for human TAP1; and (IV) DC that is positive for human TAP1. (D) ATP-dependent TAP activity was measured in splenocytes taken 24 h after the mice were infected with VV-hTAP1,2 or VV-PJS-5 (negative control). Active transport activity was measured in the presence or absence of ATP by a peptide-transport assay that determined the translocation of radioactive peptides from the cytosol into the ER. Normal uninfected mice, uninfected TAP −/− mice, and mice infected with VV-PJS-5 were used as negative controls when assessing the effect of VV-hTAP1,2 infections on peptide-transport activity. The bars represent the mean value ± standard error of the mean of triplicate measurements. The data are representative of the experiment performed in duplicate.

    Techniques Used: Expressing, Activity Assay, Mouse Assay, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Confocal Microscopy, Isolation, Fluorescence, Negative Control, Transport Assay, Translocation Assay

    VV-hTAP1,2 Restores Antigen Processing in the TAP-Deficient Cell Line T2-K b and Increases Immune Responses to VSV (A) A standard chromium-release assay was performed to establish the ability of VV-hTAP1,2 to restore antigen processing in the TAP-deficient cell line T2-K b . T2-K b cells coinfected with VV-hTAP1,2 and VV-NP-VSV were used as targets, and splenocytes from VSV-infected mice were used as effectors. Targets coinfected with both VV-PJS-5 and VV-NP-VSV or infected with VV-NP-VSV alone, or uninfected cells, were used as negative controls for VV-hTAP1,2. (B) A standard chromium-release assay was performed to measure the ability of VV-hTAP1,2 to increase specific CTL activity in immunized mice. RMA cells pulsed with VSV-NP 55–59 peptide were used as targets, and effectors were obtained from mice coinfected with VV-hTAP1,2 and low-dose VSV. Effectors from mice coinfected with VSV and VV-PJS-5 or a low dose of VSV alone were used as negative controls for the presence of VV-hTAP1,2 in the coinfections. Effectors from mice infected with a high dose of VSV demonstrated maximal CTL activity and were used as a positive control. (C) A standard chromium-release assay was used to confirm that the increase in immune responses was due to TAP-dependent transport of NP-VSV rather than to nonspecific effects of VV infection on antigen processing. RMA cells pulsed with VSV-NP 55–59 peptide were used as targets, and effectors were obtained from mice coinfected with VV-hTAP1,2 and VV-NP-VSV. Effectors from mice infected with a high dose of VSV were used as positive controls for maximal CTL activity. Effectors from mice coinfected with VV-PJS-5 and VV-NP-VSV or from mice infected with VV-NP-VSV alone were negative controls for the presence of VV-hTAP1,2. Values represent the mean of triplicate measurements ± standard error of the mean.
    Figure Legend Snippet: VV-hTAP1,2 Restores Antigen Processing in the TAP-Deficient Cell Line T2-K b and Increases Immune Responses to VSV (A) A standard chromium-release assay was performed to establish the ability of VV-hTAP1,2 to restore antigen processing in the TAP-deficient cell line T2-K b . T2-K b cells coinfected with VV-hTAP1,2 and VV-NP-VSV were used as targets, and splenocytes from VSV-infected mice were used as effectors. Targets coinfected with both VV-PJS-5 and VV-NP-VSV or infected with VV-NP-VSV alone, or uninfected cells, were used as negative controls for VV-hTAP1,2. (B) A standard chromium-release assay was performed to measure the ability of VV-hTAP1,2 to increase specific CTL activity in immunized mice. RMA cells pulsed with VSV-NP 55–59 peptide were used as targets, and effectors were obtained from mice coinfected with VV-hTAP1,2 and low-dose VSV. Effectors from mice coinfected with VSV and VV-PJS-5 or a low dose of VSV alone were used as negative controls for the presence of VV-hTAP1,2 in the coinfections. Effectors from mice infected with a high dose of VSV demonstrated maximal CTL activity and were used as a positive control. (C) A standard chromium-release assay was used to confirm that the increase in immune responses was due to TAP-dependent transport of NP-VSV rather than to nonspecific effects of VV infection on antigen processing. RMA cells pulsed with VSV-NP 55–59 peptide were used as targets, and effectors were obtained from mice coinfected with VV-hTAP1,2 and VV-NP-VSV. Effectors from mice infected with a high dose of VSV were used as positive controls for maximal CTL activity. Effectors from mice coinfected with VV-PJS-5 and VV-NP-VSV or from mice infected with VV-NP-VSV alone were negative controls for the presence of VV-hTAP1,2. Values represent the mean of triplicate measurements ± standard error of the mean.

    Techniques Used: Release Assay, Infection, Mouse Assay, CTL Assay, Activity Assay, Positive Control

    5) Product Images from "Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway"

    Article Title: Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway

    Journal: Cellular and Molecular Immunology

    doi: 10.1038/cmi.2016.23

    The OVA-Texo vaccine converts CTL exhaustion through CD40L signaling via the mTORC1 pathway. ( a ) AdVova-infected C57BL/6 mice were immunized with OVA-Texo 60 days after primary infection. Prior to and 4 days post immunization, mouse peripheral blood was analyzed for OVA-specific CTLs by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total CD8 + T-cell population. ( b ) The percentage of IFN-γ producing cells in the PE-tetramer + and FITC-CD8 + T-cell population was analyzed in each treatment group. ( c ) The mouse splenocytes from ( a ) were triple-stained with PE-Tetramer, FITC-CD8 and PE/Cy5-labeled Abs. The OVA-specific CD8 + T cells with positive PE-tetramer and FITC-CD8 staining were gated (arrow) and assessed for the expression of pAKT, pelF4E, pS6, T-bet and Ki67 (solid lines on the right). The mean fluorescence intensity (MFI) numbers of solid lines are indicated. Dotted lines (on the left) represent isotype-matched controls. The MFI numbers of the dotted lines in the upper panels were similar to those in the lower panels. ( d ) Rapamycin-treated or untreated CTLs purified from chronically AdVova-infected B6 mice were transferred to B6 mice with AdV Gal -induced chronic infection, followed by OVA-Texo vaccination 1 day post transfer. The OVA-specific CTLs were detected by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T-cell population. * P
    Figure Legend Snippet: The OVA-Texo vaccine converts CTL exhaustion through CD40L signaling via the mTORC1 pathway. ( a ) AdVova-infected C57BL/6 mice were immunized with OVA-Texo 60 days after primary infection. Prior to and 4 days post immunization, mouse peripheral blood was analyzed for OVA-specific CTLs by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total CD8 + T-cell population. ( b ) The percentage of IFN-γ producing cells in the PE-tetramer + and FITC-CD8 + T-cell population was analyzed in each treatment group. ( c ) The mouse splenocytes from ( a ) were triple-stained with PE-Tetramer, FITC-CD8 and PE/Cy5-labeled Abs. The OVA-specific CD8 + T cells with positive PE-tetramer and FITC-CD8 staining were gated (arrow) and assessed for the expression of pAKT, pelF4E, pS6, T-bet and Ki67 (solid lines on the right). The mean fluorescence intensity (MFI) numbers of solid lines are indicated. Dotted lines (on the left) represent isotype-matched controls. The MFI numbers of the dotted lines in the upper panels were similar to those in the lower panels. ( d ) Rapamycin-treated or untreated CTLs purified from chronically AdVova-infected B6 mice were transferred to B6 mice with AdV Gal -induced chronic infection, followed by OVA-Texo vaccination 1 day post transfer. The OVA-specific CTLs were detected by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T-cell population. * P

    Techniques Used: CTL Assay, Infection, Mouse Assay, Flow Cytometry, Cytometry, Staining, Labeling, Expressing, Fluorescence, Purification

    OVA-Texo counteracts CD8 + T-cell anergy via CD40L signaling. AdV Gal -infected C57BL/6 mice were immunized with ( a ) the OVA-Texo vaccine or the OVA-Texo vaccine deficient for one of several molecules or ( b ) the OVA-Texo vaccine plus anti-CD40L antibody treatment. Six days after the immunization, mouse peripheral blood samples were stained with PE-tetramer and FITC-CD8, and analyzed for the assessment of OVA-specific CTLs by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T-cell population. * P
    Figure Legend Snippet: OVA-Texo counteracts CD8 + T-cell anergy via CD40L signaling. AdV Gal -infected C57BL/6 mice were immunized with ( a ) the OVA-Texo vaccine or the OVA-Texo vaccine deficient for one of several molecules or ( b ) the OVA-Texo vaccine plus anti-CD40L antibody treatment. Six days after the immunization, mouse peripheral blood samples were stained with PE-tetramer and FITC-CD8, and analyzed for the assessment of OVA-specific CTLs by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T-cell population. * P

    Techniques Used: Infection, Mouse Assay, Staining, Flow Cytometry, Cytometry

    6) Product Images from "Essential role of the Na+-Ca2+ exchanger (NCX) in glutamate-enhanced cell survival in cardiac cells exposed to hypoxia/reoxygenation"

    Article Title: Essential role of the Na+-Ca2+ exchanger (NCX) in glutamate-enhanced cell survival in cardiac cells exposed to hypoxia/reoxygenation

