rnase treatment ssc ics  (Worthington Biochemical)


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    Neonatal Cardiomyocyte Isolation System
    Description:
    Kit for performing five separate tissue dissociations each containing up to twelve hearts Contains single use vials of purified collagenase and trypsin CMF HBSS Leibovitz L 15 media and Falcon cell strainers along with a detailed protocol The kit is use tested by Worthington to assure performance
    Catalog Number:
    lk003300
    Price:
    256
    Size:
    1 kt
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    see components
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    Structured Review

    Worthington Biochemical rnase treatment ssc ics
    et-1 , ifn-α and ifn-β mRNA expression levels in fibroblasts stimulated with <t>SSc-ICs</t> or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a et-1 ; b ifn-α ; c ifn-β . * p
    Kit for performing five separate tissue dissociations each containing up to twelve hearts Contains single use vials of purified collagenase and trypsin CMF HBSS Leibovitz L 15 media and Falcon cell strainers along with a detailed protocol The kit is use tested by Worthington to assure performance
    https://www.bioz.com/result/rnase treatment ssc ics/product/Worthington Biochemical
    Average 92 stars, based on 1245 article reviews
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    rnase treatment ssc ics - by Bioz Stars, 2020-10
    92/100 stars

    Images

    1) Product Images from "Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts"

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1689-6

    et-1 , ifn-α and ifn-β mRNA expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a et-1 ; b ifn-α ; c ifn-β . * p
    Figure Legend Snippet: et-1 , ifn-α and ifn-β mRNA expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a et-1 ; b ifn-α ; c ifn-β . * p

    Techniques Used: Expressing, Concentration Assay

    Intracellular signaling pathways in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. * p
    Figure Legend Snippet: Intracellular signaling pathways in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. * p

    Techniques Used: Software, Western Blot

    TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. * p
    Figure Legend Snippet: TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. * p

    Techniques Used: Incubation, Inhibition

    TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p
    Figure Legend Snippet: TGF-β1 and Pro-CollagenIα1 secretion and colIα1 and mmp-1 mRNA expression in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). TGF-β1 (10 ng/ml) used as positive control for collagen synthesis and secretion. a TGF-β1; b Pro-CollagenIα1; c colIα1 ; d mmp-1 . * p

    Techniques Used: Expressing, Positive Control

    TGF-β1, Pro-collagenIα1 and IL-8 in fibroblasts pretreated with NFκB inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with MG-132 (20 μmol), an NFκB inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1 and c IL-8 in untreated versus MG-132-treated cells. *** p
    Figure Legend Snippet: TGF-β1, Pro-collagenIα1 and IL-8 in fibroblasts pretreated with NFκB inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with MG-132 (20 μmol), an NFκB inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1 and c IL-8 in untreated versus MG-132-treated cells. *** p

    Techniques Used: Incubation, Inhibition

    et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p
    Figure Legend Snippet: et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p

    Techniques Used: Expressing, Acetylene Reduction Assay

    tlr mRNA expression levels in fibroblasts stimulated with SSc-ICs or control NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a tlr2 ; b tlr3 ; c tlr4 ; d tlr9 . ** p
    Figure Legend Snippet: tlr mRNA expression levels in fibroblasts stimulated with SSc-ICs or control NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as controls. a tlr2 ; b tlr3 ; c tlr4 ; d tlr9 . ** p

    Techniques Used: Expressing, Concentration Assay

    TaqMan® Gene Expression assays against SSc-specific antigens of PEG-precipitated ICs and corresponding sera evaluated by EUROLINE-SSc profile kit. One ATA-IC and one NHS-IC presented as representative assay. CTR+, assay-positive control. a, Ro-52; b, PDGF receptor; c, Ku; d, PM-Scl75; e, PM-Scl100; f, Th/To; g, NOR90; h, Fibrillarin; i, RP155; l, RP11; m, CENP B; n, CENP A; o, Scl-70 (DNA topoisomerase I). ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects
    Figure Legend Snippet: TaqMan® Gene Expression assays against SSc-specific antigens of PEG-precipitated ICs and corresponding sera evaluated by EUROLINE-SSc profile kit. One ATA-IC and one NHS-IC presented as representative assay. CTR+, assay-positive control. a, Ro-52; b, PDGF receptor; c, Ku; d, PM-Scl75; e, PM-Scl100; f, Th/To; g, NOR90; h, Fibrillarin; i, RP155; l, RP11; m, CENP B; n, CENP A; o, Scl-70 (DNA topoisomerase I). ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects

    Techniques Used: Expressing, Positive Control

    IL-6 and IL-8 in culture supernatants from dcSSc fibroblasts incubated with SSc-ICs or NHS-ICs. dcSSc fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8. * p
    Figure Legend Snippet: IL-6 and IL-8 in culture supernatants from dcSSc fibroblasts incubated with SSc-ICs or NHS-ICs. dcSSc fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8. * p

    Techniques Used: Incubation, Concentration Assay

    Dose–response dilution curve for ICAM-1 expression on fibroblast cell surface. Fibroblasts exposed to serial two-fold dilutions (from 1:2 to 1:64) of SSc-ICs and NHS-ICs, and ICAM-1 evaluated by cell ELISA. anti-Th/To anti-Th/To antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects, OD optical density
    Figure Legend Snippet: Dose–response dilution curve for ICAM-1 expression on fibroblast cell surface. Fibroblasts exposed to serial two-fold dilutions (from 1:2 to 1:64) of SSc-ICs and NHS-ICs, and ICAM-1 evaluated by cell ELISA. anti-Th/To anti-Th/To antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, NHS normal healthy subjects, OD optical density

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    Confirmation of efficacy of NFκB and p38MAPK inhibitors by western blot analysis. Cells preincubated for 1 h at 37 °C with inhibitors of NFκB and p38MAPK. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. Results expressed as percentage of inhibition of activated ( a ) NFκB and ( b ) p38MAPK (expressed as ratio of phosphorylated to nonphosphorylated forms). * p
    Figure Legend Snippet: Confirmation of efficacy of NFκB and p38MAPK inhibitors by western blot analysis. Cells preincubated for 1 h at 37 °C with inhibitors of NFκB and p38MAPK. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. Results expressed as percentage of inhibition of activated ( a ) NFκB and ( b ) p38MAPK (expressed as ratio of phosphorylated to nonphosphorylated forms). * p

    Techniques Used: Western Blot, Inhibition

    IL-6, IL-8, MMP-2 and MCP-1 levels in culture supernatants from fibroblasts incubated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8; c MMP-2; d MCP-1. ** p
    Figure Legend Snippet: IL-6, IL-8, MMP-2 and MCP-1 levels in culture supernatants from fibroblasts incubated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. a IL-6; b IL-8; c MMP-2; d MCP-1. ** p

    Techniques Used: Incubation, Concentration Assay

    ICAM-1 expression on fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. *** p
    Figure Legend Snippet: ICAM-1 expression on fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). Poly(I:C) and LPS, at concentration of 1 μg/ml, used as positive controls. *** p

    Techniques Used: Expressing, Concentration Assay

    2) Product Images from "RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer"

    Article Title: RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer

    Journal: Genes & Development

    doi: 10.1101/gad.304162.117

    RANK is expressed in human lung tumors and controls growth of human lung cancer. ( A ) Representative RANK IHC in primary human NSCLC lung adenocarcinomas. Bars, 100 µm. ( B ) Cross-correlation matrixes to compare RANK protein expression (determined by IHC) in human lung tumors with KRAS mutational status and smoking. n = 364, ”Uppsala” cohort with early stage treatment-naïve resected lung cancer, including squamous cell carcinoma, adenocarcinoma, SCLC, and large-cell carcinoma. P -values are indicated, calculated using the Fisher's exact test. ( C ) Prediction of overall survival probability in the human Affymetrix lung adenocarcinoma data set stratified for high (red lines) and low (black lines) RANK, RANKL, OPG, and LGR4 mRNA expression based on the best fit algorithm. Data were obtained using KM plotter. P -values (log rank test) are indicated. ( D ) RANK IHC in a patient-derived KRAS mutant adenocarcinoma used for xenograft experiments. Bars, 100 µm. ( E ) Growth of a patient-derived lung adenocarcinoma xenograft (PDX). PDX tumor fragments were implanted subcutaneously in the right flanks of 6- to 8-wk-old female NSG (NOD- scid IL2Rγ null ) mice. At day 45 (when tumors had reached ∼200 mm 3 ), mice were treated with 10 mg/kg OPG-Fc or PBS subcutaneously twice per week, and tumor volumes were measured twice per week using digital calipers. OPG-Fc treatment significantly reduced tumor burden. P = 0.024, repeated measures analysis of variance (RMANOVA) followed by Dunnett's post-hoc test. Data represent the mean ± SEM for each group. n = 10 per group. ( F ) Proliferation of PDX tumor cells in PBS and 10 mg/kg OPG-Fc-treated mice was determined using Ki67 immunostaining, analyzed at the termination of the experiment for ethical reasons. Data are shown as mean percentage of Ki67 + cells (±SEM) among tumor cells, comparing the two groups. n = 10 per group. P = 0.0143, unpaired t -test.
    Figure Legend Snippet: RANK is expressed in human lung tumors and controls growth of human lung cancer. ( A ) Representative RANK IHC in primary human NSCLC lung adenocarcinomas. Bars, 100 µm. ( B ) Cross-correlation matrixes to compare RANK protein expression (determined by IHC) in human lung tumors with KRAS mutational status and smoking. n = 364, ”Uppsala” cohort with early stage treatment-naïve resected lung cancer, including squamous cell carcinoma, adenocarcinoma, SCLC, and large-cell carcinoma. P -values are indicated, calculated using the Fisher's exact test. ( C ) Prediction of overall survival probability in the human Affymetrix lung adenocarcinoma data set stratified for high (red lines) and low (black lines) RANK, RANKL, OPG, and LGR4 mRNA expression based on the best fit algorithm. Data were obtained using KM plotter. P -values (log rank test) are indicated. ( D ) RANK IHC in a patient-derived KRAS mutant adenocarcinoma used for xenograft experiments. Bars, 100 µm. ( E ) Growth of a patient-derived lung adenocarcinoma xenograft (PDX). PDX tumor fragments were implanted subcutaneously in the right flanks of 6- to 8-wk-old female NSG (NOD- scid IL2Rγ null ) mice. At day 45 (when tumors had reached ∼200 mm 3 ), mice were treated with 10 mg/kg OPG-Fc or PBS subcutaneously twice per week, and tumor volumes were measured twice per week using digital calipers. OPG-Fc treatment significantly reduced tumor burden. P = 0.024, repeated measures analysis of variance (RMANOVA) followed by Dunnett's post-hoc test. Data represent the mean ± SEM for each group. n = 10 per group. ( F ) Proliferation of PDX tumor cells in PBS and 10 mg/kg OPG-Fc-treated mice was determined using Ki67 immunostaining, analyzed at the termination of the experiment for ethical reasons. Data are shown as mean percentage of Ki67 + cells (±SEM) among tumor cells, comparing the two groups. n = 10 per group. P = 0.0143, unpaired t -test.

    Techniques Used: Immunohistochemistry, Expressing, Derivative Assay, Mutagenesis, Mouse Assay, Immunostaining

    3) Product Images from "Reduced Apaf-1 levels in cardiomyocytes engage strict regulation of apoptosis by endogenous XIAP"

    Article Title: Reduced Apaf-1 levels in cardiomyocytes engage strict regulation of apoptosis by endogenous XIAP

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200504082

    Restoring Apaf-1 levels eliminates the strict control of XIAP and permits cytochrome c to induce apoptosis in cardiomyocytes. Rat cardiomyocytes were transfected with plasmids expressing GFP alone (Vector/GFP), Apaf-1 and GFP (Apaf-1/GFP), or procaspase-9 and GFP (caspase-9/GFP). After 24 h, the transfected cells (identifiable by GFP expression) were microinjected with 25 μg/μl of bovine or yeast cytochrome c and rhodamine dextran (Cyt c/Rhod). (A) Fluorescence photographs (for the GFP- or Rhodamine-selective channels) of representative cells taken 2 h after the cytochrome c injections. Arrows point to the GFP-positive cells that were injected with cytochrome c and rhodamine. (B) Quantitation of cell survival (2 h after the injections) for the various conditions. Data shown are the mean ± SEM of three independent experiments.
    Figure Legend Snippet: Restoring Apaf-1 levels eliminates the strict control of XIAP and permits cytochrome c to induce apoptosis in cardiomyocytes. Rat cardiomyocytes were transfected with plasmids expressing GFP alone (Vector/GFP), Apaf-1 and GFP (Apaf-1/GFP), or procaspase-9 and GFP (caspase-9/GFP). After 24 h, the transfected cells (identifiable by GFP expression) were microinjected with 25 μg/μl of bovine or yeast cytochrome c and rhodamine dextran (Cyt c/Rhod). (A) Fluorescence photographs (for the GFP- or Rhodamine-selective channels) of representative cells taken 2 h after the cytochrome c injections. Arrows point to the GFP-positive cells that were injected with cytochrome c and rhodamine. (B) Quantitation of cell survival (2 h after the injections) for the various conditions. Data shown are the mean ± SEM of three independent experiments.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Fluorescence, Injection, Quantitation Assay

    Apaf-1 but not caspase-9 levels are markedly reduced in cardiomyocytes in comparison to fibroblasts. (A) Western blots showing levels of Apaf-1 and caspase-9 proteins (and LDH and Troponin I as controls) in cultures of rat dermal fibroblasts and cardiomyocytes. (B) Quantitation of the data in which Apaf-1 and caspase-9 protein levels detected in cardiomyocyte cultures are expressed as a percentage of the levels (normalized to LDH) seen in fibroblast cultures. Data shown are the mean ± SEM of three independent experiments.
    Figure Legend Snippet: Apaf-1 but not caspase-9 levels are markedly reduced in cardiomyocytes in comparison to fibroblasts. (A) Western blots showing levels of Apaf-1 and caspase-9 proteins (and LDH and Troponin I as controls) in cultures of rat dermal fibroblasts and cardiomyocytes. (B) Quantitation of the data in which Apaf-1 and caspase-9 protein levels detected in cardiomyocyte cultures are expressed as a percentage of the levels (normalized to LDH) seen in fibroblast cultures. Data shown are the mean ± SEM of three independent experiments.