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13478-x

    Effect of NCX inhibition on glutamate-induced protection against H/R injury. Extracellular LDH activity measured 5 h after the hypoxic insult (3 h) in H9c2 cells ( a , b ) and 2 h after the hypoxic insult (1.5 h) in rat adult cardiomyocytes ( d ) in different experimental conditions. 1 mM glutamate, alone or in combination with 1 μM SN-6, was added during the reoxygenation phase. Differences among means were assessed by one-way ANOVA followed by Dunnet’s post hoc test. Each column represents the mean ± S.E.M. of almost 5 independent experiments performed in duplicate. ( a ) *p
    Figure Legend Snippet: Effect of NCX inhibition on glutamate-induced protection against H/R injury. Extracellular LDH activity measured 5 h after the hypoxic insult (3 h) in H9c2 cells ( a , b ) and 2 h after the hypoxic insult (1.5 h) in rat adult cardiomyocytes ( d ) in different experimental conditions. 1 mM glutamate, alone or in combination with 1 μM SN-6, was added during the reoxygenation phase. Differences among means were assessed by one-way ANOVA followed by Dunnet’s post hoc test. Each column represents the mean ± S.E.M. of almost 5 independent experiments performed in duplicate. ( a ) *p

    Techniques Used: Inhibition, Activity Assay

    Effect of EAATs inhibition on glutamate-induced protection against H/R injury. Extracellular LDH activity measured 5 h after the hypoxic insult (3 h) in H9c2-NCX1 cells ( a ) and 2 h after the hypoxic insult (1.5 h) in rat adult cardiomyocytes ( b ) in different experimental conditions. 1 mM glutamate, alone or in combination with 300 μM DL-TBOA, was added during the reoxygenation phase. Differences among means were assessed by one-way ANOVA followed by Dunnet’s post hoc test. ( a ) Each column represents the mean ± S.E.M. of almost 6 independent experiments performed in triplicate. *p
    Figure Legend Snippet: Effect of EAATs inhibition on glutamate-induced protection against H/R injury. Extracellular LDH activity measured 5 h after the hypoxic insult (3 h) in H9c2-NCX1 cells ( a ) and 2 h after the hypoxic insult (1.5 h) in rat adult cardiomyocytes ( b ) in different experimental conditions. 1 mM glutamate, alone or in combination with 300 μM DL-TBOA, was added during the reoxygenation phase. Differences among means were assessed by one-way ANOVA followed by Dunnet’s post hoc test. ( a ) Each column represents the mean ± S.E.M. of almost 6 independent experiments performed in triplicate. *p

    Techniques Used: Inhibition, Activity Assay

    Timeline of the experimental protocols (H/R). Schematic diagram showing the H/R timeline protocol in H9c2 cells ( a ) and in isolated rat adult cardiomyocytes ( b ). Control groups were incubated under normoxic conditions at 37 °C for the entire protocol. Glutamate (1 mM)-alone or in combination with 1 µM SN-6, 300 µM DL-TBOA or 3 μg/ml oligomycin (for ATP experiments conducted in H9c2-NCX1 cells)-was administered during the reoxygenation phase. Cell viability (assessed by extracellular LDH measurement) and ROS were evaluated at the end of the reoxygenation phase in both experimental protocols. ATP content, OCR and ECAR were assessed after the first hour of reoxygenation in H9c2-NCX1 cells. CTL = control; H/R = hypoxia/reoxygenation; G = glutamate.
    Figure Legend Snippet: Timeline of the experimental protocols (H/R). Schematic diagram showing the H/R timeline protocol in H9c2 cells ( a ) and in isolated rat adult cardiomyocytes ( b ). Control groups were incubated under normoxic conditions at 37 °C for the entire protocol. Glutamate (1 mM)-alone or in combination with 1 µM SN-6, 300 µM DL-TBOA or 3 μg/ml oligomycin (for ATP experiments conducted in H9c2-NCX1 cells)-was administered during the reoxygenation phase. Cell viability (assessed by extracellular LDH measurement) and ROS were evaluated at the end of the reoxygenation phase in both experimental protocols. ATP content, OCR and ECAR were assessed after the first hour of reoxygenation in H9c2-NCX1 cells. CTL = control; H/R = hypoxia/reoxygenation; G = glutamate.

    Techniques Used: Isolation, Incubation, CTL Assay

    7) Product Images from "Programming of Schwann Cells by Lats1/2-TAZ/YAP Signaling Drives Malignant Peripheral Nerve Sheath Tumorigenesis"

    Article Title: Programming of Schwann Cells by Lats1/2-TAZ/YAP Signaling Drives Malignant Peripheral Nerve Sheath Tumorigenesis

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2018.01.005

    Genetic TAZ/YAP inactivation reduces tumor burden and extends life span in Lats1/2-deficient mice (A) qRT-PCR analysis of TAZ/YAP targets between control SNs, Lats1/2-deficient and Lats1/2-def- Taz Lo/Lo Yap1 Lo/Lo paraspinal SNs. Data are as mean ± SEM (n = 3 independent experiments) (B) Kaplan-Meier survival curves for control (n = 38), Lats1/2-deficient (n = 32) and Lats1/2-def- Taz Lo/Lo Yap1 Lo/Lo mice (n = 25). p
    Figure Legend Snippet: Genetic TAZ/YAP inactivation reduces tumor burden and extends life span in Lats1/2-deficient mice (A) qRT-PCR analysis of TAZ/YAP targets between control SNs, Lats1/2-deficient and Lats1/2-def- Taz Lo/Lo Yap1 Lo/Lo paraspinal SNs. Data are as mean ± SEM (n = 3 independent experiments) (B) Kaplan-Meier survival curves for control (n = 38), Lats1/2-deficient (n = 32) and Lats1/2-def- Taz Lo/Lo Yap1 Lo/Lo mice (n = 25). p

    Techniques Used: Mouse Assay, Quantitative RT-PCR

    Mice with Lats1/2 deficiency in the SC lineage develop low and high grade GEM PNST tumors (A–B) Appearance and H E staining of low-grade tumors on the dermis (A) and high-grade GEMPNST tumors from spinal nerve roots (B) from Lats1 fl/fl Lats2 fl/+ ;Dhh -Cre ( Lats1 KO) mice at 3 months. Arrows: atypical nuclei. (C) Spinal nerve root dissection harboring a tumor (arrow). (D) An enlarged sciatic nerve (arrow) from Lats1 KO. Scale bars in A,B, 100 µm; C,D; 2 mm. (E) Appearance and/or H E staining of dermal tumors and paraspinal/nerve-associated GEM-PNST from Lats fl/+ Lats2 fl/fl :Dhh -Cre ( Lats2 KO) at 2 months. Red arrows: tumor masses. Scale bar, 100 µm. (F) Kaplan-Meier survival curves for control (n = 38), Lats1 KO (n = 37) and Lats2 KO (n = 42) mice. Control vs Lats1 or Lats2 KO mice, p
    Figure Legend Snippet: Mice with Lats1/2 deficiency in the SC lineage develop low and high grade GEM PNST tumors (A–B) Appearance and H E staining of low-grade tumors on the dermis (A) and high-grade GEMPNST tumors from spinal nerve roots (B) from Lats1 fl/fl Lats2 fl/+ ;Dhh -Cre ( Lats1 KO) mice at 3 months. Arrows: atypical nuclei. (C) Spinal nerve root dissection harboring a tumor (arrow). (D) An enlarged sciatic nerve (arrow) from Lats1 KO. Scale bars in A,B, 100 µm; C,D; 2 mm. (E) Appearance and/or H E staining of dermal tumors and paraspinal/nerve-associated GEM-PNST from Lats fl/+ Lats2 fl/fl :Dhh -Cre ( Lats2 KO) at 2 months. Red arrows: tumor masses. Scale bar, 100 µm. (F) Kaplan-Meier survival curves for control (n = 38), Lats1 KO (n = 37) and Lats2 KO (n = 42) mice. Control vs Lats1 or Lats2 KO mice, p

    Techniques Used: Mouse Assay, Staining, Dissection

    Lats1/2-deficient SCs are highly tumorigenic (A) A tamoxifen administration scheme to Lats1/2-iDeficient mice. Tumors were harvested (SAC) 9–16 weeks post injection (wpi). (B) Tomato reporter expression in Krox20 + mature SCs in adult sciatic nerves of Plp1 -CreERT at 5 weeks post tamoxifen induction. Scale bar, 50 µm. (C) Formation of a GEM-PNST tumor (arrow) in Lats1/2-iDeficient after tamoxifen-induction at 10 wpi. Scale bar, 2 cm. (D) Kaplan-Meier survival curves for control (n = 7) and Lats1/2-iDeficient (n = 8). Log-rank test was used to calculate p value. (E–F) Dermal (E) or paraspinal/nerve (F) tumors in control (n = 7), Lats1/2-iDeficient (n = 8) mice. Each data point is presented with mean ± SEM (p = 0.0002; Mann-Whitney test). (G) H E and immunostaining for Ki67, Sox10, and TAZ/YAP in a paraspinal tumor from a Lats1/2-iDeficient mouse (9 wpi). Scale bars, 25 µm. (H) Tumor phenotype in Lats1/2-iDeficient mice (n = 7). Low grade tumors are cellular and invading surrounding muscles/fat; high grade tumors are highly cellular, invasive and harbour mitotic figures. (I) Diagram of allograft transplantation with Lats1/2-deficient cells. Bottom: tumor latency per number of transplanted Lats1/2-deficient cells for the primary recipients. (J) Primary tumor growth in nude mice implanted with 1 × 10 5 Lats1/2-deficient cells into the flanks. n = 9 mice. (K) H E-staining and IHC for Ki67, Sox10 or TAZ/YAP of primary Lats1/2-deficient allografts. Scale bar, 50 µm. (L) Secondary tumor growth in nude mice implanted with 1 × 10 6 Lats1/2-deficient tumor cells into the flanks. Data are as mean ± SEM in J and L; n = 10 mice. (M) H E-staining and IHC for Ki67, Sox10 or TAZ/YAP of secondary Lats1/2-deficient allografts. Scale bar, 50 µm. .
    Figure Legend Snippet: Lats1/2-deficient SCs are highly tumorigenic (A) A tamoxifen administration scheme to Lats1/2-iDeficient mice. Tumors were harvested (SAC) 9–16 weeks post injection (wpi). (B) Tomato reporter expression in Krox20 + mature SCs in adult sciatic nerves of Plp1 -CreERT at 5 weeks post tamoxifen induction. Scale bar, 50 µm. (C) Formation of a GEM-PNST tumor (arrow) in Lats1/2-iDeficient after tamoxifen-induction at 10 wpi. Scale bar, 2 cm. (D) Kaplan-Meier survival curves for control (n = 7) and Lats1/2-iDeficient (n = 8). Log-rank test was used to calculate p value. (E–F) Dermal (E) or paraspinal/nerve (F) tumors in control (n = 7), Lats1/2-iDeficient (n = 8) mice. Each data point is presented with mean ± SEM (p = 0.0002; Mann-Whitney test). (G) H E and immunostaining for Ki67, Sox10, and TAZ/YAP in a paraspinal tumor from a Lats1/2-iDeficient mouse (9 wpi). Scale bars, 25 µm. (H) Tumor phenotype in Lats1/2-iDeficient mice (n = 7). Low grade tumors are cellular and invading surrounding muscles/fat; high grade tumors are highly cellular, invasive and harbour mitotic figures. (I) Diagram of allograft transplantation with Lats1/2-deficient cells. Bottom: tumor latency per number of transplanted Lats1/2-deficient cells for the primary recipients. (J) Primary tumor growth in nude mice implanted with 1 × 10 5 Lats1/2-deficient cells into the flanks. n = 9 mice. (K) H E-staining and IHC for Ki67, Sox10 or TAZ/YAP of primary Lats1/2-deficient allografts. Scale bar, 50 µm. (L) Secondary tumor growth in nude mice implanted with 1 × 10 6 Lats1/2-deficient tumor cells into the flanks. Data are as mean ± SEM in J and L; n = 10 mice. (M) H E-staining and IHC for Ki67, Sox10 or TAZ/YAP of secondary Lats1/2-deficient allografts. Scale bar, 50 µm. .

    Techniques Used: Mouse Assay, Injection, Expressing, MANN-WHITNEY, Immunostaining, Transplantation Assay, Staining, Immunohistochemistry

    siRNA-mediated and pharmacological TAZ/YAP inhibition effectively reduces Lats1/2-deficient tumor cell growth in vitro (A) Immunostaining for Ki67 and Sox10 in rat SCs transfected with vectors expressing GFP, TAZ 4SA or YAP S112A for 48 hr. DAPI stains nuclei. Arrowheads: reduced Sox10 immunoreactivity. Scale bars, 50 µm. (B–C) Proportions of Sox10 + (B) and Ki67 + (C) cells among transfected cells. (D) qRT-PCR analysis of HIPPO effectors between si-control (si-ctl) and si-Taz/Yap1 knockdown Lats1/2-deficient paraspinal tumor cells. (E) Immunolabeling for BrdU + (pulse-labeling for 4 hr), Ki67 + and Sox10 + cells among tumor cells dissociated from Lats1/2-deficient paraspinal tumors. Scale bars, 100 µm. (F–G) Proportions of Ki67 + (F) and BrdU + (G) cells among Sox10 + Lats1/2-deficient tumor cells with knockdown of TAZ, YAP or both. (H–I) Quantification of cleaved caspase 3 + (H) and cell density (I) of Lats1/2-deficient tumor cells with combined si-Taz and si-Yap1 knockdown after 72 hr in vitro . (J) Immunolabeling for BrdU + (4 hr pulse), Ki67 + and Sox10 + cells among Lats1/2-deficient tumor cells treated with verteporfin 2 µM or dobutamine 30 µM, and a control drug MK-0752 20 nM for 72 hr in vitro . Scale bars, 50 µm. (K) Immunoblots for YAP and TAZ protein levels in Lats1/2-deficient tumor cells treated with verteporfin for 3 hr. (L–M) Quantification of Ki67 + (L) and BrdU + (M) cells in Lats1/2-deficient tumor cells treated with DMSO, verteporfin (V) 2 µM, dobutamine (D) 30 µM, and MK-0752 20 nM for 72 hr. (N) qRT-PCR analysis of HIPPO effector genes between si-Ctrl and si-TAZ/YAP1 knockdown in SNF02.2 cells. (O) Quantification of BrdU + cells (12 hr pulse) in SNF02.2 cells with si-TAZ/YAP1 knockdown. (P) qRT-PCR analysis of HIPPO effector genes between si-Ctrl and si-TAZ/YAP1 knockdown in SNF96.2 cells. (Q) Quantification of BrdU + cells (12 hr pulse) in SNF96.2 cells with si-TAZ/YAP1 knockdown. Data are as mean ± SEM from at least 3 independent experiments (*p
    Figure Legend Snippet: siRNA-mediated and pharmacological TAZ/YAP inhibition effectively reduces Lats1/2-deficient tumor cell growth in vitro (A) Immunostaining for Ki67 and Sox10 in rat SCs transfected with vectors expressing GFP, TAZ 4SA or YAP S112A for 48 hr. DAPI stains nuclei. Arrowheads: reduced Sox10 immunoreactivity. Scale bars, 50 µm. (B–C) Proportions of Sox10 + (B) and Ki67 + (C) cells among transfected cells. (D) qRT-PCR analysis of HIPPO effectors between si-control (si-ctl) and si-Taz/Yap1 knockdown Lats1/2-deficient paraspinal tumor cells. (E) Immunolabeling for BrdU + (pulse-labeling for 4 hr), Ki67 + and Sox10 + cells among tumor cells dissociated from Lats1/2-deficient paraspinal tumors. Scale bars, 100 µm. (F–G) Proportions of Ki67 + (F) and BrdU + (G) cells among Sox10 + Lats1/2-deficient tumor cells with knockdown of TAZ, YAP or both. (H–I) Quantification of cleaved caspase 3 + (H) and cell density (I) of Lats1/2-deficient tumor cells with combined si-Taz and si-Yap1 knockdown after 72 hr in vitro . (J) Immunolabeling for BrdU + (4 hr pulse), Ki67 + and Sox10 + cells among Lats1/2-deficient tumor cells treated with verteporfin 2 µM or dobutamine 30 µM, and a control drug MK-0752 20 nM for 72 hr in vitro . Scale bars, 50 µm. (K) Immunoblots for YAP and TAZ protein levels in Lats1/2-deficient tumor cells treated with verteporfin for 3 hr. (L–M) Quantification of Ki67 + (L) and BrdU + (M) cells in Lats1/2-deficient tumor cells treated with DMSO, verteporfin (V) 2 µM, dobutamine (D) 30 µM, and MK-0752 20 nM for 72 hr. (N) qRT-PCR analysis of HIPPO effector genes between si-Ctrl and si-TAZ/YAP1 knockdown in SNF02.2 cells. (O) Quantification of BrdU + cells (12 hr pulse) in SNF02.2 cells with si-TAZ/YAP1 knockdown. (P) qRT-PCR analysis of HIPPO effector genes between si-Ctrl and si-TAZ/YAP1 knockdown in SNF96.2 cells. (Q) Quantification of BrdU + cells (12 hr pulse) in SNF96.2 cells with si-TAZ/YAP1 knockdown. Data are as mean ± SEM from at least 3 independent experiments (*p

    Techniques Used: Inhibition, In Vitro, Immunostaining, Transfection, Expressing, Quantitative RT-PCR, CTL Assay, Immunolabeling, Labeling, Western Blot

    TAZ/YAP activation drives an oncogenic growth program in Lats1/2-deficient SCs (A) Isolation of GFP + reporter-expressing SC cells by FACS from dissociated control SNs and Lats1/2-deficient paraspinal or nerve-associated tumors. (B) Heatmap shows differentially expressed genes in Lats1/2-deficient GFP + SCs (n = 6 mice) compared with control GFP + SCs (n = 3 mice). (C) Volcano plot of transcriptome profiles between control GFP + SCs and Lats1/2-deficient GFP + SCs. Red and blue dots represent genes significantly upregulated and downregulated in Lats1/2-deficient GFP + SCs (p
    Figure Legend Snippet: TAZ/YAP activation drives an oncogenic growth program in Lats1/2-deficient SCs (A) Isolation of GFP + reporter-expressing SC cells by FACS from dissociated control SNs and Lats1/2-deficient paraspinal or nerve-associated tumors. (B) Heatmap shows differentially expressed genes in Lats1/2-deficient GFP + SCs (n = 6 mice) compared with control GFP + SCs (n = 3 mice). (C) Volcano plot of transcriptome profiles between control GFP + SCs and Lats1/2-deficient GFP + SCs. Red and blue dots represent genes significantly upregulated and downregulated in Lats1/2-deficient GFP + SCs (p