    Techniques Used: Western Blot, Quantitation Assay

    XIAP-deficient cardiomyocytes die with injection of cytochrome c . Cardiomyocytes isolated from XIAP-deficient (−/−) or wild-type (+/+) littermate mice were microinjected with 25 μg/μl of bovine cytochrome c . As a control, XIAP-deficient cardiomyocytes were also injected with 25 μg/μl of yeast cytochrome c . (A) Phase-contrast and fluorescence photographs of representative cells. Arrows mark the injected cells, identified by coinjection of rhodamine dextran along with cytochrome c . (B) Quantitation of cell survival at multiple times after the injections. Data shown are the mean ± SEM of three independent experiments.
    Figure Legend Snippet: XIAP-deficient cardiomyocytes die with injection of cytochrome c . Cardiomyocytes isolated from XIAP-deficient (−/−) or wild-type (+/+) littermate mice were microinjected with 25 μg/μl of bovine cytochrome c . As a control, XIAP-deficient cardiomyocytes were also injected with 25 μg/μl of yeast cytochrome c . (A) Phase-contrast and fluorescence photographs of representative cells. Arrows mark the injected cells, identified by coinjection of rhodamine dextran along with cytochrome c . (B) Quantitation of cell survival at multiple times after the injections. Data shown are the mean ± SEM of three independent experiments.

    Techniques Used: Injection, Isolation, Mouse Assay, Fluorescence, Quantitation Assay

    Cytosolic microinjection of cytochrome c induces death in fibroblasts but not cardiomyocytes. Neonatal rat cardiomyocytes and dermal fibroblasts were microinjected with 25 μg/μl of bovine or yeast cytochrome c , and cell survival (using morphological criteria) was assessed at multiple times after the injections. (A) Phase-contrast and fluorescence photographs of the cells 3 h after the injections of cytochrome c . The injected cells (arrows) were identified by the presence of rhodamine dextran coinjected with the cytochrome c . (B) Quantitation of cell survival. Data shown are the mean ± SEM of three independent experiments.
    Figure Legend Snippet: Cytosolic microinjection of cytochrome c induces death in fibroblasts but not cardiomyocytes. Neonatal rat cardiomyocytes and dermal fibroblasts were microinjected with 25 μg/μl of bovine or yeast cytochrome c , and cell survival (using morphological criteria) was assessed at multiple times after the injections. (A) Phase-contrast and fluorescence photographs of the cells 3 h after the injections of cytochrome c . The injected cells (arrows) were identified by the presence of rhodamine dextran coinjected with the cytochrome c . (B) Quantitation of cell survival. Data shown are the mean ± SEM of three independent experiments.

    Techniques Used: Fluorescence, Injection, Quantitation Assay

    Cardiomyocyte resistance to cytochrome c can be overcome with the exogenous addition of the IAP inhibitor Smac. (A) Western blots showing that the rat cardiomyocyte cultures express the core apoptotic components (Apaf-1, caspase-9, and caspase-3) and various IAPs (XIAP, cIAP-1, and cIAP-2). LDH and Troponin I are shown as controls. (B) Rat cardiomyocytes were microinjected with 25 μg/μl of bovine cytochrome c , 1 μg/μl of wild-type mature Smac, or both, and cell survival was assessed at multiple times after the injections. (C) Rat cardiomyocytes were injected with 25 μg/μl of bovine cytochrome c and 1 μg/μl of either mature wild-type AVPI-Smac or mature mutant MVPI-Smac in the presence or absence of 50 μM of the pan caspase inhibitor zVAD-FMK. Cell survival was assessed at multiple times after the injections. Data shown are the mean ± SEM of three independent experiments.
    Figure Legend Snippet: Cardiomyocyte resistance to cytochrome c can be overcome with the exogenous addition of the IAP inhibitor Smac. (A) Western blots showing that the rat cardiomyocyte cultures express the core apoptotic components (Apaf-1, caspase-9, and caspase-3) and various IAPs (XIAP, cIAP-1, and cIAP-2). LDH and Troponin I are shown as controls. (B) Rat cardiomyocytes were microinjected with 25 μg/μl of bovine cytochrome c , 1 μg/μl of wild-type mature Smac, or both, and cell survival was assessed at multiple times after the injections. (C) Rat cardiomyocytes were injected with 25 μg/μl of bovine cytochrome c and 1 μg/μl of either mature wild-type AVPI-Smac or mature mutant MVPI-Smac in the presence or absence of 50 μM of the pan caspase inhibitor zVAD-FMK. Cell survival was assessed at multiple times after the injections. Data shown are the mean ± SEM of three independent experiments.

    Techniques Used: Western Blot, Injection, Mutagenesis

    4) Product Images from "DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers"

    Article Title: DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.200910191

    Representative confocal x-y images (1/second) obtained from fluo-4–loaded FDB fibers before, during, and after exposure to solutions of increased or decreased osmolarity. For each cell, the number of LCR events observed per frame throughout the experiment is indicated in the graph on the right. (A) The application of a hyperosmotic sucrose solution was often associated with a marked elevation of [Ca 2+ ], and LCR was difficult to identify (cell 1). However, where the rise in [Ca 2+ ] was less pronounced, individual LCR events were apparent during the application of sucrose (cell 2). Prior exposure to 100 µM nifedipine, to inhibit the DHPR, prevented LCR upon sucrose exposure (cell 3). (B) The introduction of a hyperosmotic CaCl 2 solution induced LCR (cell 1), which was markedly inhibited in the presence of nifedipine (cell 2). (C) The application of a hypoosmotic (254 mOsm) solution had no apparent effect on [Ca 2+ ] i . However, returning to the isoosmotic solution precipitated LCR events (cell 1). In the presence of nifedipine, transient exposure to a hypoosmotic solution failed to induce LCR (cell 2). Fibers subject to hypoosmotic Tyrode's exposure were initially exposed to isoosmotic Tyrode for 30 s, and then for 2 min to a hypoosmotic before returning to isoosmotic Tyrode's solution for 4 min. Horizontal bar, 20 µm.
    Figure Legend Snippet: Representative confocal x-y images (1/second) obtained from fluo-4–loaded FDB fibers before, during, and after exposure to solutions of increased or decreased osmolarity. For each cell, the number of LCR events observed per frame throughout the experiment is indicated in the graph on the right. (A) The application of a hyperosmotic sucrose solution was often associated with a marked elevation of [Ca 2+ ], and LCR was difficult to identify (cell 1). However, where the rise in [Ca 2+ ] was less pronounced, individual LCR events were apparent during the application of sucrose (cell 2). Prior exposure to 100 µM nifedipine, to inhibit the DHPR, prevented LCR upon sucrose exposure (cell 3). (B) The introduction of a hyperosmotic CaCl 2 solution induced LCR (cell 1), which was markedly inhibited in the presence of nifedipine (cell 2). (C) The application of a hypoosmotic (254 mOsm) solution had no apparent effect on [Ca 2+ ] i . However, returning to the isoosmotic solution precipitated LCR events (cell 1). In the presence of nifedipine, transient exposure to a hypoosmotic solution failed to induce LCR (cell 2). Fibers subject to hypoosmotic Tyrode's exposure were initially exposed to isoosmotic Tyrode for 30 s, and then for 2 min to a hypoosmotic before returning to isoosmotic Tyrode's solution for 4 min. Horizontal bar, 20 µm.

    Techniques Used:

    5) Product Images from "A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation"

    Article Title: A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20132267

    TCR- and SAP-dependent Tfh formation. (a) FACS analysis of splenic CD4 + T cells from 10-wk-old OTII Tg with intact or deleted Cul3 as indicated. CD4 + cells gated as TCR Tg + or TCR Tg − were stained for ICOS and PD1 to measure the frequency of Tfh cells. Numbers represent mean ± SEM of five mice per group from two independent experiments. (b) FACS analysis of gated CD4 + splenocytes from 10–15-wk-old littermate (LM) controls, Sh2d1a (SAP) −/− , Cul3cKO, and Sh2d1a −/− Cul3cKO mice, stained for PD1 and CXCR5 (top) or PD1 and ICOS (bottom). (c) FACS analysis of the same spleens as in b, gated on B220 + IgD − cells to identify GC B cells. Numbers represent the percentage (mean ± SEM) of five mice/group from three independent experiments in corresponding gates. For CD4 + T cells, GC-Tfh cells are identified as PD1 hi ICOS hi CXCR5 hi and pre-Tfh cells as intermediate for the same markers as shown.
    Figure Legend Snippet: TCR- and SAP-dependent Tfh formation. (a) FACS analysis of splenic CD4 + T cells from 10-wk-old OTII Tg with intact or deleted Cul3 as indicated. CD4 + cells gated as TCR Tg + or TCR Tg − were stained for ICOS and PD1 to measure the frequency of Tfh cells. Numbers represent mean ± SEM of five mice per group from two independent experiments. (b) FACS analysis of gated CD4 + splenocytes from 10–15-wk-old littermate (LM) controls, Sh2d1a (SAP) −/− , Cul3cKO, and Sh2d1a −/− Cul3cKO mice, stained for PD1 and CXCR5 (top) or PD1 and ICOS (bottom). (c) FACS analysis of the same spleens as in b, gated on B220 + IgD − cells to identify GC B cells. Numbers represent the percentage (mean ± SEM) of five mice/group from three independent experiments in corresponding gates. For CD4 + T cells, GC-Tfh cells are identified as PD1 hi ICOS hi CXCR5 hi and pre-Tfh cells as intermediate for the same markers as shown.

    Techniques Used: FACS, Staining, Mouse Assay

    Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4 + SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10 −6 ). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4 + SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P
    Figure Legend Snippet: Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4 + SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10 −6 ). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4 + SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P

    Techniques Used: Expressing, Quantitative RT-PCR, Genome Wide, Generated, Magnetic Cell Separation, FACS, Irradiation, Purification

    Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP 16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1 hi CXCR5 hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4 + SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP 41 (left) and BSA-NP 4 (right) at days 0 and 21 after immunization with OVA-NP 16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P
    Figure Legend Snippet: Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP 16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1 hi CXCR5 hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4 + SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP 41 (left) and BSA-NP 4 (right) at days 0 and 21 after immunization with OVA-NP 16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P

    Techniques Used: Injection, Mouse Assay, FACS, Staining, Fluorescence, In Vivo

    Exaggerated Tfh responses without alteration of Th1 or Th2 programs. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII or OTII Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before injection of OVA + CFA s.c., OVA + alum i.p., or PBS, as indicated. OTII cells were analyzed at day 7 after immunization in inguinal lymph nodes (CFA; a) or spleen (alum; b). (first row) Expression of CXCR5 and PD1. Numbers above the dot plots are the absolute numbers of OTII cells recovered from the lymph nodes or spleen. Numbers in top right quadrants are the percentage of CXCR5 + PD1 + (mean ± SEM). (second row) Expression of Ki67 and CXCR5. (third row) Expression of Tbet and Bcl6. Numbers indicate the percentage (mean ± SEM) of Tbet hi Bcl6 hi cells (right box) or Tbet hi Bcl6 int cells (left box). Background staining is shown after preincubation with an excess of unconjugated antibodies (cold Tbet+Bcl6). (fourth row) Expression of Gata3 and CXCR5 with quadrant statistics (mean ± SEM). Background staining is shown for fluorochrome-conjugated isotype control. Data are representative of two independent experiments with total n = 6 mice.
    Figure Legend Snippet: Exaggerated Tfh responses without alteration of Th1 or Th2 programs. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII or OTII Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before injection of OVA + CFA s.c., OVA + alum i.p., or PBS, as indicated. OTII cells were analyzed at day 7 after immunization in inguinal lymph nodes (CFA; a) or spleen (alum; b). (first row) Expression of CXCR5 and PD1. Numbers above the dot plots are the absolute numbers of OTII cells recovered from the lymph nodes or spleen. Numbers in top right quadrants are the percentage of CXCR5 + PD1 + (mean ± SEM). (second row) Expression of Ki67 and CXCR5. (third row) Expression of Tbet and Bcl6. Numbers indicate the percentage (mean ± SEM) of Tbet hi Bcl6 hi cells (right box) or Tbet hi Bcl6 int cells (left box). Background staining is shown after preincubation with an excess of unconjugated antibodies (cold Tbet+Bcl6). (fourth row) Expression of Gata3 and CXCR5 with quadrant statistics (mean ± SEM). Background staining is shown for fluorochrome-conjugated isotype control. Data are representative of two independent experiments with total n = 6 mice.

    Techniques Used: Injection, Expressing, Staining, Mouse Assay

    6) Product Images from "Elevated VEGF-D Modulates Tumor Inflammation and Reduces the Growth of Carcinogen-Induced Skin Tumors"

    Article Title: Elevated VEGF-D Modulates Tumor Inflammation and Reduces the Growth of Carcinogen-Induced Skin Tumors

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2016.05.002

    The TG overexpression of VEGF-D affects inflammatory cell accumulation in skin tumors. Representative photographs of CD4 + (A) and CD8 + (C) T-cells and F4/80 + macrophages (E) in the peritumoral area of WT and TG papillomas at week 20. The number of CD4 + (B) and CD8 + T-cells (D), and macrophages (F) in the UT skin, TPA-treated skin, PAPs and cSCCs collected at different time points of skin carcinogenesis. VEGF-D overexpression resulted in significantly less CD4 + T-cells and macrophages, and more CD8 + T-cells in skin tumors at several time points of skin carcinogenesis. (G) FACS analysis revealed significantly less CD4 + T-cells (CD3 + CD4 + Gr-1 − ) and macrophages (Gr-1 − CD11b + F4/80 + ), and significantly more CD8 + T-cells (CD3 + CD8 + Gr-1 − ) in the DMBA-TPA treated skin of the TG mice (N = 5) than in the WT mice (N = 6) at week 10 of carcinogenesis. Scale bars: 100 μm (A, E) and 50 μm (C). Error bars: SD. *: P
    Figure Legend Snippet: The TG overexpression of VEGF-D affects inflammatory cell accumulation in skin tumors. Representative photographs of CD4 + (A) and CD8 + (C) T-cells and F4/80 + macrophages (E) in the peritumoral area of WT and TG papillomas at week 20. The number of CD4 + (B) and CD8 + T-cells (D), and macrophages (F) in the UT skin, TPA-treated skin, PAPs and cSCCs collected at different time points of skin carcinogenesis. VEGF-D overexpression resulted in significantly less CD4 + T-cells and macrophages, and more CD8 + T-cells in skin tumors at several time points of skin carcinogenesis. (G) FACS analysis revealed significantly less CD4 + T-cells (CD3 + CD4 + Gr-1 − ) and macrophages (Gr-1 − CD11b + F4/80 + ), and significantly more CD8 + T-cells (CD3 + CD8 + Gr-1 − ) in the DMBA-TPA treated skin of the TG mice (N = 5) than in the WT mice (N = 6) at week 10 of carcinogenesis. Scale bars: 100 μm (A, E) and 50 μm (C). Error bars: SD. *: P

    Techniques Used: Over Expression, Papanicolaou Stain, FACS, Mouse Assay

    DMBA-TPA-induced skin carcinogenesis in VEGF-D knockout mice. Tumor incidence (A) and cumulative multiplicity (B) in the KO males (N = 26) and the WT males (N = 27) did not show statistically significant differences between the genotypes. ( C-D ) Expression of VEGF-D in mouse skin tumors. Representative immunohistochemical stainings showing VEGF-D expression in mouse cSCCs. (C) Consecutive sections of the WT cSCC stained with antibodies against VEGF-D and F4/80 demonstrate that VEGF-D is expressed by F4/80-positive macrophages (arrowheads) within the WT cSCC stroma. Asterisks indicate vessels that facilitate the identification of co-stained cells in the consecutive sections. (D) VEGF-D expression by invasive carcinoma cells (arrowheads) within the WT cSCC. The KO cSCC is used as a negative control for VEGF-D staining. (E) The quantification of lymphatic vessel density in the untreated (UT) skin, TPA-treated skin, and papillomas (PAP) and cSCCs collected at different time points of skin carcinogenesis. The KO tumors showed a trend for reduced lymphangiogenesis. In the UT skin, and in cSCCs at week 29, the difference between the genotypes was significant. (F) No significant differences in blood vessel densities between the KO and WT males in any type of sample. Scale bars: 50 μm (C) and 100 μm (D): Error bars: SD. *: P
    Figure Legend Snippet: DMBA-TPA-induced skin carcinogenesis in VEGF-D knockout mice. Tumor incidence (A) and cumulative multiplicity (B) in the KO males (N = 26) and the WT males (N = 27) did not show statistically significant differences between the genotypes. ( C-D ) Expression of VEGF-D in mouse skin tumors. Representative immunohistochemical stainings showing VEGF-D expression in mouse cSCCs. (C) Consecutive sections of the WT cSCC stained with antibodies against VEGF-D and F4/80 demonstrate that VEGF-D is expressed by F4/80-positive macrophages (arrowheads) within the WT cSCC stroma. Asterisks indicate vessels that facilitate the identification of co-stained cells in the consecutive sections. (D) VEGF-D expression by invasive carcinoma cells (arrowheads) within the WT cSCC. The KO cSCC is used as a negative control for VEGF-D staining. (E) The quantification of lymphatic vessel density in the untreated (UT) skin, TPA-treated skin, and papillomas (PAP) and cSCCs collected at different time points of skin carcinogenesis. The KO tumors showed a trend for reduced lymphangiogenesis. In the UT skin, and in cSCCs at week 29, the difference between the genotypes was significant. (F) No significant differences in blood vessel densities between the KO and WT males in any type of sample. Scale bars: 50 μm (C) and 100 μm (D): Error bars: SD. *: P

    Techniques Used: Knock-Out, Mouse Assay, Expressing, Immunohistochemistry, Staining, Negative Control

    Impaired skin tumor formation in TG K14-mVEGF-D mice in a chemical skin carcinogenesis model. Tumors were induced in the mouse skin using a multistage DMBA-TPA protocol and their growth was monitored for up to 30 weeks (Wk). Data from two separate experimental groups of males (WT N = 23, TG N = 25) are combined. (A) Representative photographs of the WT and K14-mVEGF-D TG male mice at different time points. Black arrowheads indicate exophytic papillomas and white arrowheads regressing papillomas. (B) Tumor incidence showed a significant delay in the TG mice. (C) The TG mice developed markedly less tumors than the WT mice. Cumulative tumor multiplicity is shown. (D) The percentage of regressing papillomas (the black portion of the column) was higher in the TG mice (red) than in the WT mice (green). (E) Smaller tumor size in the TG mice than in the WT mice. Tumor sizes are shown as a percentage of tumors in three different size categories. Statistical significance for each size category is shown in between the column pairs. (F) At weeks 20–30 the percentage of TG male mice with clinically apparent cSCC was lower than that of WT control males. (G) A trend towards less cSCCs per mouse was observed in TG males. N = number of mice. Error bars: SD.*: P
    Figure Legend Snippet: Impaired skin tumor formation in TG K14-mVEGF-D mice in a chemical skin carcinogenesis model. Tumors were induced in the mouse skin using a multistage DMBA-TPA protocol and their growth was monitored for up to 30 weeks (Wk). Data from two separate experimental groups of males (WT N = 23, TG N = 25) are combined. (A) Representative photographs of the WT and K14-mVEGF-D TG male mice at different time points. Black arrowheads indicate exophytic papillomas and white arrowheads regressing papillomas. (B) Tumor incidence showed a significant delay in the TG mice. (C) The TG mice developed markedly less tumors than the WT mice. Cumulative tumor multiplicity is shown. (D) The percentage of regressing papillomas (the black portion of the column) was higher in the TG mice (red) than in the WT mice (green). (E) Smaller tumor size in the TG mice than in the WT mice. Tumor sizes are shown as a percentage of tumors in three different size categories. Statistical significance for each size category is shown in between the column pairs. (F) At weeks 20–30 the percentage of TG male mice with clinically apparent cSCC was lower than that of WT control males. (G) A trend towards less cSCCs per mouse was observed in TG males. N = number of mice. Error bars: SD.*: P

    Techniques Used: Mouse Assay

    7) Product Images from "Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice"

    Article Title: Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice

    Journal: Molecular Pain

    doi: 10.1177/1744806916666284

    GM3S depletion prevents increased intracellular calcium influx in DRG neurons in diet-induced diabetes. (a–d) Representative tracings of intracellular calcium responses ([Ca++] i ) to different concentration of potassium (K + ) (10–50 mM) of acutely cultured DRG sensory neurons from control WT RD (a, n = 56), WT HFD (b, n = 66), GM3S KO RD (c, n = 108), and GM3S KO HFD (d, n = 41) mice. (e) Percentage of DRG neurons from each group responding to different concentration of potassium (K + ) (10, 25, and 50 mM). Values are expressed as mean ± SE. *** p
    Figure Legend Snippet: GM3S depletion prevents increased intracellular calcium influx in DRG neurons in diet-induced diabetes. (a–d) Representative tracings of intracellular calcium responses ([Ca++] i ) to different concentration of potassium (K + ) (10–50 mM) of acutely cultured DRG sensory neurons from control WT RD (a, n = 56), WT HFD (b, n = 66), GM3S KO RD (c, n = 108), and GM3S KO HFD (d, n = 41) mice. (e) Percentage of DRG neurons from each group responding to different concentration of potassium (K + ) (10, 25, and 50 mM). Values are expressed as mean ± SE. *** p

    Techniques Used: Concentration Assay, Cell Culture, Mouse Assay

    8) Product Images from "Single-Particle Detection of Transcription following Rotavirus Entry"

    Article Title: Single-Particle Detection of Transcription following Rotavirus Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.00651-17

    Infectivity of RRV and fluorescently labeled rcTLPs. Focus-forming assays comparing native RRV (TLP) to the two differently labeled rcTLP preparations employed in this work were performed. For rcTLP-1, the DLPs and VP7 were labeled with Atto 647N and Atto 488 dyes, respectively. For rcTLP-2, the DLPs and VP7 were labeled with Atto 488 and Atto 390 dyes, respectively. Infectivity is shown in focus-forming units per milliliter from triplicate experiments in the BSC-1 (dark gray) and MA104 (light gray) cell lines. Standard deviations of the three measurements are shown as error bars. The specific infectivity (particle per focus-forming unit [P/FFU]) of the virus in each sample is shown above the respective bars along with its standard deviation.
    Figure Legend Snippet: Infectivity of RRV and fluorescently labeled rcTLPs. Focus-forming assays comparing native RRV (TLP) to the two differently labeled rcTLP preparations employed in this work were performed. For rcTLP-1, the DLPs and VP7 were labeled with Atto 647N and Atto 488 dyes, respectively. For rcTLP-2, the DLPs and VP7 were labeled with Atto 488 and Atto 390 dyes, respectively. Infectivity is shown in focus-forming units per milliliter from triplicate experiments in the BSC-1 (dark gray) and MA104 (light gray) cell lines. Standard deviations of the three measurements are shown as error bars. The specific infectivity (particle per focus-forming unit [P/FFU]) of the virus in each sample is shown above the respective bars along with its standard deviation.

    Techniques Used: Infection, Labeling, Standard Deviation

    9) Product Images from "Nano-enabled pancreas cancer immunotherapy using immunogenic cell death and reversing immunosuppression"

    Article Title: Nano-enabled pancreas cancer immunotherapy using immunogenic cell death and reversing immunosuppression

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01651-9

    Dual delivery of OX plus IND-NV by MSNP induced effective anti-PDAC immunity in the orthotopic tumor model. a Orthotopic tumor-bearing B6/129 mice ( n = 7) were IV injected with the OX/IND-MSNP to deliver the equivalent 5 mg/kg OX and 50 mg/kg IND every 4 days, for a total of 4 administrations. The 1 st injection started on day 10. Free OX, OX/LB-MSNP, IND-NV, IND-NV + free OX, and OX/IND-MSNP were used for comparison at the equivalent doses. Interval IVIS imaging monitored tumor growth, which was quantitatively expressed as image intensity at the ROI. b Representative IVIS imaging on days 10, 18, 27, and 36, according to which the normalized tumor burden was plotted as fold-increase compared to the non-treated control. c Representative ex vivo bioluminescence imaging on day 36 to show the effect of treatment on metastatic tumor spread to the stomach, intestines, liver, spleen, kidneys, diaphragm, and abdominal wall, but not the heart or lung. We also included in the same experiment, treatment with anti-CD8 and anti-TLR4 antibodies, as well as an injectable pool of siRNAs for knockdown of CD91. The effect of interference in the immune response is shown in Supplementary Fig. 12a . The corresponding heat map display of the ex vivo imaging is summarized in Supplementary Fig. 10b . d Assessment of the survival effect of OX/IND-MSNP ( n = 7) vs. the controls was conducted by repeating the experiment in ( a ). e IHC staining for CD8 + and Foxp3 + T cells in tumor tissue, collected in c (left panel). Scale bar is 100 μm. CD8/Tregs ratio in tumor tissue determined by flow cytometry (right panel). f Real-time PCR measurement of P-S6K and IL-6 mRNA expression as a result of interference in the IDO pathway in vivo. The results are expressed as mean ± SEM. * p
    Figure Legend Snippet: Dual delivery of OX plus IND-NV by MSNP induced effective anti-PDAC immunity in the orthotopic tumor model. a Orthotopic tumor-bearing B6/129 mice ( n = 7) were IV injected with the OX/IND-MSNP to deliver the equivalent 5 mg/kg OX and 50 mg/kg IND every 4 days, for a total of 4 administrations. The 1 st injection started on day 10. Free OX, OX/LB-MSNP, IND-NV, IND-NV + free OX, and OX/IND-MSNP were used for comparison at the equivalent doses. Interval IVIS imaging monitored tumor growth, which was quantitatively expressed as image intensity at the ROI. b Representative IVIS imaging on days 10, 18, 27, and 36, according to which the normalized tumor burden was plotted as fold-increase compared to the non-treated control. c Representative ex vivo bioluminescence imaging on day 36 to show the effect of treatment on metastatic tumor spread to the stomach, intestines, liver, spleen, kidneys, diaphragm, and abdominal wall, but not the heart or lung. We also included in the same experiment, treatment with anti-CD8 and anti-TLR4 antibodies, as well as an injectable pool of siRNAs for knockdown of CD91. The effect of interference in the immune response is shown in Supplementary Fig. 12a . The corresponding heat map display of the ex vivo imaging is summarized in Supplementary Fig. 10b . d Assessment of the survival effect of OX/IND-MSNP ( n = 7) vs. the controls was conducted by repeating the experiment in ( a ). e IHC staining for CD8 + and Foxp3 + T cells in tumor tissue, collected in c (left panel). Scale bar is 100 μm. CD8/Tregs ratio in tumor tissue determined by flow cytometry (right panel). f Real-time PCR measurement of P-S6K and IL-6 mRNA expression as a result of interference in the IDO pathway in vivo. The results are expressed as mean ± SEM. * p

    Techniques Used: Mouse Assay, Injection, Imaging, Ex Vivo, Immunohistochemistry, Staining, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Expressing, In Vivo