    Techniques Used: Activation Assay, Isolation, Expressing, FACS, Mouse Assay

    8) Product Images from "Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity"

    Article Title: Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity

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

    doi: 10.1073/pnas.0900969106

    Antigen storage organelles are electron dense with lysosomal characteristics. ( A–C ) Immunoelectron microscopy images of D1 DCs at 48 h after pulse loading with IgG–OVA AlexaFluor488 . Sections were double ImmunoGold labeled for MHC class II ( A ), LAMP1 ( B ), Invariant chain (IC) ( C ), and Alexa Fluor 488 ( A–C ) with gold particle sizes as indicated in nanometers in superscript. AD indicate antigen depots (AD); arrows indicate LAMP1. PM, plasma membrane; M, mitochondrion; EE, early endosome; G, Golgi complex. (Scale bars, 200 nm.)
    Figure Legend Snippet: Antigen storage organelles are electron dense with lysosomal characteristics. ( A–C ) Immunoelectron microscopy images of D1 DCs at 48 h after pulse loading with IgG–OVA AlexaFluor488 . Sections were double ImmunoGold labeled for MHC class II ( A ), LAMP1 ( B ), Invariant chain (IC) ( C ), and Alexa Fluor 488 ( A–C ) with gold particle sizes as indicated in nanometers in superscript. AD indicate antigen depots (AD); arrows indicate LAMP1. PM, plasma membrane; M, mitochondrion; EE, early endosome; G, Golgi complex. (Scale bars, 200 nm.)

    Techniques Used: Immuno-Electron Microscopy, Labeling

    Characterization of antigen-containing compartments by confocal microscopy. ( A–D ) High-resolution confocal images of DCs, 48 h after pulse incubation with IgG–OVA AlexaFluor488 . Cells were fixed, permeabilized, and incubated with Abs specific for LAMP1 ( A ), EEA1 ( B ), MHC class I ( C ), or TAP1 ( D ). Single scans are representative for multiple cells analyzed in at least 2 experiments. Both D1 DCs and BM DCs were used. ( E ) Confocal images of BM DCs derived from the MHC class II-EGFP knock-in mouse 48 h after pulse incubation with IgG–OVA AlexaFluor647 .
    Figure Legend Snippet: Characterization of antigen-containing compartments by confocal microscopy. ( A–D ) High-resolution confocal images of DCs, 48 h after pulse incubation with IgG–OVA AlexaFluor488 . Cells were fixed, permeabilized, and incubated with Abs specific for LAMP1 ( A ), EEA1 ( B ), MHC class I ( C ), or TAP1 ( D ). Single scans are representative for multiple cells analyzed in at least 2 experiments. Both D1 DCs and BM DCs were used. ( E ) Confocal images of BM DCs derived from the MHC class II-EGFP knock-in mouse 48 h after pulse incubation with IgG–OVA AlexaFluor647 .

    Techniques Used: Confocal Microscopy, Incubation, Derivative Assay, Knock-In

    Sustained MHC class I antigen presentation from an internal antigen source in DCs. ( A and B ) In vitro CD8 + T cell activation of DCs at subsequent days after pulse incubation with 20 nM IgG–OVA ( A , black bars) or 0.5 μM TLR ligand-long peptide conjugate (TLRL-PEP) ( B , black bars), compared with equimolar amounts of OVA8 (white bars). Values depicted are relative to day 0. Experiment was repeated twice with similar results in both D1 DCs and BM DCs. ( C–E ) In vitro CD8 + T cell activation by DCs pulse loaded with 10 nM IgG–OVA ( C ), 0.5 μM TLRL-PEP ( D ), or 10 nM OVA8 ( E ). CD8 + T cell activation was assessed 48 h after pulse loading with medium or the different compounds before (black bar) or after treatment with elution buffer (white bar) and after 16 h recovery in the absence of antigen (gray bar). Error bars represent SD of triplicates. Experiments were performed 6 times with similar results in both D1 DCs and BM DCs. ( F ) CD8 + T cell activation by DCs 48 h after pulse loading with medium or after pulse loading with 10 nM IgG–OVA with or without treatment with elution buffer and proteasome inhibitor epoxomicin (epox). CD8 + T cell activation was assessed directly after elution (white bar), or after 2 and 4 h of recovery with or without 5 mM epoxomicin. Experiment was performed 3 times with similar results.
    Figure Legend Snippet: Sustained MHC class I antigen presentation from an internal antigen source in DCs. ( A and B ) In vitro CD8 + T cell activation of DCs at subsequent days after pulse incubation with 20 nM IgG–OVA ( A , black bars) or 0.5 μM TLR ligand-long peptide conjugate (TLRL-PEP) ( B , black bars), compared with equimolar amounts of OVA8 (white bars). Values depicted are relative to day 0. Experiment was repeated twice with similar results in both D1 DCs and BM DCs. ( C–E ) In vitro CD8 + T cell activation by DCs pulse loaded with 10 nM IgG–OVA ( C ), 0.5 μM TLRL-PEP ( D ), or 10 nM OVA8 ( E ). CD8 + T cell activation was assessed 48 h after pulse loading with medium or the different compounds before (black bar) or after treatment with elution buffer (white bar) and after 16 h recovery in the absence of antigen (gray bar). Error bars represent SD of triplicates. Experiments were performed 6 times with similar results in both D1 DCs and BM DCs. ( F ) CD8 + T cell activation by DCs 48 h after pulse loading with medium or after pulse loading with 10 nM IgG–OVA with or without treatment with elution buffer and proteasome inhibitor epoxomicin (epox). CD8 + T cell activation was assessed directly after elution (white bar), or after 2 and 4 h of recovery with or without 5 mM epoxomicin. Experiment was performed 3 times with similar results.

    Techniques Used: In Vitro, Activation Assay, Incubation

    Long-lasting CTL priming capacity of DCs after a short antigen pulse. ( A ) Priming of OVA-specific CTL in mice that were injected i.v. with DCs continuously incubated with 1 μg/mL (20 nM) IgG–OVA for 48 h (c48); or pulse incubated for 1 h with 1 μg/mL IgG–OVA and cultured for 48 h (p48) or 96 h (p96) in the absence of antigen. Each symbol represents the percentage of tetramer (TM)-specific CD8 + T cells per mouse. ( B ) Priming of OVA-specific CTL in mice with matured DCs 48 h after pulse loading with the following: 20 nM of the minimal MHC class I binding peptide SIINFEKL (OVA8) plus 10 μg/mL LPS; 20 nM OVA protein (OVA) plus 10 μg/mL LPS; or 20 nM IgG–OVA. ( C ) In vivo proliferation of CFSE-labeled OVA-specific TCR transgenic CD8 + T cells 2 days after i.v. injection of DCs that were pulse incubated with 1 μg/mL IgG–OVA. ( D ) In vivo T cell proliferation (as in C ) in spleen (black bars) and lymph nodes (gray bars) at different days after i.v. injection of pulse-incubated DC.
    Figure Legend Snippet: Long-lasting CTL priming capacity of DCs after a short antigen pulse. ( A ) Priming of OVA-specific CTL in mice that were injected i.v. with DCs continuously incubated with 1 μg/mL (20 nM) IgG–OVA for 48 h (c48); or pulse incubated for 1 h with 1 μg/mL IgG–OVA and cultured for 48 h (p48) or 96 h (p96) in the absence of antigen. Each symbol represents the percentage of tetramer (TM)-specific CD8 + T cells per mouse. ( B ) Priming of OVA-specific CTL in mice with matured DCs 48 h after pulse loading with the following: 20 nM of the minimal MHC class I binding peptide SIINFEKL (OVA8) plus 10 μg/mL LPS; 20 nM OVA protein (OVA) plus 10 μg/mL LPS; or 20 nM IgG–OVA. ( C ) In vivo proliferation of CFSE-labeled OVA-specific TCR transgenic CD8 + T cells 2 days after i.v. injection of DCs that were pulse incubated with 1 μg/mL IgG–OVA. ( D ) In vivo T cell proliferation (as in C ) in spleen (black bars) and lymph nodes (gray bars) at different days after i.v. injection of pulse-incubated DC.

    Techniques Used: CTL Assay, Mouse Assay, Injection, Incubation, Cell Culture, Binding Assay, In Vivo, Labeling, Transgenic Assay

    MHC class I–peptide complexes are short-lived on DCs compared with stable MHC class II–peptide complexes. ( A ) ( Lower ) Decrease of cell surface MHC class I (black bars) and β-chain of MHC class II (white bars) 3 consecutive days after biotinylation of D1 DCs that were pulse loaded with IgG–OVA 1 day earlier. ( Upper ) Immunoprecipitated MHC class I and II molecules detected by Western blot analysis. This experiment was performed 2 times with similar results. ( B ) Decrease of MHC class I and MHC class II antigen presentation by DCs pulse incubated with minimal peptides. D1 DCs were pretreated for 24 h with 10 μg/mL LPS and pulse incubated for 2 h with 100 ng/mL MHC class I (OVA8) (black bars) and 20 μg/mL MHC class II binding peptides (MuLV19) (white bars) at different days before analysis of specific T cell activation. Experiment was performed 2 times with similar results.
    Figure Legend Snippet: MHC class I–peptide complexes are short-lived on DCs compared with stable MHC class II–peptide complexes. ( A ) ( Lower ) Decrease of cell surface MHC class I (black bars) and β-chain of MHC class II (white bars) 3 consecutive days after biotinylation of D1 DCs that were pulse loaded with IgG–OVA 1 day earlier. ( Upper ) Immunoprecipitated MHC class I and II molecules detected by Western blot analysis. This experiment was performed 2 times with similar results. ( B ) Decrease of MHC class I and MHC class II antigen presentation by DCs pulse incubated with minimal peptides. D1 DCs were pretreated for 24 h with 10 μg/mL LPS and pulse incubated for 2 h with 100 ng/mL MHC class I (OVA8) (black bars) and 20 μg/mL MHC class II binding peptides (MuLV19) (white bars) at different days before analysis of specific T cell activation. Experiment was performed 2 times with similar results.

    Techniques Used: Immunoprecipitation, Western Blot, Incubation, Binding Assay, Activation Assay

    Intracellular conservation of antigen after receptor-mediated uptake. ( A ) Persistence of fluorescence in DCs at subsequent days after pulse incubation with IgG–OVA AlexaFluor488 as measured by flow cytometry. Experiment was performed 4 times with similar results in both D1 DCs and BM DCs. ( B ) Persistence of OVA protein fragments in IgG–OVA AlexaFluor488 pulse-loaded DCs determined by SDS/PAGE visualized directly in gel. Right lanes, Total cell lysates of 2 × 10 5 DCs collected at subsequent days after pulse loading. Left lanes, One or 10 ng of OVA AlexaFluor488 . Experiment was performed 4 times with similar results in both D1 DCs and BM DCs.
    Figure Legend Snippet: Intracellular conservation of antigen after receptor-mediated uptake. ( A ) Persistence of fluorescence in DCs at subsequent days after pulse incubation with IgG–OVA AlexaFluor488 as measured by flow cytometry. Experiment was performed 4 times with similar results in both D1 DCs and BM DCs. ( B ) Persistence of OVA protein fragments in IgG–OVA AlexaFluor488 pulse-loaded DCs determined by SDS/PAGE visualized directly in gel. Right lanes, Total cell lysates of 2 × 10 5 DCs collected at subsequent days after pulse loading. Left lanes, One or 10 ng of OVA AlexaFluor488 . Experiment was performed 4 times with similar results in both D1 DCs and BM DCs.

    Techniques Used: Fluorescence, Incubation, Flow Cytometry, Cytometry, SDS Page

    9) Product Images from "Effect of Oxidative Stress on Homer Scaffolding Proteins"

    Article Title: Effect of Oxidative Stress on Homer Scaffolding Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026128

    Oxidative modification of Homer results in loss of solubility. A) Western blot of lysates from C2C12 myotubes under control conditions (-) and conditions of oxidative stress (+200 µM menadione for 30 min.). Myotubes were lysed in 1% Triton and in the presence of 20 mM n-ethylmaleimide and run under non-reducing (−TCEP) and reducing conditions (+TCEP). B) Quantification of detectable Homer expression under control conditions (CTL) and after exposure to menadione (Mena) by non-reducing Western blot shown in (A). C) The addition of a reducing agent, either 10 mM reduced glutathione or 10 mM DTT, to the media prior to menadione exposure successfully blocked the loss in detectable Homer observed after prolonged (1 hour) exposure to menadione ( Figure 3C ). D) Adult myofibers were exposed to control conditions or oxidative stress by addition of 200 µM H 2 O 2 . Western blot of Homer protein expression showing a decrease in detectable Homer in the Triton soluble fraction in response to oxidative stress and an increase in Homer detected in Triton insoluble fraction. E) Lysates from HEK 293 cells transfected with WT Homer 1b or the double mutant (C246G, C365G) and exposed to intracellular oxidative stress (200 µM menadione for 30 min.) were separated into Triton soluble and insoluble fractions. WT Homer 1b was detected in the Triton insoluble fraction only in response to oxidative stress, but no evidence of the double mutant (C246G, C365G) was detected in the insoluble fraction in response to oxidative stress. F) C2C12 myotubes were exposed to 200 µM menadione for 0, 30, and 60 minutes respectively. Cells were lysed in buffer containing 1% Triton, 8 M guanidine HCL, and 50 mM TCEP. Analysis of whole lysates showed a decrease in detectable Homer over time.
    Figure Legend Snippet: Oxidative modification of Homer results in loss of solubility. A) Western blot of lysates from C2C12 myotubes under control conditions (-) and conditions of oxidative stress (+200 µM menadione for 30 min.). Myotubes were lysed in 1% Triton and in the presence of 20 mM n-ethylmaleimide and run under non-reducing (−TCEP) and reducing conditions (+TCEP). B) Quantification of detectable Homer expression under control conditions (CTL) and after exposure to menadione (Mena) by non-reducing Western blot shown in (A). C) The addition of a reducing agent, either 10 mM reduced glutathione or 10 mM DTT, to the media prior to menadione exposure successfully blocked the loss in detectable Homer observed after prolonged (1 hour) exposure to menadione ( Figure 3C ). D) Adult myofibers were exposed to control conditions or oxidative stress by addition of 200 µM H 2 O 2 . Western blot of Homer protein expression showing a decrease in detectable Homer in the Triton soluble fraction in response to oxidative stress and an increase in Homer detected in Triton insoluble fraction. E) Lysates from HEK 293 cells transfected with WT Homer 1b or the double mutant (C246G, C365G) and exposed to intracellular oxidative stress (200 µM menadione for 30 min.) were separated into Triton soluble and insoluble fractions. WT Homer 1b was detected in the Triton insoluble fraction only in response to oxidative stress, but no evidence of the double mutant (C246G, C365G) was detected in the insoluble fraction in response to oxidative stress. F) C2C12 myotubes were exposed to 200 µM menadione for 0, 30, and 60 minutes respectively. Cells were lysed in buffer containing 1% Triton, 8 M guanidine HCL, and 50 mM TCEP. Analysis of whole lysates showed a decrease in detectable Homer over time.

    Techniques Used: Modification, Solubility, Western Blot, Expressing, CTL Assay, Transfection, Mutagenesis

    10) Product Images from "Modulation of Intracellular Calcium Waves and Triggered Activities by Mitochondrial Ca Flux in Mouse Cardiomyocytes"

    Article Title: Modulation of Intracellular Calcium Waves and Triggered Activities by Mitochondrial Ca Flux in Mouse Cardiomyocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080574

    Effect of FCCP on High [Ca 2+ ] o -induced Ca i 2+ waves. A . FCCP (100nM) induced spontaneous CaWs (indicated by arrows) under normal excitation contraction coupling (ECC). The cells were paced by field stimulation at 0.5 Hz in the presence of 1 mM Ca 2+ concentration under control (ctl) condition and ~ 5 min after perfusion with 100 nM FCCP. B . A Ca i 2+ fluorescence trace recorded from a mouse ventricular myocytes. The cell was first perfused with the normal Tyrode's solution (1 mM Ca 2+ ) and then with a high Ca 2+ Tyrode's solution (4 mM Ca 2+ ). Ca i 2+ waves (CaWs) were consistently observed in the presence of high external Ca 2+ (Ca o 2+ ; 4 mM). Spontaneous Ca 2+ CaW were eliminated by Tetracaine (2 mM). C - a . A representative Ca i 2+ fluorescence trace showing the dose-dependent effects of FCCP on the SCWs. C - b . Effect of FCCP on CaW frequency in a dose-dependent and biphasic manner. C - c . Summary data showing the effect of FCCP on basal Ca i 2+ . ∗ p
    Figure Legend Snippet: Effect of FCCP on High [Ca 2+ ] o -induced Ca i 2+ waves. A . FCCP (100nM) induced spontaneous CaWs (indicated by arrows) under normal excitation contraction coupling (ECC). The cells were paced by field stimulation at 0.5 Hz in the presence of 1 mM Ca 2+ concentration under control (ctl) condition and ~ 5 min after perfusion with 100 nM FCCP. B . A Ca i 2+ fluorescence trace recorded from a mouse ventricular myocytes. The cell was first perfused with the normal Tyrode's solution (1 mM Ca 2+ ) and then with a high Ca 2+ Tyrode's solution (4 mM Ca 2+ ). Ca i 2+ waves (CaWs) were consistently observed in the presence of high external Ca 2+ (Ca o 2+ ; 4 mM). Spontaneous Ca 2+ CaW were eliminated by Tetracaine (2 mM). C - a . A representative Ca i 2+ fluorescence trace showing the dose-dependent effects of FCCP on the SCWs. C - b . Effect of FCCP on CaW frequency in a dose-dependent and biphasic manner. C - c . Summary data showing the effect of FCCP on basal Ca i 2+ . ∗ p