    Oxaliplatin-induced ICD provides a successful anti-PDAC vaccination approach. a Confocal microscopy showing the induction of the ICD marker, CRT, in KPC cells in the presence of PBS, Cis (100 µM), OX (50 µM), and DOX (1 µM) for 4 h. The cell nuclei, surface membrane and CRT were detected by Hoechst 33342, Alexa Fluor ® 488-Conjugated Wheat Germ Agglutinin, and Alexa Fluor ® 647-conjugated anti-CRT antibody staining, respectively. Scale bar is 20 μm. b CRT surface detection by flow cytometry, using the same conditions and reagents as in a (3 independent experiments). c Animal experimentation using 2 rounds of vaccination one week apart, followed by injecting live KPC cells SC on the contralateral side. The details of the animal vaccination experiment are provided in the methods section. Tumors were collected on day 29 for IHC and flow cytometry analysis. d Spaghetti curves to show KPC tumor growth in the contralateral flank. e Tumor collection was performed after euthanizing the animal to conduct IHC. Representative images are shown for the IHC staining of CD8 (upper panel) and Foxp3 (lower panel) T cells. The tumor tissues were also analyzed by flow cytometry to determine the CD8/Tregs ratio (see experimental section for details) (right panel). f IHC staining for cleaved caspase-3 (CC-3) and IFN-γ to demonstrate recruitment of cytotoxic T cells in response to ICD. Scale bar in IHC is 100 μm. g The 3 surviving animals in the OX-treated group, described in c , received orthotopic implant of live KPC cells on day 74. Animals maintained their tumor-free status up to 132 days, whereupon they were euthanized for collecting the immune splenocytes to perform an adoptive transfer experiment. IV injection of the immune splenocytes into the tail vein of B6/129 mice prevented the growth of KPC cells, implanted SC. The controls included IV administration of non-immune splenocytes or saline. The same experiment was also carried out in mice receiving SC injection of B16 melanoma cells. In this case, there was no interference in tumor growth by immune splenocytes, demonstrating the antigen specificity of the adoptive transfer response (Supplementary Fig. 3 ). The results are expressed as mean ± SEM. * p
    Figure Legend Snippet: Oxaliplatin-induced ICD provides a successful anti-PDAC vaccination approach. a Confocal microscopy showing the induction of the ICD marker, CRT, in KPC cells in the presence of PBS, Cis (100 µM), OX (50 µM), and DOX (1 µM) for 4 h. The cell nuclei, surface membrane and CRT were detected by Hoechst 33342, Alexa Fluor ® 488-Conjugated Wheat Germ Agglutinin, and Alexa Fluor ® 647-conjugated anti-CRT antibody staining, respectively. Scale bar is 20 μm. b CRT surface detection by flow cytometry, using the same conditions and reagents as in a (3 independent experiments). c Animal experimentation using 2 rounds of vaccination one week apart, followed by injecting live KPC cells SC on the contralateral side. The details of the animal vaccination experiment are provided in the methods section. Tumors were collected on day 29 for IHC and flow cytometry analysis. d Spaghetti curves to show KPC tumor growth in the contralateral flank. e Tumor collection was performed after euthanizing the animal to conduct IHC. Representative images are shown for the IHC staining of CD8 (upper panel) and Foxp3 (lower panel) T cells. The tumor tissues were also analyzed by flow cytometry to determine the CD8/Tregs ratio (see experimental section for details) (right panel). f IHC staining for cleaved caspase-3 (CC-3) and IFN-γ to demonstrate recruitment of cytotoxic T cells in response to ICD. Scale bar in IHC is 100 μm. g The 3 surviving animals in the OX-treated group, described in c , received orthotopic implant of live KPC cells on day 74. Animals maintained their tumor-free status up to 132 days, whereupon they were euthanized for collecting the immune splenocytes to perform an adoptive transfer experiment. IV injection of the immune splenocytes into the tail vein of B6/129 mice prevented the growth of KPC cells, implanted SC. The controls included IV administration of non-immune splenocytes or saline. The same experiment was also carried out in mice receiving SC injection of B16 melanoma cells. In this case, there was no interference in tumor growth by immune splenocytes, demonstrating the antigen specificity of the adoptive transfer response (Supplementary Fig. 3 ). The results are expressed as mean ± SEM. * p

    Techniques Used: Confocal Microscopy, Marker, Staining, Flow Cytometry, Cytometry, Immunohistochemistry, Adoptive Transfer Assay, IV Injection, Mouse Assay, Injection

    Co-administration of IND-NV with OX at the tumor site augments anti-PDAC immunity. a Local co-administration in KPC tumors established by SC injection in syngeneic mice. Treatment details are provided in the methods section. b KPC tumor growth curve after a single IT injection of the various drugs at a tumor size of 60–80 mm 3 . OX was injected at 1.25 mg/kg 15 . Low (L, 2.5 mg IND/kg) and High (H, 12.5 mg IND/kg) refer to the IND or IND-NV doses. c Representative tumor images from each group after euthanizing the animal on day 31. d IHC depicting CD8 and Foxp3 biomarkers in the collected tumor tissue. A full set of panels of the IHC staining data are shown in Supplementary Fig. 7a–j . e Flow cytometry determination of CD8/Tregs ratio, as described in d . f Flow cytometry analysis to determine CD91 expression in the population of CD45 + /CD11b + /CD11c + cells in the tumor tissue. g IHC to depict CRT and HMGB-1 expression in the collected tumor tissues. The results are expressed as mean ± SEM. * p
    Figure Legend Snippet: Co-administration of IND-NV with OX at the tumor site augments anti-PDAC immunity. a Local co-administration in KPC tumors established by SC injection in syngeneic mice. Treatment details are provided in the methods section. b KPC tumor growth curve after a single IT injection of the various drugs at a tumor size of 60–80 mm 3 . OX was injected at 1.25 mg/kg 15 . Low (L, 2.5 mg IND/kg) and High (H, 12.5 mg IND/kg) refer to the IND or IND-NV doses. c Representative tumor images from each group after euthanizing the animal on day 31. d IHC depicting CD8 and Foxp3 biomarkers in the collected tumor tissue. A full set of panels of the IHC staining data are shown in Supplementary Fig. 7a–j . e Flow cytometry determination of CD8/Tregs ratio, as described in d . f Flow cytometry analysis to determine CD91 expression in the population of CD45 + /CD11b + /CD11c + cells in the tumor tissue. g IHC to depict CRT and HMGB-1 expression in the collected tumor tissues. The results are expressed as mean ± SEM. * p

    Techniques Used: Injection, Mouse Assay, Immunohistochemistry, Staining, Flow Cytometry, Cytometry, Expressing

    10) Product Images from "Effect of Amblyomma maculatum (Acari: Ixodidae) Saliva on the Acute Cutaneous Immune Response to Rickettsia parkeri Infection in a Murine Model"

    Article Title: Effect of Amblyomma maculatum (Acari: Ixodidae) Saliva on the Acute Cutaneous Immune Response to Rickettsia parkeri Infection in a Murine Model

    Journal: Journal of Medical Entomology

    doi: 10.1093/jme/tjw125

    Concentrations of skin inoculation site inflammatory cytokines are increased in response to intradermal R. parkeri inoculation, but not significantly altered by the addition of A. maculatum saliva to the rickettsial inoculum. Concentrations of interferon γ ( A ), interleukin-6 ( B ), and interleukin-10 ( C ) at the cutaneous inoculation site were determined at the indicated time points post inoculation by a magnetic cytokine bead panel kit. These cytokines were significantly elevated at various time points both in response to R. parkeri inoculation alone as well as in response to R. parkeri inoculation with A. maculatum saliva. However, no significant differences were found between the R. parkeri only group and the R. parkeri + saliva group at any time point. The data are presented as the medians and interquartile ranges. P
    Figure Legend Snippet: Concentrations of skin inoculation site inflammatory cytokines are increased in response to intradermal R. parkeri inoculation, but not significantly altered by the addition of A. maculatum saliva to the rickettsial inoculum. Concentrations of interferon γ ( A ), interleukin-6 ( B ), and interleukin-10 ( C ) at the cutaneous inoculation site were determined at the indicated time points post inoculation by a magnetic cytokine bead panel kit. These cytokines were significantly elevated at various time points both in response to R. parkeri inoculation alone as well as in response to R. parkeri inoculation with A. maculatum saliva. However, no significant differences were found between the R. parkeri only group and the R. parkeri + saliva group at any time point. The data are presented as the medians and interquartile ranges. P

    Techniques Used:

    11) Product Images from "DNA binding by the MATα2 transcription factor controls its access to alternative ubiquitin-modification pathways"

    Article Title: DNA binding by the MATα2 transcription factor controls its access to alternative ubiquitin-modification pathways

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-10-0589

    Reduced DNA binding is not the sole cause of stabilization for the α2(N182A, R185A) mutant. (A) Degradation rates, reported as half-life, for the indicated α2 variant following pulse-chase analysis in matα2 Δ cells (MHY1147) bearing plasmid pRS314-α2 or variant. Half-life ranges shown for α2-WT and the α2(N182A, R185A) mutant are from at least three replicates. All other half-life values are from a single degradation assay in which α2-WT was also tested and had a half-life of 4–6 min. Each α2 variant was tested at least twice with similar results. (B) Assay for repression of a-specific gene transcription in MHY481 cells by α2 or the indicated variant, selected from those characterized in degradation assays (A). Error bars denote SD ( N = 3). * p = 0.025. No other α2 variant tested, besides α2(N182D, R185A), yielded statistically poorer repression than the α2(N182A, R185A) variant. (C) Representative EMSA data for the interaction of purified α2 103-210 -6His, or the indicated variants of this protein, with synthetic, Cy5-labeled DNA corresponding to regulatory sequence upstream of BAR1 (an a-specific gene). DNA used was a half operator (a single α2 binding site and single Mcm1 binding site), and no Mcm1 protein was included in assays shown. Two previous experiments, using independent protein preparations, showed comparable DNA binding efficiencies for the different α2 103-210 -6His variants.
    Figure Legend Snippet: Reduced DNA binding is not the sole cause of stabilization for the α2(N182A, R185A) mutant. (A) Degradation rates, reported as half-life, for the indicated α2 variant following pulse-chase analysis in matα2 Δ cells (MHY1147) bearing plasmid pRS314-α2 or variant. Half-life ranges shown for α2-WT and the α2(N182A, R185A) mutant are from at least three replicates. All other half-life values are from a single degradation assay in which α2-WT was also tested and had a half-life of 4–6 min. Each α2 variant was tested at least twice with similar results. (B) Assay for repression of a-specific gene transcription in MHY481 cells by α2 or the indicated variant, selected from those characterized in degradation assays (A). Error bars denote SD ( N = 3). * p = 0.025. No other α2 variant tested, besides α2(N182D, R185A), yielded statistically poorer repression than the α2(N182A, R185A) variant. (C) Representative EMSA data for the interaction of purified α2 103-210 -6His, or the indicated variants of this protein, with synthetic, Cy5-labeled DNA corresponding to regulatory sequence upstream of BAR1 (an a-specific gene). DNA used was a half operator (a single α2 binding site and single Mcm1 binding site), and no Mcm1 protein was included in assays shown. Two previous experiments, using independent protein preparations, showed comparable DNA binding efficiencies for the different α2 103-210 -6His variants.

    Techniques Used: Binding Assay, Mutagenesis, Variant Assay, Pulse Chase, Plasmid Preparation, Degradation Assay, Purification, Labeling, Sequencing

    12) Product Images from "Muscle development and regeneration controlled by AUF1-mediated stage-specific degradation of fate-determining checkpoint mRNAs"

    Article Title: Muscle development and regeneration controlled by AUF1-mediated stage-specific degradation of fate-determining checkpoint mRNAs

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

    doi: 10.1073/pnas.1901165116

    Auf1 −/− satellite cells show aberrant terminal differentiation. ( A ) Representative cultured mass preparation of hindlimb skeletal muscles harvested from 4-mo-old mice, 10 d in culture. n = 3. Proliferating myoblasts (MyoD), white arrows; elongated myocytes (Myogenin), orange arrows; myofibers, yellow arrows. Nuclei stained with DAPI. ( B ) Representative IF staining of MyoD (green) and Myogenin (red) in WT and AUF1 KO-isolated myofibers, cultured for 72 h. Ten myofibers analyzed per mouse, n = 3. ( C ) Representative IF staining of Flag-AUF1 (red) and nuclei (DAPI, blue) in WT and AUF1 KO myofibers. Myofibers from mass preparations of the TA muscle as in A transduced with lentivirus vectors expressing AUF1 cDNAs for 72 h. Ten myofibers per group analyzed, n = 3. ( D ) Quantification of nuclei number and AUF1 in myofibers transduced with individual AUF1 isoforms, as in C , n = 3. (Scale bars: 100 μm.)
    Figure Legend Snippet: Auf1 −/− satellite cells show aberrant terminal differentiation. ( A ) Representative cultured mass preparation of hindlimb skeletal muscles harvested from 4-mo-old mice, 10 d in culture. n = 3. Proliferating myoblasts (MyoD), white arrows; elongated myocytes (Myogenin), orange arrows; myofibers, yellow arrows. Nuclei stained with DAPI. ( B ) Representative IF staining of MyoD (green) and Myogenin (red) in WT and AUF1 KO-isolated myofibers, cultured for 72 h. Ten myofibers analyzed per mouse, n = 3. ( C ) Representative IF staining of Flag-AUF1 (red) and nuclei (DAPI, blue) in WT and AUF1 KO myofibers. Myofibers from mass preparations of the TA muscle as in A transduced with lentivirus vectors expressing AUF1 cDNAs for 72 h. Ten myofibers per group analyzed, n = 3. ( D ) Quantification of nuclei number and AUF1 in myofibers transduced with individual AUF1 isoforms, as in C , n = 3. (Scale bars: 100 μm.)

    Techniques Used: Cell Culture, Mouse Assay, Staining, Isolation, Transduction, Expressing

    13) Product Images from "Transforming growth factor-? modulates the expression of nitric oxide signaling enzymes in the injured developing lung and in vascular smooth muscle cells"

    Article Title: Transforming growth factor-? modulates the expression of nitric oxide signaling enzymes in the injured developing lung and in vascular smooth muscle cells

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00181.2009

    Interstitial lung cells with smooth muscle cell (SMC) lineage isolated from the periphery of mouse pup lungs. A : a ∼1-mm-thick section of peripheral mouse pup lung tissue, containing terminal airway and microvascular structures as shown here in
    Figure Legend Snippet: Interstitial lung cells with smooth muscle cell (SMC) lineage isolated from the periphery of mouse pup lungs. A : a ∼1-mm-thick section of peripheral mouse pup lung tissue, containing terminal airway and microvascular structures as shown here in

    Techniques Used: Isolation

    14) Product Images from "Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity"

    Article Title: Deficiency in adipocyte chemokine receptor CXCR4 exacerbates obesity and compromises thermoregulatory responses of brown adipose tissue in a mouse model of diet-induced obesity

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-249797

    HFD feeding does not potentiate obesity in MyeCXCR4ko mice. WT C57BL/6 control ( n =40) and MyeCXCR4ko ( n =40) mice were fed a CD for 18 wk or an HFD for 24 wk. Animals were weighed 1×/wk and euthanized at the end of the feeding regimen. A ) Visceral
    Figure Legend Snippet: HFD feeding does not potentiate obesity in MyeCXCR4ko mice. WT C57BL/6 control ( n =40) and MyeCXCR4ko ( n =40) mice were fed a CD for 18 wk or an HFD for 24 wk. Animals were weighed 1×/wk and euthanized at the end of the feeding regimen. A ) Visceral

    Techniques Used: Mouse Assay

    Obesity in AdCXCR4ko mice is not a result of hyperphagia but positively correlates with increased adiposity, mass, and hypertrophy of BAT and WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed a CD or an HFD for 24 wk.
    Figure Legend Snippet: Obesity in AdCXCR4ko mice is not a result of hyperphagia but positively correlates with increased adiposity, mass, and hypertrophy of BAT and WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed a CD or an HFD for 24 wk.