    Techniques Used: Concentration Assay, CTL Assay, Fluorescence

    11) Product Images from "Using the TAP Component of the Antigen-Processing Machinery as a Molecular Adjuvant"

    Article Title: Using the TAP Component of the Antigen-Processing Machinery as a Molecular Adjuvant

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0010036

    A Viral-Challenge Experiment Was Used to Measure the Protection Provided by Low-Dose Vaccination with VV-hTAP1,2 (A) Three groups of mice were vaccinated with escalating doses of VV-hTAP1,2 (3e3 PFU, 3e4 PFU, 3e5 PFU) and were challenged 14 d later with a lethal dose of VV-WR (1e5 PFU). Percentage weight change was measured as an indication of death and morbidity. Three doses of low-dose VV were administered. (B) Three Groups of mice were vaccinated with escalating doses of VV-PJS-5 (3e3 PFU, 3e4 PFU, 3e5 PFU), and were challenged 14 d later with a lethal dose of VV-WR (1e5 PFU). These groups served as negative controls for the effect of VV-hTAP1,2 on protection from lethal viral challenge. Mice vaccinated with PBS served as positive controls for lethal viral challenge. Data points represent mean weight changes ± standard error of the mean ( n = 6) recorded daily.
    Figure Legend Snippet: A Viral-Challenge Experiment Was Used to Measure the Protection Provided by Low-Dose Vaccination with VV-hTAP1,2 (A) Three groups of mice were vaccinated with escalating doses of VV-hTAP1,2 (3e3 PFU, 3e4 PFU, 3e5 PFU) and were challenged 14 d later with a lethal dose of VV-WR (1e5 PFU). Percentage weight change was measured as an indication of death and morbidity. Three doses of low-dose VV were administered. (B) Three Groups of mice were vaccinated with escalating doses of VV-PJS-5 (3e3 PFU, 3e4 PFU, 3e5 PFU), and were challenged 14 d later with a lethal dose of VV-WR (1e5 PFU). These groups served as negative controls for the effect of VV-hTAP1,2 on protection from lethal viral challenge. Mice vaccinated with PBS served as positive controls for lethal viral challenge. Data points represent mean weight changes ± standard error of the mean ( n = 6) recorded daily.

    Techniques Used: Mouse Assay

    Antigen-Specific Tetramer Staining Was Used to Determine T-Cell Responses in Coinfections with VV-hTAP1,2 and VSV The percentage of CD8 + splenocytes specific for H-2K b –VSV-NP 52–59 was determined by flow cytometry using double labeling with an anti-CD8 + antibody and a VSV-NP–specific tetramer. The value in the upper-right quadrant of the scatter-plots represents the percentage of CD8 + cells specific for H-2K b –VSV-NP 52–59 for mice infected with a low dose of VSV and VV-hTAP1,2. The mice coinfected with both VSV and VV-PJS-5 or with a low dose of VSV, or uninfected mice, were used as negative controls for VV-hTAP1,2. The mice infected with a high dose of VSV alone were used as a positive control.
    Figure Legend Snippet: Antigen-Specific Tetramer Staining Was Used to Determine T-Cell Responses in Coinfections with VV-hTAP1,2 and VSV The percentage of CD8 + splenocytes specific for H-2K b –VSV-NP 52–59 was determined by flow cytometry using double labeling with an anti-CD8 + antibody and a VSV-NP–specific tetramer. The value in the upper-right quadrant of the scatter-plots represents the percentage of CD8 + cells specific for H-2K b –VSV-NP 52–59 for mice infected with a low dose of VSV and VV-hTAP1,2. The mice coinfected with both VSV and VV-PJS-5 or with a low dose of VSV, or uninfected mice, were used as negative controls for VV-hTAP1,2. The mice infected with a high dose of VSV alone were used as a positive control.

    Techniques Used: Staining, Flow Cytometry, Cytometry, Labeling, Mouse Assay, Infection, Positive Control

    VV-hTAP1,2 Increases Antigen Presentation and Immune Responses to SV and VV in Mice (A) A standard chromium-release assay was used to determine the ability of VV-hTAP1,2 to increase immune responses to SV. RMA cells pulsed with SV-NP peptides were used as targets, and effectors were obtained from the mice coinfected with a low dose of SV and VV-hTAP1,2. The mice coinfected with a low dose of SV and VV-PJS-5 or with a low dose of SV alone were used as negative controls. Effectors from the mice infected with a high dose of SV were used as positive controls for maximal SV-specific CTL activity. (B) A standard chromium-release assay was used to determine the ability of VV-hTAP1,2 to stimulate VV-specific CTL responses. RMA cells infected with VV-PJS-5 were used as targets, and effectors were obtained from the mice vaccinated with a low dose of VV-hTAP1,2. Effectors from the mice vaccinated with an equivalent low dose of VV-PJS-5 were used as negative controls, and effectors from the mice vaccinated with a high dose of VV-PJS-5 were used as positive controls for maximal CTL activity. (C) A standard chromium-release assay was used to measure the ability of human TAP expression to increase antigen presentation in normal mouse splenocytes. Naïve splenocytes, which had been stimulated overnight with LPS (LPS blasts) and infected with VV-hTAP1,2, were used as targets for VV-specific effectors; VV-specific effectors were obtained from mice infected with VV-PJS-5. LPS blasts infected with VV-PJS-5 were used as negative controls. Values represent mean of triplicate measurements ± standard error of the mean.
    Figure Legend Snippet: VV-hTAP1,2 Increases Antigen Presentation and Immune Responses to SV and VV in Mice (A) A standard chromium-release assay was used to determine the ability of VV-hTAP1,2 to increase immune responses to SV. RMA cells pulsed with SV-NP peptides were used as targets, and effectors were obtained from the mice coinfected with a low dose of SV and VV-hTAP1,2. The mice coinfected with a low dose of SV and VV-PJS-5 or with a low dose of SV alone were used as negative controls. Effectors from the mice infected with a high dose of SV were used as positive controls for maximal SV-specific CTL activity. (B) A standard chromium-release assay was used to determine the ability of VV-hTAP1,2 to stimulate VV-specific CTL responses. RMA cells infected with VV-PJS-5 were used as targets, and effectors were obtained from the mice vaccinated with a low dose of VV-hTAP1,2. Effectors from the mice vaccinated with an equivalent low dose of VV-PJS-5 were used as negative controls, and effectors from the mice vaccinated with a high dose of VV-PJS-5 were used as positive controls for maximal CTL activity. (C) A standard chromium-release assay was used to measure the ability of human TAP expression to increase antigen presentation in normal mouse splenocytes. Naïve splenocytes, which had been stimulated overnight with LPS (LPS blasts) and infected with VV-hTAP1,2, were used as targets for VV-specific effectors; VV-specific effectors were obtained from mice infected with VV-PJS-5. LPS blasts infected with VV-PJS-5 were used as negative controls. Values represent mean of triplicate measurements ± standard error of the mean.

    Techniques Used: Mouse Assay, Release Assay, Infection, CTL Assay, Activity Assay, Expressing

    The Effect of VV-hTAP1,2 and VV-mTAP1 Infection on the Cross-Presentation Activity of OVA/SIINFEKL by Normal Spleen-Derived DCs DCs infected with VV-hTAP1,2 expressed greater (A) H-2K b –SIINFEKL and (B) total H-2K b than DCs infected with VV-PJS-5. DCs infected with VV-mTAP1 also expressed greater (C) H-2K b –SIINFEKL and (D) total H-2K b than DCs infected with VV-PJS-5. DCs infected with VV-PJS-5, but not incubated with OVA, served as negative controls for cross-presentation. The data are representative of the experiment performed in duplicate.
    Figure Legend Snippet: The Effect of VV-hTAP1,2 and VV-mTAP1 Infection on the Cross-Presentation Activity of OVA/SIINFEKL by Normal Spleen-Derived DCs DCs infected with VV-hTAP1,2 expressed greater (A) H-2K b –SIINFEKL and (B) total H-2K b than DCs infected with VV-PJS-5. DCs infected with VV-mTAP1 also expressed greater (C) H-2K b –SIINFEKL and (D) total H-2K b than DCs infected with VV-PJS-5. DCs infected with VV-PJS-5, but not incubated with OVA, served as negative controls for cross-presentation. The data are representative of the experiment performed in duplicate.