    Techniques Used: Mouse Assay

    Adipocyte CXCR4 deficiency alters ATM and lymphocyte contents in WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed an HFD for 24 wk and then euthanized. Blood was collected and plasma separated. Visceral mesenteric,
    Figure Legend Snippet: Adipocyte CXCR4 deficiency alters ATM and lymphocyte contents in WAT. WT C57BL/6 control ( n =15), AdCXCR4ko ( n =15), and MyeCXCR4ko ( n =15) mice were fed an HFD for 24 wk and then euthanized. Blood was collected and plasma separated. Visceral mesenteric,

    Techniques Used: Mouse Assay

    AdCXCR4ko mice display impaired adaptive thermogenesis. WT C57BL/6 control ( n =5), AdCXCR4ko ( n =5), and MyeCXCR4ko mice were fed either a CD or an HFD for 24 wk. Body temperature was measured at 25°C or at the indicated times ( D ) at 4°C.
    Figure Legend Snippet: AdCXCR4ko mice display impaired adaptive thermogenesis. WT C57BL/6 control ( n =5), AdCXCR4ko ( n =5), and MyeCXCR4ko mice were fed either a CD or an HFD for 24 wk. Body temperature was measured at 25°C or at the indicated times ( D ) at 4°C.

    Techniques Used: Mouse Assay

    15) Product Images from "Concerted regulation of skeletal muscle contractility by oxygen tension and endogenous nitric oxide"

    Article Title: Concerted regulation of skeletal muscle contractility by oxygen tension and endogenous nitric oxide

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

    doi: 10.1073/pnas.2433468100

    Ca 2+ transients ( A ) and sarcomere shortening ( B ) induced by depolarization of myocytes prepared from FDB of WT or nNOS -/- mice and stimulated at 1% or 20% O 2 . Both measures are significantly greater at 1% vs. 20% O 2 in myocytes from WT mice ( * ), but there is no effect of pO 2 on either measure in nNOS -/- myocytes. Note also that the magnitudes of both Ca 2+ transients and sarcomere shortening are significantly greater at 20% O 2 , but not at 1% O 2 , in myocytes from nNOS -/- vs. WT mice ( ** ). ( C ) Representative records of cytosolic Ca 2+ and sarcomere length from an individual WT myocyte and an individual nNOS -/- myocyte. Note that the amplitudes of Ca 2+ transients and of sarcomere shortening are independent of pO 2 in the nNOS -/- myocyte.
    Figure Legend Snippet: Ca 2+ transients ( A ) and sarcomere shortening ( B ) induced by depolarization of myocytes prepared from FDB of WT or nNOS -/- mice and stimulated at 1% or 20% O 2 . Both measures are significantly greater at 1% vs. 20% O 2 in myocytes from WT mice ( * ), but there is no effect of pO 2 on either measure in nNOS -/- myocytes. Note also that the magnitudes of both Ca 2+ transients and sarcomere shortening are significantly greater at 20% O 2 , but not at 1% O 2 , in myocytes from nNOS -/- vs. WT mice ( ** ). ( C ) Representative records of cytosolic Ca 2+ and sarcomere length from an individual WT myocyte and an individual nNOS -/- myocyte. Note that the amplitudes of Ca 2+ transients and of sarcomere shortening are independent of pO 2 in the nNOS -/- myocyte.

    Techniques Used: Mouse Assay

    16) Product Images from "Systemic Immune Deficiency Necessary for Cytomegalovirus Invasion of the Mature Brain"

    Article Title: Systemic Immune Deficiency Necessary for Cytomegalovirus Invasion of the Mature Brain

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.3.1473-1487.2004

    Temporal quantification of neurotropic mCMV infection in SCID mice following peripheral inoculation. (A) Mean number of CMV foci per brain; no significant differences were found in localization to the fore-, mid-, or hindbrain. (B) Mean number of infected cells per infection focus. The median is displayed as a line across the bar; the range is given in parentheses. (C) Total number of infected cells per brain divided by fore-, mid-, and hindbrain regions. The x axis depicts days following inoculation. Error bars, SEM. Symbols: *, significance at P
    Figure Legend Snippet: Temporal quantification of neurotropic mCMV infection in SCID mice following peripheral inoculation. (A) Mean number of CMV foci per brain; no significant differences were found in localization to the fore-, mid-, or hindbrain. (B) Mean number of infected cells per infection focus. The median is displayed as a line across the bar; the range is given in parentheses. (C) Total number of infected cells per brain divided by fore-, mid-, and hindbrain regions. The x axis depicts days following inoculation. Error bars, SEM. Symbols: *, significance at P

    Techniques Used: Infection, Mouse Assay

    Temporal quantification of mCMV infection in tissue following peripheral inoculation. As more cells became infected with the GFP-expressing CMV, organ fluorescence increased. Significant multifocal, diffuse fluorescence is present in multiple systemic organs in SCID mice but not control mice 21 days following inoculation. Shown are photomicrographs of GFP-labeled cells in spleen (A and D), lung (B and E), and liver (C and F). (A to C) Control mouse; (D to F) SCID mouse. The mean numbers of infected cells in peripheral organs are similar in both control and SCID phenotypes in acute infection but significantly increase over time in SCID mice (G) while disappearing completely in control mice (I) by 21 days following inoculation. Group sample sizes are indicated (N). Dissemination to the CNS occurred only in SCID mice, beginning 21 days dpi and increasing through 35 dpi. (H) Counts of fluorescent cell number per unit area in the lung after mCMV-GFP infection. A strong positive correlation was found between organ green fluorescence and the number of infected cells. The x axis depicts days following peripheral inoculation. (G and I) Error bars, SEM. Scale bar = 100 μm.
    Figure Legend Snippet: Temporal quantification of mCMV infection in tissue following peripheral inoculation. As more cells became infected with the GFP-expressing CMV, organ fluorescence increased. Significant multifocal, diffuse fluorescence is present in multiple systemic organs in SCID mice but not control mice 21 days following inoculation. Shown are photomicrographs of GFP-labeled cells in spleen (A and D), lung (B and E), and liver (C and F). (A to C) Control mouse; (D to F) SCID mouse. The mean numbers of infected cells in peripheral organs are similar in both control and SCID phenotypes in acute infection but significantly increase over time in SCID mice (G) while disappearing completely in control mice (I) by 21 days following inoculation. Group sample sizes are indicated (N). Dissemination to the CNS occurred only in SCID mice, beginning 21 days dpi and increasing through 35 dpi. (H) Counts of fluorescent cell number per unit area in the lung after mCMV-GFP infection. A strong positive correlation was found between organ green fluorescence and the number of infected cells. The x axis depicts days following peripheral inoculation. (G and I) Error bars, SEM. Scale bar = 100 μm.

    Techniques Used: Infection, Expressing, Fluorescence, Mouse Assay, Labeling

    Mouse CMV infects different brain regions. mCMV infection of the CNS in SCID mice 21 to 35 days following peripheral inoculation, detected with GFP reporter. (A) Olfactory bulb (granular cell and external plexiform layers). (B) Dorsal peduncle. (C) Focal striatial infection, asymmetrical and localized. Multiple cell types are infected with mCMV, including astrocytes, neurons, and glial cells. (D) Extension of GFP from neuronal cell body to dendritic processes. (E) Infected cells of the choroid plexus (arrows). (F) Localized region of infection near the central canal above the medulla; infected tanycyte processes (arrow). Unstained sections. Bar = 25 μm (C), 50 μm (B and D), or 100 μm (A, E, and F). (G) Schematic diagrams of coronal sections through immunodeficient mouse brain illustrating CMV-infected foci following peripheral inoculation. CMV-infected cells (dots) are asymmetrical in location and extend by local expansion rather than following neuronal tracts. Brains typically have several independent and unrelated sites of infection, suggesting that a unique event is occurring at each location, allowing virus to penetrate the CNS and replicate locally.
    Figure Legend Snippet: Mouse CMV infects different brain regions. mCMV infection of the CNS in SCID mice 21 to 35 days following peripheral inoculation, detected with GFP reporter. (A) Olfactory bulb (granular cell and external plexiform layers). (B) Dorsal peduncle. (C) Focal striatial infection, asymmetrical and localized. Multiple cell types are infected with mCMV, including astrocytes, neurons, and glial cells. (D) Extension of GFP from neuronal cell body to dendritic processes. (E) Infected cells of the choroid plexus (arrows). (F) Localized region of infection near the central canal above the medulla; infected tanycyte processes (arrow). Unstained sections. Bar = 25 μm (C), 50 μm (B and D), or 100 μm (A, E, and F). (G) Schematic diagrams of coronal sections through immunodeficient mouse brain illustrating CMV-infected foci following peripheral inoculation. CMV-infected cells (dots) are asymmetrical in location and extend by local expansion rather than following neuronal tracts. Brains typically have several independent and unrelated sites of infection, suggesting that a unique event is occurring at each location, allowing virus to penetrate the CNS and replicate locally.

    Techniques Used: Infection, Mouse Assay

    17) Product Images from "Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1"

    Article Title: Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19041215

    High glucose-induces endothelial cell senescence. ( A ) Senescence associated β-Gal activity (black arrows) and quantification ( B ) showing a significant increase in SA β-Gal positive cells with high glucose (HG, 25 mM) treatment when compared to normal glucose (NG, 5 mM). ABH treatment prevented this increase. * p
    Figure Legend Snippet: High glucose-induces endothelial cell senescence. ( A ) Senescence associated β-Gal activity (black arrows) and quantification ( B ) showing a significant increase in SA β-Gal positive cells with high glucose (HG, 25 mM) treatment when compared to normal glucose (NG, 5 mM). ABH treatment prevented this increase. * p

    Techniques Used: Activity Assay

    Diabetes induces senescence in retinal tissue. Senescence associated β-galactosidase (SA β-Gal) activity images of frozen retinal sections showing positive signal in cells of the large vessels (white arrows) as well as in the surrounding tissues (red arrows) of the central retina. Scale bar = 20 µM.
    Figure Legend Snippet: Diabetes induces senescence in retinal tissue. Senescence associated β-galactosidase (SA β-Gal) activity images of frozen retinal sections showing positive signal in cells of the large vessels (white arrows) as well as in the surrounding tissues (red arrows) of the central retina. Scale bar = 20 µM.

    Techniques Used: Activity Assay

    A1 deletion prevents diabetes-induced senescence in retinal vessels. SA β-Gal activity images of isolated retinal vessels showing increased activity (arrows) in vessels isolated from WT diabetic mice. The vessels isolated from A1+/− diabetic retinas are negative for SA β-Gal staining. The non-diabetic control retinas from both WT and A1+/− mice are also negative for SA β-Gal staining. n = 3–5. Scale bar = 50 µM.
    Figure Legend Snippet: A1 deletion prevents diabetes-induced senescence in retinal vessels. SA β-Gal activity images of isolated retinal vessels showing increased activity (arrows) in vessels isolated from WT diabetic mice. The vessels isolated from A1+/− diabetic retinas are negative for SA β-Gal staining. The non-diabetic control retinas from both WT and A1+/− mice are also negative for SA β-Gal staining. n = 3–5. Scale bar = 50 µM.

    Techniques Used: Activity Assay, Isolation, Mouse Assay, Staining

    18) Product Images from "Origin of Oligodendrocytes in the Subventricular Zone of the Adult Brain"

    Article Title: Origin of Oligodendrocytes in the Subventricular Zone of the Adult Brain

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.1299-06.2006

    Oligodendrocyte progenitors exist in the SVZ and in the CC of the adult mouse brain. Replication-incompetent murine β-actin p –GFP virus was injected into the SVZ ( A–D ) or the CC ( E , F ) of adult CD-1 mice. At 21 d after injection, GFP-labeled mature myelinating ( A , E ) and nonmyelinating ( B–D , F ) oligodendrocytes were identified in the CC. C , D , Double immunofluorescence with GFP (green) and Olig2 ( C , red) or NG2 ( D , red). Note that highly branched GFP + nonmyelinating oligodendrocytes express both markers. G , Schematic representation of an adult mouse brain hemisphere showing the location of GFP-labeled cells in the CC after β-actin p –GFP injection into the SVZ (red) or the CC (blue). Injection tracks for the SVZ (red) and CC (blue) are represented. Note that GFP-labeled cells are widely dispersed in the adult CC after SVZ retroviral injection in contrast to those generated after injection into the CC. Ctx, Cortex; LV, lateral ventricle; Sep, septum; Str, striatum. Scale bar, 60 μm.
    Figure Legend Snippet: Oligodendrocyte progenitors exist in the SVZ and in the CC of the adult mouse brain. Replication-incompetent murine β-actin p –GFP virus was injected into the SVZ ( A–D ) or the CC ( E , F ) of adult CD-1 mice. At 21 d after injection, GFP-labeled mature myelinating ( A , E ) and nonmyelinating ( B–D , F ) oligodendrocytes were identified in the CC. C , D , Double immunofluorescence with GFP (green) and Olig2 ( C , red) or NG2 ( D , red). Note that highly branched GFP + nonmyelinating oligodendrocytes express both markers. G , Schematic representation of an adult mouse brain hemisphere showing the location of GFP-labeled cells in the CC after β-actin p –GFP injection into the SVZ (red) or the CC (blue). Injection tracks for the SVZ (red) and CC (blue) are represented. Note that GFP-labeled cells are widely dispersed in the adult CC after SVZ retroviral injection in contrast to those generated after injection into the CC. Ctx, Cortex; LV, lateral ventricle; Sep, septum; Str, striatum. Scale bar, 60 μm.