    Techniques Used: Infection, Activity Assay, Derivative Assay, Incubation

    Human TAP Expression and Activity Was Determined in Splenocytes 24 h after the Mice Were Infected with VV-hTAP1,2 (A) Human TAP1 protein expression in mouse splenocytes was determined by Western blot. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression. (B) The expression of human TAP1 and human TAP2 was detected by RT-PCR 24 h after the mice were infected with VV-hTAP1,2. The mice infected with VV-PJS-5 were negative for human TAP1 and TAP2. (C) Immunofluorescence visualized with confocal microscopy identified human TAP1 expression in antigen-presenting splenocytes isolated from mice 24 h after infection with VV-hTAP1,2. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression (green fluorescence) (I). Cell-surface markers (red fluorescence) identified cell types. Representative images show the following cell types: (I) B cell from a mouse infected with VV-PJS-5 (negative control); (II) B cell that is positive for human TAP1; (III) macrophage that is positive for human TAP1; and (IV) DC that is positive for human TAP1. (D) ATP-dependent TAP activity was measured in splenocytes taken 24 h after the mice were infected with VV-hTAP1,2 or VV-PJS-5 (negative control). Active transport activity was measured in the presence or absence of ATP by a peptide-transport assay that determined the translocation of radioactive peptides from the cytosol into the ER. Normal uninfected mice, uninfected TAP −/− mice, and mice infected with VV-PJS-5 were used as negative controls when assessing the effect of VV-hTAP1,2 infections on peptide-transport activity. The bars represent the mean value ± standard error of the mean of triplicate measurements. The data are representative of the experiment performed in duplicate.
    Figure Legend Snippet: Human TAP Expression and Activity Was Determined in Splenocytes 24 h after the Mice Were Infected with VV-hTAP1,2 (A) Human TAP1 protein expression in mouse splenocytes was determined by Western blot. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression. (B) The expression of human TAP1 and human TAP2 was detected by RT-PCR 24 h after the mice were infected with VV-hTAP1,2. The mice infected with VV-PJS-5 were negative for human TAP1 and TAP2. (C) Immunofluorescence visualized with confocal microscopy identified human TAP1 expression in antigen-presenting splenocytes isolated from mice 24 h after infection with VV-hTAP1,2. The mice infected with VV-PJS-5 were used as negative controls for human TAP1 expression (green fluorescence) (I). Cell-surface markers (red fluorescence) identified cell types. Representative images show the following cell types: (I) B cell from a mouse infected with VV-PJS-5 (negative control); (II) B cell that is positive for human TAP1; (III) macrophage that is positive for human TAP1; and (IV) DC that is positive for human TAP1. (D) ATP-dependent TAP activity was measured in splenocytes taken 24 h after the mice were infected with VV-hTAP1,2 or VV-PJS-5 (negative control). Active transport activity was measured in the presence or absence of ATP by a peptide-transport assay that determined the translocation of radioactive peptides from the cytosol into the ER. Normal uninfected mice, uninfected TAP −/− mice, and mice infected with VV-PJS-5 were used as negative controls when assessing the effect of VV-hTAP1,2 infections on peptide-transport activity. The bars represent the mean value ± standard error of the mean of triplicate measurements. The data are representative of the experiment performed in duplicate.

    Techniques Used: Expressing, Activity Assay, Mouse Assay, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Confocal Microscopy, Isolation, Fluorescence, Negative Control, Transport Assay, Translocation Assay

    VV-hTAP1,2 Restores Antigen Processing in the TAP-Deficient Cell Line T2-K b and Increases Immune Responses to VSV (A) A standard chromium-release assay was performed to establish the ability of VV-hTAP1,2 to restore antigen processing in the TAP-deficient cell line T2-K b . T2-K b cells coinfected with VV-hTAP1,2 and VV-NP-VSV were used as targets, and splenocytes from VSV-infected mice were used as effectors. Targets coinfected with both VV-PJS-5 and VV-NP-VSV or infected with VV-NP-VSV alone, or uninfected cells, were used as negative controls for VV-hTAP1,2. (B) A standard chromium-release assay was performed to measure the ability of VV-hTAP1,2 to increase specific CTL activity in immunized mice. RMA cells pulsed with VSV-NP 55–59 peptide were used as targets, and effectors were obtained from mice coinfected with VV-hTAP1,2 and low-dose VSV. Effectors from mice coinfected with VSV and VV-PJS-5 or a low dose of VSV alone were used as negative controls for the presence of VV-hTAP1,2 in the coinfections. Effectors from mice infected with a high dose of VSV demonstrated maximal CTL activity and were used as a positive control. (C) A standard chromium-release assay was used to confirm that the increase in immune responses was due to TAP-dependent transport of NP-VSV rather than to nonspecific effects of VV infection on antigen processing. RMA cells pulsed with VSV-NP 55–59 peptide were used as targets, and effectors were obtained from mice coinfected with VV-hTAP1,2 and VV-NP-VSV. Effectors from mice infected with a high dose of VSV were used as positive controls for maximal CTL activity. Effectors from mice coinfected with VV-PJS-5 and VV-NP-VSV or from mice infected with VV-NP-VSV alone were negative controls for the presence of VV-hTAP1,2. Values represent the mean of triplicate measurements ± standard error of the mean.
    Figure Legend Snippet: VV-hTAP1,2 Restores Antigen Processing in the TAP-Deficient Cell Line T2-K b and Increases Immune Responses to VSV (A) A standard chromium-release assay was performed to establish the ability of VV-hTAP1,2 to restore antigen processing in the TAP-deficient cell line T2-K b . T2-K b cells coinfected with VV-hTAP1,2 and VV-NP-VSV were used as targets, and splenocytes from VSV-infected mice were used as effectors. Targets coinfected with both VV-PJS-5 and VV-NP-VSV or infected with VV-NP-VSV alone, or uninfected cells, were used as negative controls for VV-hTAP1,2. (B) A standard chromium-release assay was performed to measure the ability of VV-hTAP1,2 to increase specific CTL activity in immunized mice. RMA cells pulsed with VSV-NP 55–59 peptide were used as targets, and effectors were obtained from mice coinfected with VV-hTAP1,2 and low-dose VSV. Effectors from mice coinfected with VSV and VV-PJS-5 or a low dose of VSV alone were used as negative controls for the presence of VV-hTAP1,2 in the coinfections. Effectors from mice infected with a high dose of VSV demonstrated maximal CTL activity and were used as a positive control. (C) A standard chromium-release assay was used to confirm that the increase in immune responses was due to TAP-dependent transport of NP-VSV rather than to nonspecific effects of VV infection on antigen processing. RMA cells pulsed with VSV-NP 55–59 peptide were used as targets, and effectors were obtained from mice coinfected with VV-hTAP1,2 and VV-NP-VSV. Effectors from mice infected with a high dose of VSV were used as positive controls for maximal CTL activity. Effectors from mice coinfected with VV-PJS-5 and VV-NP-VSV or from mice infected with VV-NP-VSV alone were negative controls for the presence of VV-hTAP1,2. Values represent the mean of triplicate measurements ± standard error of the mean.

    Techniques Used: Release Assay, Infection, Mouse Assay, CTL Assay, Activity Assay, Positive Control

    12) Product Images from "Targeted Programming of the Lymph Node Environment Causes Evolution of Local and Systemic Immunity"

    Article Title: Targeted Programming of the Lymph Node Environment Causes Evolution of Local and Systemic Immunity

    Journal: Cellular and Molecular Bioengineering

    doi: 10.1007/s12195-016-0455-6

    CpG MPs induce superior tumor-specific CTL responses compared to PolyIC MPs. Mice were primed at day 0 i.LN. will either PolyIC MPs or CpG MPs, and either a model antigen (OVA) or a melanoma associated antigen (Trp2) in a soluble form. Mice were boosted at day 21, and antigen-specific MHC-I tetramer was used to measure antigen specific CD8 + T cell responses compared to a sham injection. (a) 7 days after priming, PolyIC and CpG MPs both induced potent levels of SIINFEKL-specific CD8 + , but no differences were observed as a function of TLRa. In the Trp2 model, both PolyIC and CpG MPs increased the levels of Trp2-specific CD8 + T-cells, with CpG exhibiting a statistically significant increase compared to both the sham and PolyIC MP injections. (b) At day 28, 7 days after the boost, a similar response was seen with a robust response in the OVA model for both PolyIC and CpG MPs, but without dependence on the specific TLRa included in the particles. In the Trp2 studies, only CpG MPs induced a significant, potent recall response. (* p
    Figure Legend Snippet: CpG MPs induce superior tumor-specific CTL responses compared to PolyIC MPs. Mice were primed at day 0 i.LN. will either PolyIC MPs or CpG MPs, and either a model antigen (OVA) or a melanoma associated antigen (Trp2) in a soluble form. Mice were boosted at day 21, and antigen-specific MHC-I tetramer was used to measure antigen specific CD8 + T cell responses compared to a sham injection. (a) 7 days after priming, PolyIC and CpG MPs both induced potent levels of SIINFEKL-specific CD8 + , but no differences were observed as a function of TLRa. In the Trp2 model, both PolyIC and CpG MPs increased the levels of Trp2-specific CD8 + T-cells, with CpG exhibiting a statistically significant increase compared to both the sham and PolyIC MP injections. (b) At day 28, 7 days after the boost, a similar response was seen with a robust response in the OVA model for both PolyIC and CpG MPs, but without dependence on the specific TLRa included in the particles. In the Trp2 studies, only CpG MPs induced a significant, potent recall response. (* p

    Techniques Used: CTL Assay, Mouse Assay, Injection

    13) Product Images from "Involvement of Mitochondrial Permeability Transition Pore (mPTP) in Cardiac Arrhythmias: Evidence from Cyclophilin D Knockout Mice"

    Article Title: Involvement of Mitochondrial Permeability Transition Pore (mPTP) in Cardiac Arrhythmias: Evidence from Cyclophilin D Knockout Mice

    Journal: Cell calcium

    doi: 10.1016/j.ceca.2016.09.001

    Lower Incidence of FCCP-Induced ST-T Wave Alternans was Observed in CypD KO Mouse ex-vivo Hearts (A) Pseudo-Lead II ECG signals recorded from Langendorff-perfused WT or CypD KO hearts in the absence (Ctl) and presence of 30 nM FCCP (FCCP). Arrows indicated alternating ST level alternans. (B) Overlapped ECG waveforms; a and b represent individual complexes appearing in the ECG traces as indicated in A. (C) Summarized Data showing incidences of ST alternans in WT and CypD KO hearts. *p
    Figure Legend Snippet: Lower Incidence of FCCP-Induced ST-T Wave Alternans was Observed in CypD KO Mouse ex-vivo Hearts (A) Pseudo-Lead II ECG signals recorded from Langendorff-perfused WT or CypD KO hearts in the absence (Ctl) and presence of 30 nM FCCP (FCCP). Arrows indicated alternating ST level alternans. (B) Overlapped ECG waveforms; a and b represent individual complexes appearing in the ECG traces as indicated in A. (C) Summarized Data showing incidences of ST alternans in WT and CypD KO hearts. *p

    Techniques Used: Ex Vivo, CTL Assay

    Related Articles

    Incubation:

    Article Title: Differential Responses of Human Fetal Brain Neural Stem Cells to Zika Virus Infection
    Article Snippet: .. After a 2-day incubation, 8 × 104 hNSCs (small spheres) were seeded into 24-well plates pre-coated with 0.01% poly-D-lysine and 1 μg/cm2 laminin (Invitrogen/Gibco) and incubated for 4 days with priming medium containing EGF (20 ng/mL), LIF (10 ng/mL), and laminin (1 μg/mL), followed by 9 days incubation with a differentiation medium consisting of N2 plus glutathione (1 μg/mL) (Sigma), biotin (0.1 μg/mL) (Sigma), superoxide dismutase (2.5 μg/mL) (Worthington), DL-α-tocopherol (1 μg/mL) (Sigma), DL-α-tocopherol acetate (1 μg/mL) (Sigma), and catalase (Sigma). .. BrdU Labeling BrdU was acquired from Sigma (catalog no. B9285-16) and prepared in a natural saline solution of 0.9% sodium chloride (Sigma).

    Recombinant:

    Article Title: Racial differences in tumor necrosis factor-?-induced endothelial microparticles and interleukin-6 production
    Article Snippet: .. Experimental procedures Human recombinant TNF-α was purchased from Sigma (St Louis, MO) and SOD was purchased from Worthington Biochemical Corporation (Lakewood, NJ). ..

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    Worthington Biochemical ova specific ctls
    The <t>OVA-Texo</t> vaccine converts CTL exhaustion through CD40L signaling via the mTORC1 pathway. ( a ) AdVova-infected C57BL/6 mice were immunized with OVA-Texo 60 days after primary infection. Prior to and 4 days post immunization, mouse peripheral blood was analyzed for OVA-specific <t>CTLs</t> by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total CD8 + T-cell population. ( b ) The percentage of IFN-γ producing cells in the PE-tetramer + and FITC-CD8 + T-cell population was analyzed in each treatment group. ( c ) The mouse splenocytes from ( a ) were triple-stained with PE-Tetramer, FITC-CD8 and PE/Cy5-labeled Abs. The OVA-specific CD8 + T cells with positive PE-tetramer and FITC-CD8 staining were gated (arrow) and assessed for the expression of pAKT, pelF4E, pS6, T-bet and Ki67 (solid lines on the right). The mean fluorescence intensity (MFI) numbers of solid lines are indicated. Dotted lines (on the left) represent isotype-matched controls. The MFI numbers of the dotted lines in the upper panels were similar to those in the lower panels. ( d ) Rapamycin-treated or untreated CTLs purified from chronically AdVova-infected B6 mice were transferred to B6 mice with AdV Gal -induced chronic infection, followed by OVA-Texo vaccination 1 day post transfer. The OVA-specific CTLs were detected by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T-cell population. * P
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    The OVA-Texo vaccine converts CTL exhaustion through CD40L signaling via the mTORC1 pathway. ( a ) AdVova-infected C57BL/6 mice were immunized with OVA-Texo 60 days after primary infection. Prior to and 4 days post immunization, mouse peripheral blood was analyzed for OVA-specific CTLs by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total CD8 + T-cell population. ( b ) The percentage of IFN-γ producing cells in the PE-tetramer + and FITC-CD8 + T-cell population was analyzed in each treatment group. ( c ) The mouse splenocytes from ( a ) were triple-stained with PE-Tetramer, FITC-CD8 and PE/Cy5-labeled Abs. The OVA-specific CD8 + T cells with positive PE-tetramer and FITC-CD8 staining were gated (arrow) and assessed for the expression of pAKT, pelF4E, pS6, T-bet and Ki67 (solid lines on the right). The mean fluorescence intensity (MFI) numbers of solid lines are indicated. Dotted lines (on the left) represent isotype-matched controls. The MFI numbers of the dotted lines in the upper panels were similar to those in the lower panels. ( d ) Rapamycin-treated or untreated CTLs purified from chronically AdVova-infected B6 mice were transferred to B6 mice with AdV Gal -induced chronic infection, followed by OVA-Texo vaccination 1 day post transfer. The OVA-specific CTLs were detected by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T-cell population. * P

    Journal: Cellular and Molecular Immunology

    Article Title: Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway

    doi: 10.1038/cmi.2016.23

    Figure Lengend Snippet: The OVA-Texo vaccine converts CTL exhaustion through CD40L signaling via the mTORC1 pathway. ( a ) AdVova-infected C57BL/6 mice were immunized with OVA-Texo 60 days after primary infection. Prior to and 4 days post immunization, mouse peripheral blood was analyzed for OVA-specific CTLs by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total CD8 + T-cell population. ( b ) The percentage of IFN-γ producing cells in the PE-tetramer + and FITC-CD8 + T-cell population was analyzed in each treatment group. ( c ) The mouse splenocytes from ( a ) were triple-stained with PE-Tetramer, FITC-CD8 and PE/Cy5-labeled Abs. The OVA-specific CD8 + T cells with positive PE-tetramer and FITC-CD8 staining were gated (arrow) and assessed for the expression of pAKT, pelF4E, pS6, T-bet and Ki67 (solid lines on the right). The mean fluorescence intensity (MFI) numbers of solid lines are indicated. Dotted lines (on the left) represent isotype-matched controls. The MFI numbers of the dotted lines in the upper panels were similar to those in the lower panels. ( d ) Rapamycin-treated or untreated CTLs purified from chronically AdVova-infected B6 mice were transferred to B6 mice with AdV Gal -induced chronic infection, followed by OVA-Texo vaccination 1 day post transfer. The OVA-specific CTLs were detected by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T-cell population. * P

    Article Snippet: To assess OVA-specific CTLs in the spleens and lungs, mouse splenocytes and lung cell suspensions prepared by mincing lung tissues into small fragments and digesting them with collagenase D (1 mg/ml, Worthington Biochemical, Freehold, NJ, USA) at 37 °C for 30 min were stained with FITC-CD8 Ab and PE-tetramer and analyzed by flow cytometry.

    Techniques: CTL Assay, Infection, Mouse Assay, Flow Cytometry, Cytometry, Staining, Labeling, Expressing, Fluorescence, Purification

    OVA-Texo counteracts CD8 + T-cell anergy via CD40L signaling. AdV Gal -infected C57BL/6 mice were immunized with ( a ) the OVA-Texo vaccine or the OVA-Texo vaccine deficient for one of several molecules or ( b ) the OVA-Texo vaccine plus anti-CD40L antibody treatment. Six days after the immunization, mouse peripheral blood samples were stained with PE-tetramer and FITC-CD8, and analyzed for the assessment of OVA-specific CTLs by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T-cell population. * P

    Journal: Cellular and Molecular Immunology

    Article Title: Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway

    doi: 10.1038/cmi.2016.23

    Figure Lengend Snippet: OVA-Texo counteracts CD8 + T-cell anergy via CD40L signaling. AdV Gal -infected C57BL/6 mice were immunized with ( a ) the OVA-Texo vaccine or the OVA-Texo vaccine deficient for one of several molecules or ( b ) the OVA-Texo vaccine plus anti-CD40L antibody treatment. Six days after the immunization, mouse peripheral blood samples were stained with PE-tetramer and FITC-CD8, and analyzed for the assessment of OVA-specific CTLs by flow cytometry. The value in each panel represents the percentage of PE-tetramer-positive CD8 + T cells in the total peripheral CD8 + T-cell population. * P

    Article Snippet: To assess OVA-specific CTLs in the spleens and lungs, mouse splenocytes and lung cell suspensions prepared by mincing lung tissues into small fragments and digesting them with collagenase D (1 mg/ml, Worthington Biochemical, Freehold, NJ, USA) at 37 °C for 30 min were stained with FITC-CD8 Ab and PE-tetramer and analyzed by flow cytometry.

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