    Techniques Used: Injection, Mouse Assay, Labeling, Immunofluorescence, Generated

    SVZ astrocytes produce oligodendrocytes in vitro , and some produce both neurons and oligodendrocytes. A–F , Ten thousand SVZ cells isolated from adult CD-1 mice were allowed to differentiate on top of an astrocytic monolayer for 3 div ( A–D ) and 5 div ( E , F ). A–D , At 3 div, clusters derived from adult SVZ precursor cells are mixed colonies which contain both O 4 + and Tuj1 + cells. E , F , At 5 div, Tuj1 + ( E ) and O 4 + ( F ) cells present typical morphologies of differentiated neurons and oligodendrocytes, respectively. A , Differential interference contrast (DIC) image of a typical colony. B , E , Immunoreactivity for the neuronal marker Tuj1. C , F , Immunoreactivity for the oligodendroglial marker O 4 . D , Merged field showing combined immunostaining for Tuj1 and O 4 . G–I , Single SVZ astrocytes give rise to both neurons and oligodendrocytes in vitro . Adult SVZ astrocytes were specifically labeled in vivo or in vitro with an adenovirus GFAP p –GFP. G , Schematic showing the GFAP promoter reporter construct used for the adenoviral vector. H , I , Single GFP + SVZ astrocytes were purified by FACS, plated on top of an astrocytic monolayer, and grown in culture for 5 div. H , Density plots showing gates for selecting SVZ astrocytes [GFP and propidium iodide (PI)]. The right plot corresponds to the sorting of SVZ cells isolated at 1.5 d after injection of the adenovirus GFAP p –GFP; the left plot is a control non-injected animal. I , The number of GFP + cells initially plated is indicated for each experiment (3 independent cultures). The total number of colonies that arose from these cells is presented. These colonies contained only O 4 + , only Tuj1 + , or both O 4 + and Tuj1 + cells. Note that 61 of the 233 single GFP + SVZ cells initially plated gave rise to colonies. Among them, seven generated mixed colonies. Scale bar: A–F , 10 μm. SD, Splice donor; SA, splice acceptor.
    Figure Legend Snippet: SVZ astrocytes produce oligodendrocytes in vitro , and some produce both neurons and oligodendrocytes. A–F , Ten thousand SVZ cells isolated from adult CD-1 mice were allowed to differentiate on top of an astrocytic monolayer for 3 div ( A–D ) and 5 div ( E , F ). A–D , At 3 div, clusters derived from adult SVZ precursor cells are mixed colonies which contain both O 4 + and Tuj1 + cells. E , F , At 5 div, Tuj1 + ( E ) and O 4 + ( F ) cells present typical morphologies of differentiated neurons and oligodendrocytes, respectively. A , Differential interference contrast (DIC) image of a typical colony. B , E , Immunoreactivity for the neuronal marker Tuj1. C , F , Immunoreactivity for the oligodendroglial marker O 4 . D , Merged field showing combined immunostaining for Tuj1 and O 4 . G–I , Single SVZ astrocytes give rise to both neurons and oligodendrocytes in vitro . Adult SVZ astrocytes were specifically labeled in vivo or in vitro with an adenovirus GFAP p –GFP. G , Schematic showing the GFAP promoter reporter construct used for the adenoviral vector. H , I , Single GFP + SVZ astrocytes were purified by FACS, plated on top of an astrocytic monolayer, and grown in culture for 5 div. H , Density plots showing gates for selecting SVZ astrocytes [GFP and propidium iodide (PI)]. The right plot corresponds to the sorting of SVZ cells isolated at 1.5 d after injection of the adenovirus GFAP p –GFP; the left plot is a control non-injected animal. I , The number of GFP + cells initially plated is indicated for each experiment (3 independent cultures). The total number of colonies that arose from these cells is presented. These colonies contained only O 4 + , only Tuj1 + , or both O 4 + and Tuj1 + cells. Note that 61 of the 233 single GFP + SVZ cells initially plated gave rise to colonies. Among them, seven generated mixed colonies. Scale bar: A–F , 10 μm. SD, Splice donor; SA, splice acceptor.

    Techniques Used: In Vitro, Isolation, Mouse Assay, Derivative Assay, Marker, Immunostaining, Labeling, In Vivo, Construct, Plasmid Preparation, Purification, FACS, Injection, Generated

    19) Product Images from "Structural Requirements for Basolateral Sorting of the Human Transferrin Receptor in the Biosynthetic and Endocytic Pathways of Madin-Darby Canine Kidney Cells"

    Article Title: Structural Requirements for Basolateral Sorting of the Human Transferrin Receptor in the Biosynthetic and Endocytic Pathways of Madin-Darby Canine Kidney Cells

    Journal: The Journal of Cell Biology

    doi:

    TR is sorted efficiently to the basolateral surface upon transplantation of residues 29–41 to a more membrane- distal position in the cytoplasmic tail. ( A ) The amino acid sequence of the TR cytoplasmic tail depicting the transplantation of residues 29– 41 to a more membrane- distal position located between Pro-17 and Leu-18. ( B ) The fraction of newly synthesized and internalized mutant 29–41 transplant TR sorted basolaterally was determined as described in the legend to Fig. 2 .
    Figure Legend Snippet: TR is sorted efficiently to the basolateral surface upon transplantation of residues 29–41 to a more membrane- distal position in the cytoplasmic tail. ( A ) The amino acid sequence of the TR cytoplasmic tail depicting the transplantation of residues 29– 41 to a more membrane- distal position located between Pro-17 and Leu-18. ( B ) The fraction of newly synthesized and internalized mutant 29–41 transplant TR sorted basolaterally was determined as described in the legend to Fig. 2 .

    Techniques Used: Transplantation Assay, Sequencing, Synthesized, Mutagenesis

    Residues 19–41 of the TR cytoplasmic tail are necessary and sufficient for basolateral sorting in both the biosynthetic and endocytic pathways. ( A ) Newly synthesized wild-type and mutant TR expressed in filter-grown MDCK cells were pulse- labeled with 35 S-labeled methionine/cysteine for 30 min and then chased to the cell surface for 20 min at 37°C. Receptors at the apical or basolateral surface were then cleaved at 4°C for 30 min with trypsin (100 μg/ml). Trypsin inhibitor (100 μg/ml) was included in the opposite media. B3/25 mAb immunoprecipitates of the ∼70-kD TR extracellular fragment in the media collected from each surface were analyzed on SDS–polyacrylamide gels and quantitated by phosphorimage analysis (mean ± standard error of three independent experiments). No material could be detected in immunoprecipitates from the media containing trypsin inhibitor. ( B) Newly synthesized receptors were pulse-labeled as described above and chased at 37°C for 30 min ( black bars ), 60 min ( gray bars ), or 120 min ( open bars ). Trypsin was added to the apical or basolateral surface of the monolayers at 4°C as described above or was included in the apical or basolateral media during the 37°C chase. TR external domain fragments were immunoprecipitated and analyzed by SDS-PAGE as described above. ( C) Filter-grown MDCK cells expressing wild-type and mutant TR were incubated either apically or basolaterally at 37°C for 1 h with 125 I-labeled Tf. Monolayers were then washed at 4°C, and surface-bound 125 I-labeled Tf was removed with deferoxamine mesylate. Cells were then incubated at 37°C for 90 min, and the appearance of 125 I-labeled Tf in the apical and basolateral media was determined (mean ± standard error of three independent experiments). More than 90% of the internalized 125 I-labeled Tf recycled after 90 min at 37°C.
    Figure Legend Snippet: Residues 19–41 of the TR cytoplasmic tail are necessary and sufficient for basolateral sorting in both the biosynthetic and endocytic pathways. ( A ) Newly synthesized wild-type and mutant TR expressed in filter-grown MDCK cells were pulse- labeled with 35 S-labeled methionine/cysteine for 30 min and then chased to the cell surface for 20 min at 37°C. Receptors at the apical or basolateral surface were then cleaved at 4°C for 30 min with trypsin (100 μg/ml). Trypsin inhibitor (100 μg/ml) was included in the opposite media. B3/25 mAb immunoprecipitates of the ∼70-kD TR extracellular fragment in the media collected from each surface were analyzed on SDS–polyacrylamide gels and quantitated by phosphorimage analysis (mean ± standard error of three independent experiments). No material could be detected in immunoprecipitates from the media containing trypsin inhibitor. ( B) Newly synthesized receptors were pulse-labeled as described above and chased at 37°C for 30 min ( black bars ), 60 min ( gray bars ), or 120 min ( open bars ). Trypsin was added to the apical or basolateral surface of the monolayers at 4°C as described above or was included in the apical or basolateral media during the 37°C chase. TR external domain fragments were immunoprecipitated and analyzed by SDS-PAGE as described above. ( C) Filter-grown MDCK cells expressing wild-type and mutant TR were incubated either apically or basolaterally at 37°C for 1 h with 125 I-labeled Tf. Monolayers were then washed at 4°C, and surface-bound 125 I-labeled Tf was removed with deferoxamine mesylate. Cells were then incubated at 37°C for 90 min, and the appearance of 125 I-labeled Tf in the apical and basolateral media was determined (mean ± standard error of three independent experiments). More than 90% of the internalized 125 I-labeled Tf recycled after 90 min at 37°C.

    Techniques Used: Synthesized, Mutagenesis, Labeling, Immunoprecipitation, SDS Page, Expressing, Incubation

    Residues 29–41 of the TR cytoplasmic tail are the most important for basolateral sorting of newly synthesized TR. Newly synthesized wild-type and mutant TR expressed in filter-grown MDCK cells were pulse-labeled and chased to the cell surface, and the ∼70-kD TR extracellular fragment derived from receptors at either the apical or basolateral surface was immunoprecipitated and quantitated as described in the legend to Fig. 2 A.
    Figure Legend Snippet: Residues 29–41 of the TR cytoplasmic tail are the most important for basolateral sorting of newly synthesized TR. Newly synthesized wild-type and mutant TR expressed in filter-grown MDCK cells were pulse-labeled and chased to the cell surface, and the ∼70-kD TR extracellular fragment derived from receptors at either the apical or basolateral surface was immunoprecipitated and quantitated as described in the legend to Fig. 2 A.

    Techniques Used: Synthesized, Mutagenesis, Labeling, Derivative Assay, Immunoprecipitation

    The structural requirements for basolateral sorting of TR in the endocytic pathway differ from the structural requirements for sorting in the biosynthetic pathway. The endocytic pathways of MDCK cells expressing wild-type or mutant TR were loaded with 125 I-labeled Tf for 1 h at 37°C from either the apical ( A ) or basolateral ( B ) border. Monolayers were then washed at 4°C, and the amount of radiolabel recycled to the apical or basolateral surface after 90 min at 37°C was determined as described in the legend to Fig. 2 C.
    Figure Legend Snippet: The structural requirements for basolateral sorting of TR in the endocytic pathway differ from the structural requirements for sorting in the biosynthetic pathway. The endocytic pathways of MDCK cells expressing wild-type or mutant TR were loaded with 125 I-labeled Tf for 1 h at 37°C from either the apical ( A ) or basolateral ( B ) border. Monolayers were then washed at 4°C, and the amount of radiolabel recycled to the apical or basolateral surface after 90 min at 37°C was determined as described in the legend to Fig. 2 C.

    Techniques Used: Expressing, Mutagenesis, Labeling

    Features of the TR basolateral sorting signal. Residues 19–41, which are necessary and sufficient for basolateral sorting of TR, are shown. Each of the two four-residue sequences within this region that are predicted to adopt a tight turn conformation, the YTRF internalization signal and the sequence GDNS ( Collawn et al., 1990 ), are underlined. The region most important for basolateral sorting is within residues 29–41, and the TR basolateral sorting signal is neither tyrosine based nor colinear with the YTRF internalization signal. Also indicated is the four-residue sequence RQVD, which is similar to the sequence RNVD found to be important for basolateral sorting of pIgR ( Aroeti et al., 1993 ). However, the sequence RQVD is not required for basolateral sorting of TR, and neither Val-29 nor Val-36 is important for activity of the TR basolateral sorting signal.
    Figure Legend Snippet: Features of the TR basolateral sorting signal. Residues 19–41, which are necessary and sufficient for basolateral sorting of TR, are shown. Each of the two four-residue sequences within this region that are predicted to adopt a tight turn conformation, the YTRF internalization signal and the sequence GDNS ( Collawn et al., 1990 ), are underlined. The region most important for basolateral sorting is within residues 29–41, and the TR basolateral sorting signal is neither tyrosine based nor colinear with the YTRF internalization signal. Also indicated is the four-residue sequence RQVD, which is similar to the sequence RNVD found to be important for basolateral sorting of pIgR ( Aroeti et al., 1993 ). However, the sequence RQVD is not required for basolateral sorting of TR, and neither Val-29 nor Val-36 is important for activity of the TR basolateral sorting signal.

    Techniques Used: Sequencing, Activity Assay

    Residues 19–41 of the TR cytoplasmic tail are necessary and sufficient to target TR to the basolateral surface of MDCK cells. Filter-grown MDCK cells expressing wild-type and mutant TR were incubated either apically or basolaterally for 1 h at 4°C with 125 I-labeled Tf (4 μg/ml). After washing away unbound Tf, the amount of 125 I-labeled Tf specifically bound on each surface was determined (mean ± standard error of three independent experiments).
    Figure Legend Snippet: Residues 19–41 of the TR cytoplasmic tail are necessary and sufficient to target TR to the basolateral surface of MDCK cells. Filter-grown MDCK cells expressing wild-type and mutant TR were incubated either apically or basolaterally for 1 h at 4°C with 125 I-labeled Tf (4 μg/ml). After washing away unbound Tf, the amount of 125 I-labeled Tf specifically bound on each surface was determined (mean ± standard error of three independent experiments).

    Techniques Used: Expressing, Mutagenesis, Incubation, Labeling

    20) Product Images from "A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation"

    Article Title: A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20132267

    Cul3 regulates Tfh responses in mature CD4 + splenocytes. CD4 + splenocytes from OTII Tg Cul3 fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3 . Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP + cells among donor cells (CD45.2 + ) at time of recovery. The second and third columns show staining of gated CD4 + donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1 hi CXCR5 hi Tfh cells based on three separate experiments for day 5 ( n = 5) and day 7 ( n = 6). Horizontal bars indicate mean. ***, P
    Figure Legend Snippet: Cul3 regulates Tfh responses in mature CD4 + splenocytes. CD4 + splenocytes from OTII Tg Cul3 fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3 . Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP + cells among donor cells (CD45.2 + ) at time of recovery. The second and third columns show staining of gated CD4 + donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1 hi CXCR5 hi Tfh cells based on three separate experiments for day 5 ( n = 5) and day 7 ( n = 6). Horizontal bars indicate mean. ***, P

    Techniques Used: Mouse Assay, Transduction, Expressing, Injection, Staining

    Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4 + SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10 −6 ). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4 + SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P
    Figure Legend Snippet: Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4 + SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4 + SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10 −6 ). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4 + SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P

    Techniques Used: Expressing, Quantitative RT-PCR, Genome Wide, Generated, Magnetic Cell Separation, FACS, Irradiation, Purification

    Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP 16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1 hi CXCR5 hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4 + SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP 41 (left) and BSA-NP 4 (right) at days 0 and 21 after immunization with OVA-NP 16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P
    Figure Legend Snippet: Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP 16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1 hi CXCR5 hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4 + SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP 41 (left) and BSA-NP 4 (right) at days 0 and 21 after immunization with OVA-NP 16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P

    Techniques Used: Injection, Mouse Assay, FACS, Staining, Fluorescence, In Vivo

    Exaggerated Tfh responses without alteration of Th1 or Th2 programs. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII or OTII Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before injection of OVA + CFA s.c., OVA + alum i.p., or PBS, as indicated. OTII cells were analyzed at day 7 after immunization in inguinal lymph nodes (CFA; a) or spleen (alum; b). (first row) Expression of CXCR5 and PD1. Numbers above the dot plots are the absolute numbers of OTII cells recovered from the lymph nodes or spleen. Numbers in top right quadrants are the percentage of CXCR5 + PD1 + (mean ± SEM). (second row) Expression of Ki67 and CXCR5. (third row) Expression of Tbet and Bcl6. Numbers indicate the percentage (mean ± SEM) of Tbet hi Bcl6 hi cells (right box) or Tbet hi Bcl6 int cells (left box). Background staining is shown after preincubation with an excess of unconjugated antibodies (cold Tbet+Bcl6). (fourth row) Expression of Gata3 and CXCR5 with quadrant statistics (mean ± SEM). Background staining is shown for fluorochrome-conjugated isotype control. Data are representative of two independent experiments with total n = 6 mice.
    Figure Legend Snippet: Exaggerated Tfh responses without alteration of Th1 or Th2 programs. (a and b) 0.5 × 10 6 CD4 + enriched SP thymocytes from OTII or OTII Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before injection of OVA + CFA s.c., OVA + alum i.p., or PBS, as indicated. OTII cells were analyzed at day 7 after immunization in inguinal lymph nodes (CFA; a) or spleen (alum; b). (first row) Expression of CXCR5 and PD1. Numbers above the dot plots are the absolute numbers of OTII cells recovered from the lymph nodes or spleen. Numbers in top right quadrants are the percentage of CXCR5 + PD1 + (mean ± SEM). (second row) Expression of Ki67 and CXCR5. (third row) Expression of Tbet and Bcl6. Numbers indicate the percentage (mean ± SEM) of Tbet hi Bcl6 hi cells (right box) or Tbet hi Bcl6 int cells (left box). Background staining is shown after preincubation with an excess of unconjugated antibodies (cold Tbet+Bcl6). (fourth row) Expression of Gata3 and CXCR5 with quadrant statistics (mean ± SEM). Background staining is shown for fluorochrome-conjugated isotype control. Data are representative of two independent experiments with total n = 6 mice.

    Techniques Used: Injection, Expressing, Staining, Mouse Assay

    21) Product Images from "Muscle development and regeneration controlled by AUF1-mediated stage-specific degradation of fate-determining checkpoint mRNAs"

    Article Title: Muscle development and regeneration controlled by AUF1-mediated stage-specific degradation of fate-determining checkpoint mRNAs

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

    doi: 10.1073/pnas.1901165116

    Auf1 −/− satellite cells show aberrant terminal differentiation. ( A ) Representative cultured mass preparation of hindlimb skeletal muscles harvested from 4-mo-old mice, 10 d in culture. n = 3. Proliferating myoblasts (MyoD), white arrows; elongated myocytes (Myogenin), orange arrows; myofibers, yellow arrows. Nuclei stained with DAPI. ( B ) Representative IF staining of MyoD (green) and Myogenin (red) in WT and AUF1 KO-isolated myofibers, cultured for 72 h. Ten myofibers analyzed per mouse, n = 3. ( C ) Representative IF staining of Flag-AUF1 (red) and nuclei (DAPI, blue) in WT and AUF1 KO myofibers. Myofibers from mass preparations of the TA muscle as in A transduced with lentivirus vectors expressing AUF1 cDNAs for 72 h. Ten myofibers per group analyzed, n = 3. ( D ) Quantification of nuclei number and AUF1 in myofibers transduced with individual AUF1 isoforms, as in C , n = 3. (Scale bars: 100 μm.)
    Figure Legend Snippet: Auf1 −/− satellite cells show aberrant terminal differentiation. ( A ) Representative cultured mass preparation of hindlimb skeletal muscles harvested from 4-mo-old mice, 10 d in culture. n = 3. Proliferating myoblasts (MyoD), white arrows; elongated myocytes (Myogenin), orange arrows; myofibers, yellow arrows. Nuclei stained with DAPI. ( B ) Representative IF staining of MyoD (green) and Myogenin (red) in WT and AUF1 KO-isolated myofibers, cultured for 72 h. Ten myofibers analyzed per mouse, n = 3. ( C ) Representative IF staining of Flag-AUF1 (red) and nuclei (DAPI, blue) in WT and AUF1 KO myofibers. Myofibers from mass preparations of the TA muscle as in A transduced with lentivirus vectors expressing AUF1 cDNAs for 72 h. Ten myofibers per group analyzed, n = 3. ( D ) Quantification of nuclei number and AUF1 in myofibers transduced with individual AUF1 isoforms, as in C , n = 3. (Scale bars: 100 μm.)

    Techniques Used: Cell Culture, Mouse Assay, Staining, Isolation, Transduction, Expressing

    22) Product Images from "A RUNX2 stabilization pathway mediates physiologic and pathologic bone formation"

    Article Title: A RUNX2 stabilization pathway mediates physiologic and pathologic bone formation

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16038-6

    HAUSP is required for maturation of osteoprogenitors in vitro and in vivo. a – e COBs isolated from P5 Hausp fl/fl pups were infected with lentiviruses expressing vector (WT) or Cre recombinase ( Hausp KO) and cultured under osteogenic conditions. RT-PCR analysis to measure mRNA levels of Hausp and osteogenic genes ( a ) and ALP activity ( b , left) and staining (b, right) were performed 7 days after the culture. Mineralization was assessed by alizarin red staining 16 days after the culture ( c ). Protein ( d ) and mRNA ( e ) levels of RUNX2 were assessed in these COBs. Scale bar, 100 μm ( b ). a , e n = 4; b , c n = 9. f shCtrl or sh Hausp -expressing C3H10T1/2 cells were transfected with OG2-luc and Renilla in the presence or absence of RUNX2 overexpression. Two days after transfection, OG2-luc activity was measured and normalized to Renilla . ( n = 6 biologically independent samples). Hausp -sufficient or -deficient COBs were infected with lentiviruses expressing vector or RUNX2 , cultured under osteogenic conditions for 7 days, and ALP activity ( g ) and mRNA levels of Bglap2 ( h ) were assessed. g n = 6; h n = 4 biologically independent samples). i Hausp mRNA levels in P0 Hausp fl/fl , Osx-Cre , and Hausp +/Osx calvaria. ( n = 4). j , l , m MicroCT analysis shows 3D-reconstruction of calvaria ( j ) and femurs ( l ) from 2-month-old Hausp fl/fl , Osx-Cre and Hausp +/Osx male mice. Quantification of femoral bone mass is displayed (m). The arrows indicate hypomineralization areas. Scale bars, 2 mm ( j ); 500 μm ( l ). m n = 10 ( Hausp fl/fl ), 5 ( Osx-Cre ) or 10 ( Hausp +/Osx ). k Alizarin red/alcian blue staining of skeletal preparations of clavicles obtained from 2-month-old Hausp fl/fl , Osx-Cre and Hausp +/Osx male mice. Scale bar, 2 mm. n H E-stained longitudinal sections of femurs from 2-month-old Osx-Cre and Hausp +/Osx male mice. Scale bar, 50 μm. Alizarin red/alcian blue staining of skeletal preparations of calvaria (top) and clavicles (bottom) from P10 Osx-Cre , Csnk2b +/Osx , Hausp +/Osx , and Csnk2b +/Osx ; Hausp +/Osx mice ( o ) and Osx-Cre , Runx2 +/Osx , Runx2 +/Osx ; Csnk2b +/Osx , and Runx2 +/Osx ; Hausp +/Osx mice ( p ). Scale bars, 2 mm ( o , p ). Data are representative of three ( a – l , n – p ) independent experiments or are pooled from two experiments ( m ). A two-tailed unpaired Student’s t test for comparing two groups ( a – c , e – i , m ; error bars, SD of biological replicates).
    Figure Legend Snippet: HAUSP is required for maturation of osteoprogenitors in vitro and in vivo. a – e COBs isolated from P5 Hausp fl/fl pups were infected with lentiviruses expressing vector (WT) or Cre recombinase ( Hausp KO) and cultured under osteogenic conditions. RT-PCR analysis to measure mRNA levels of Hausp and osteogenic genes ( a ) and ALP activity ( b , left) and staining (b, right) were performed 7 days after the culture. Mineralization was assessed by alizarin red staining 16 days after the culture ( c ). Protein ( d ) and mRNA ( e ) levels of RUNX2 were assessed in these COBs. Scale bar, 100 μm ( b ). a , e n = 4; b , c n = 9. f shCtrl or sh Hausp -expressing C3H10T1/2 cells were transfected with OG2-luc and Renilla in the presence or absence of RUNX2 overexpression. Two days after transfection, OG2-luc activity was measured and normalized to Renilla . ( n = 6 biologically independent samples). Hausp -sufficient or -deficient COBs were infected with lentiviruses expressing vector or RUNX2 , cultured under osteogenic conditions for 7 days, and ALP activity ( g ) and mRNA levels of Bglap2 ( h ) were assessed. g n = 6; h n = 4 biologically independent samples). i Hausp mRNA levels in P0 Hausp fl/fl , Osx-Cre , and Hausp +/Osx calvaria. ( n = 4). j , l , m MicroCT analysis shows 3D-reconstruction of calvaria ( j ) and femurs ( l ) from 2-month-old Hausp fl/fl , Osx-Cre and Hausp +/Osx male mice. Quantification of femoral bone mass is displayed (m). The arrows indicate hypomineralization areas. Scale bars, 2 mm ( j ); 500 μm ( l ). m n = 10 ( Hausp fl/fl ), 5 ( Osx-Cre ) or 10 ( Hausp +/Osx ). k Alizarin red/alcian blue staining of skeletal preparations of clavicles obtained from 2-month-old Hausp fl/fl , Osx-Cre and Hausp +/Osx male mice. Scale bar, 2 mm. n H E-stained longitudinal sections of femurs from 2-month-old Osx-Cre and Hausp +/Osx male mice. Scale bar, 50 μm. Alizarin red/alcian blue staining of skeletal preparations of calvaria (top) and clavicles (bottom) from P10 Osx-Cre , Csnk2b +/Osx , Hausp +/Osx , and Csnk2b +/Osx ; Hausp +/Osx mice ( o ) and Osx-Cre , Runx2 +/Osx , Runx2 +/Osx ; Csnk2b +/Osx , and Runx2 +/Osx ; Hausp +/Osx mice ( p ). Scale bars, 2 mm ( o , p ). Data are representative of three ( a – l , n – p ) independent experiments or are pooled from two experiments ( m ). A two-tailed unpaired Student’s t test for comparing two groups ( a – c , e – i , m ; error bars, SD of biological replicates).

    Techniques Used: In Vitro, In Vivo, Isolation, Infection, Expressing, Plasmid Preparation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Staining, Transfection, Over Expression, Mouse Assay, Two Tailed Test

    23) Product Images from "Wntless regulates lipogenic gene expression in adipocytes and protects against diet-induced metabolic dysfunction"

    Article Title: Wntless regulates lipogenic gene expression in adipocytes and protects against diet-induced metabolic dysfunction

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2020.100992

    Canonical Wnt signaling is active in cultured adipocytes and Wntless is upregulated by diet-induced obesity . ( A–B ) MSCs isolated from C57BL/6J mice were cultured under standard conditions and induced to differentiate. Wntless gene (n = 6) and protein expression at indicated days of differentiation. ( C ) Wntless gene expression in the stromal-vascular (SVF) and adipocyte (Ads) fractions isolated from epididymal (eWAT) and inguinal (iWAT) white adipose tissues of C57BL/6J mice (males; n = 6). ( D ) Representative immunoblot of Wntless protein expression across C57BL/6J mouse tissues (asWAT, anterior subcutaneous WAT; pWAT, perirenal WAT; and BAT, brown adipose tissue); adiponectin, laminin, and Ponceau S were included as controls. ( E–F ) Regulation of Wntless gene and protein expression in eWAT and iWAT of 16-week-old wild-type mice fed a normal chow diet (NCD) or 8 weeks of high-fat diet (HFD) (males; n = 6). ( G ) Wnt-related genes found to be significantly up- or down-regulated by RNA-seq analyses of day 12 adipocytes vs day 0 cultured MSCs (n = 4). ( H ) Select Wnt-related genes found to be significantly up- or down-regulated by RNA-seq analyses of day 12 cultured adipocytes treated with recombinant Wnt3a (20 ng/ml) for 4 h (n = 4). RNA expression normalized to PPIA. Data are presented as mean ± S.D. ∗ indicates significance at p
    Figure Legend Snippet: Canonical Wnt signaling is active in cultured adipocytes and Wntless is upregulated by diet-induced obesity . ( A–B ) MSCs isolated from C57BL/6J mice were cultured under standard conditions and induced to differentiate. Wntless gene (n = 6) and protein expression at indicated days of differentiation. ( C ) Wntless gene expression in the stromal-vascular (SVF) and adipocyte (Ads) fractions isolated from epididymal (eWAT) and inguinal (iWAT) white adipose tissues of C57BL/6J mice (males; n = 6). ( D ) Representative immunoblot of Wntless protein expression across C57BL/6J mouse tissues (asWAT, anterior subcutaneous WAT; pWAT, perirenal WAT; and BAT, brown adipose tissue); adiponectin, laminin, and Ponceau S were included as controls. ( E–F ) Regulation of Wntless gene and protein expression in eWAT and iWAT of 16-week-old wild-type mice fed a normal chow diet (NCD) or 8 weeks of high-fat diet (HFD) (males; n = 6). ( G ) Wnt-related genes found to be significantly up- or down-regulated by RNA-seq analyses of day 12 adipocytes vs day 0 cultured MSCs (n = 4). ( H ) Select Wnt-related genes found to be significantly up- or down-regulated by RNA-seq analyses of day 12 cultured adipocytes treated with recombinant Wnt3a (20 ng/ml) for 4 h (n = 4). RNA expression normalized to PPIA. Data are presented as mean ± S.D. ∗ indicates significance at p

    Techniques Used: Cell Culture, Isolation, Mouse Assay, Expressing, RNA Sequencing Assay, Recombinant, RNA Expression

    24) Product Images from "Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout"

    Article Title: Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout

    Journal: Communications Biology

    doi: 10.1038/s42003-020-1025-y

    Establishing the inhibin - DsRed transgenic rainbow trout strain and Sertoli cell line. a Localization of inhibin α mRNA in immature testes assessed by in situ hybridization. Inhibin mRNA was expressed in Sertoli cells surrounding ASGs. b Micrograph of immature testes from 13-month-old inhibin - DsRed transgenic and nontransgenic rainbow trout. c Fluorescent view of the same field as b . d , e Higher magnification views of an immature testis from an inhibin - DsRed transgenic rainbow trout. f – i Confocal microscopy of an immature testis from a 13-month-old vasa - gfp / inhibin - DsRed double transgenic rainbow trout. ASGs (green) were surrounded by Sertoli cells (red). Cell nuclei were stained with Hoechst 33342 (blue). Insets show a higher magnification of each photo. j Flow cytometry analysis of immature testes from nontransgenic (Non-TG) and inhibin - DsRed transgenic rainbow trout. The gated regions with blue lines and orange lines indicate the DsRed(−) population and DsRed(+) population, respectively. k – n Bright and U-MWIG2 filter-fluorescent views of the dissociated immature testicular cells ( k , l ) and Sertoli cells isolated by flow cytometry ( m , n ) from inhibin - DsRed transgenic rainbow trout. o The effect of fish serum on TSC growth. p The effect of FBS on TSC growth. The Sertoli cell counts were determined following 14 days of culture. Data are shown as the mean ± SEM ( n = 3). Groups with different letters are significantly different based on one-way ANOVA followed by Tukey’s multiple comparisons test ( P
    Figure Legend Snippet: Establishing the inhibin - DsRed transgenic rainbow trout strain and Sertoli cell line. a Localization of inhibin α mRNA in immature testes assessed by in situ hybridization. Inhibin mRNA was expressed in Sertoli cells surrounding ASGs. b Micrograph of immature testes from 13-month-old inhibin - DsRed transgenic and nontransgenic rainbow trout. c Fluorescent view of the same field as b . d , e Higher magnification views of an immature testis from an inhibin - DsRed transgenic rainbow trout. f – i Confocal microscopy of an immature testis from a 13-month-old vasa - gfp / inhibin - DsRed double transgenic rainbow trout. ASGs (green) were surrounded by Sertoli cells (red). Cell nuclei were stained with Hoechst 33342 (blue). Insets show a higher magnification of each photo. j Flow cytometry analysis of immature testes from nontransgenic (Non-TG) and inhibin - DsRed transgenic rainbow trout. The gated regions with blue lines and orange lines indicate the DsRed(−) population and DsRed(+) population, respectively. k – n Bright and U-MWIG2 filter-fluorescent views of the dissociated immature testicular cells ( k , l ) and Sertoli cells isolated by flow cytometry ( m , n ) from inhibin - DsRed transgenic rainbow trout. o The effect of fish serum on TSC growth. p The effect of FBS on TSC growth. The Sertoli cell counts were determined following 14 days of culture. Data are shown as the mean ± SEM ( n = 3). Groups with different letters are significantly different based on one-way ANOVA followed by Tukey’s multiple comparisons test ( P

    Techniques Used: Transgenic Assay, In Situ Hybridization, Confocal Microscopy, Staining, Flow Cytometry, Isolation, Fluorescence In Situ Hybridization

    25) Product Images from "Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells"

    Article Title: Microbiota-derived butyrate limits the autoimmune response by promoting the differentiation of follicular regulatory T cells

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2020.102913

    iT FR  cells prevent autoimmune responses and CIA development (a, b) CII-specific IgG responses in CIA mice inoculated with Foxp3-hCD2 +  cells cultured under iT FR -cell conditions in the absence or presence of 100 µM SB. Sort-purified 5 × 10 6  of Foxp3-hCD2 + CD4 + T  cells were injected intravenously into DBA/1 J mice one week before the initial CII immunization.All mice were fed the control HAMS diet ( n = 10, 11). The number of CII-specific IgG AFC cells per 1 × 10 5  DLN cells (a, each dot indicates data from 1 well,  n = 44, 32, statistical analysis was performed on the mean of each mouse.), and serum levels of CII-specific IgG 1  and IgG 2a  three weeks after booster immunization (b,  n = 5, 6). (c) Arthritis scores after booster immunization of CIA mice inoculated with Foxp3-hCD2 +  cells from iT FR  cell culture ( n = 10, 11). AUC was calculated in each mice. Results show one representative experiment of at least two experiments.** P
    Figure Legend Snippet: iT FR cells prevent autoimmune responses and CIA development (a, b) CII-specific IgG responses in CIA mice inoculated with Foxp3-hCD2 + cells cultured under iT FR -cell conditions in the absence or presence of 100 µM SB. Sort-purified 5 × 10 6 of Foxp3-hCD2 + CD4 + T cells were injected intravenously into DBA/1 J mice one week before the initial CII immunization.All mice were fed the control HAMS diet ( n = 10, 11). The number of CII-specific IgG AFC cells per 1 × 10 5 DLN cells (a, each dot indicates data from 1 well, n = 44, 32, statistical analysis was performed on the mean of each mouse.), and serum levels of CII-specific IgG 1 and IgG 2a three weeks after booster immunization (b, n = 5, 6). (c) Arthritis scores after booster immunization of CIA mice inoculated with Foxp3-hCD2 + cells from iT FR cell culture ( n = 10, 11). AUC was calculated in each mice. Results show one representative experiment of at least two experiments.** P

    Techniques Used: Mouse Assay, Cell Culture, Purification, Injection

    26) Product Images from "ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression"

    Article Title: ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31784-w

    ADAP1 does not interfere with Mek1ca-induced fetal gene program activation. ( A – I ) Analysis by RT-qPCR of different mRNA expressed in rat neonatal ventricular cardiomyocytes (RNVC) that are representative of the fetal gene program. The RNVC were infected with an β-Gal- (negative control), ADAP1-, or Mek1ca-overexpressing adenovirus, individually or in combination as indicated, and were cultured for 72 h post-infection. The histograms represent mRNA expression levels relative to the β-Gal control and normalized to the Rpl30 reporter gene ( n = 3 independent experiments). * P
    Figure Legend Snippet: ADAP1 does not interfere with Mek1ca-induced fetal gene program activation. ( A – I ) Analysis by RT-qPCR of different mRNA expressed in rat neonatal ventricular cardiomyocytes (RNVC) that are representative of the fetal gene program. The RNVC were infected with an β-Gal- (negative control), ADAP1-, or Mek1ca-overexpressing adenovirus, individually or in combination as indicated, and were cultured for 72 h post-infection. The histograms represent mRNA expression levels relative to the β-Gal control and normalized to the Rpl30 reporter gene ( n = 3 independent experiments). * P

    Techniques Used: Activation Assay, Quantitative RT-PCR, Infection, Negative Control, Cell Culture, Expressing

    ADAP1 restrains the serum-induced increase in cell size of cultured cardiomyocytes. ( A ) Western blot detection of adenovirus-mediated 3xFLAG-hADAP1 overexpression (MOI of 50) in rat neonatal ventricular cardiomyocytes (RNVC) cultured for 72 h post-infection. ( B ) RNVC were infected with either β-Gal- (negative control) or ADAP1-overexpressing adenovirus and were cultured for 72 h in the absence (0%) or presence (10%) of serum. Representative images of α-Actinin-immunostained RNVC (left) and corresponding segmented images were acquired using the Operetta High-Content Imaging System (Perkin Elmer). The scale bar represents 50 µm. ( C ) The histogram represents the cell surface areas of RNVC overexpressing either β-Gal or ADAP1 and cultured for 72 h with increasing concentrations of serum ( n = 3 independent experiments). * P
    Figure Legend Snippet: ADAP1 restrains the serum-induced increase in cell size of cultured cardiomyocytes. ( A ) Western blot detection of adenovirus-mediated 3xFLAG-hADAP1 overexpression (MOI of 50) in rat neonatal ventricular cardiomyocytes (RNVC) cultured for 72 h post-infection. ( B ) RNVC were infected with either β-Gal- (negative control) or ADAP1-overexpressing adenovirus and were cultured for 72 h in the absence (0%) or presence (10%) of serum. Representative images of α-Actinin-immunostained RNVC (left) and corresponding segmented images were acquired using the Operetta High-Content Imaging System (Perkin Elmer). The scale bar represents 50 µm. ( C ) The histogram represents the cell surface areas of RNVC overexpressing either β-Gal or ADAP1 and cultured for 72 h with increasing concentrations of serum ( n = 3 independent experiments). * P

    Techniques Used: Cell Culture, Western Blot, Over Expression, Infection, Negative Control, Imaging

    ADAP1 blocks phenylephrine- and Mek1ca-induced hypertrophy. Cell surface area measurements of rat neonatal ventricular cardiomyocytes (RNVC) overexpressing ADAP1 in the absence or presence of 50 µM phenylephrine ( A ) or Mek1ca-overexpressing adenovirus ( B ) compared with an adβ-Gal-infected control. Quantification of at least 3 independent experiments expressed as means ± SD. * P
    Figure Legend Snippet: ADAP1 blocks phenylephrine- and Mek1ca-induced hypertrophy. Cell surface area measurements of rat neonatal ventricular cardiomyocytes (RNVC) overexpressing ADAP1 in the absence or presence of 50 µM phenylephrine ( A ) or Mek1ca-overexpressing adenovirus ( B ) compared with an adβ-Gal-infected control. Quantification of at least 3 independent experiments expressed as means ± SD. * P

    Techniques Used: Infection

    ADAP1 relocalizes cytoskeletal α-Actinin. ( A ) Representative confocal images (Olympus FluoView FV1000 microscope) of α-Actinin-immunostained rat neonatal ventricular cardiomyocytes (RNVC) infected with a β-Gal- (negative control), ADAP1-, or Mek1ca-overexpressing adenovirus, individually or in combination as indicated, and cultured for 72 h post-infection. Arrows point to α-Actinin dense puncta. The scale bar represents 12 µm. ( B ) Number of α-Actinin puncta per cell measured with the Operetta High-Content Imaging System (Perkin Elmer) using the same experimental conditions as in A ( n = 4 independent experiments). **** P
    Figure Legend Snippet: ADAP1 relocalizes cytoskeletal α-Actinin. ( A ) Representative confocal images (Olympus FluoView FV1000 microscope) of α-Actinin-immunostained rat neonatal ventricular cardiomyocytes (RNVC) infected with a β-Gal- (negative control), ADAP1-, or Mek1ca-overexpressing adenovirus, individually or in combination as indicated, and cultured for 72 h post-infection. Arrows point to α-Actinin dense puncta. The scale bar represents 12 µm. ( B ) Number of α-Actinin puncta per cell measured with the Operetta High-Content Imaging System (Perkin Elmer) using the same experimental conditions as in A ( n = 4 independent experiments). **** P

    Techniques Used: Microscopy, Infection, Negative Control, Cell Culture, Imaging

    Adap1 expression in cardiac cells. ( A ) Relative levels of Adap1 mRNA expression in the rat brain (adult) and heart (adult and 2-day-old neonate) were measured by RT-qPCR and were normalized to the Rpl30 reporter gene ( n = 4 independent tissues). ( B ) Representative Western blots of Adap1 and Gapdh (loading control) detected from whole brain and heart extracts. ( C ) The histogram represents the relative expression level of Adap1 normalized to Gapdh in the respective tissues ( n = 4 independent tissues). ( D ) Representative Western blot of Adap1, α-Actinin (cardiomyocyte specific marker), and Gapdh (loading control) detected in the protein lysates of enriched rat neonatal ventricular cardiomyocytes (RNVC) and non-cardiomyocytes (Non-CM). ( E ) The histogram represents the relative expression level of Adap1 normalized to Gapdh in the respective cell lysates ( n .
    Figure Legend Snippet: Adap1 expression in cardiac cells. ( A ) Relative levels of Adap1 mRNA expression in the rat brain (adult) and heart (adult and 2-day-old neonate) were measured by RT-qPCR and were normalized to the Rpl30 reporter gene ( n = 4 independent tissues). ( B ) Representative Western blots of Adap1 and Gapdh (loading control) detected from whole brain and heart extracts. ( C ) The histogram represents the relative expression level of Adap1 normalized to Gapdh in the respective tissues ( n = 4 independent tissues). ( D ) Representative Western blot of Adap1, α-Actinin (cardiomyocyte specific marker), and Gapdh (loading control) detected in the protein lysates of enriched rat neonatal ventricular cardiomyocytes (RNVC) and non-cardiomyocytes (Non-CM). ( E ) The histogram represents the relative expression level of Adap1 normalized to Gapdh in the respective cell lysates ( n .

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Marker

    27) Product Images from "Construction of Cardiac Tissue Rings Using a Magnetic Tissue Fabrication Technique"

    Article Title: Construction of Cardiac Tissue Rings Using a Magnetic Tissue Fabrication Technique

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms11082910

    A procedure for fabrication of a cardiac tissue ring by combining Mag-TE and ECM-based techniques. A mixture of diluted ECM precursor and MCL-labeled cardiomyocytes was cast into a well of a 24-well ultra-low attachment plate containing a polycarbonate cylinder fixed in the center of each well. Immediately thereafter, a magnet was placed underneath the wells to attract the MCL-labeled cardiomyocytes to the culture bottom, enabling the removal of excess amounts of ECM precursor from the upper side of the formed cell layer. The remaining ECM within the cell layer was then hardened, and the medium was added. During the culture, the cell layer gradually shrank towards the cylinder, resulting in the formation of ring-shaped cardiac tissue.
    Figure Legend Snippet: A procedure for fabrication of a cardiac tissue ring by combining Mag-TE and ECM-based techniques. A mixture of diluted ECM precursor and MCL-labeled cardiomyocytes was cast into a well of a 24-well ultra-low attachment plate containing a polycarbonate cylinder fixed in the center of each well. Immediately thereafter, a magnet was placed underneath the wells to attract the MCL-labeled cardiomyocytes to the culture bottom, enabling the removal of excess amounts of ECM precursor from the upper side of the formed cell layer. The remaining ECM within the cell layer was then hardened, and the medium was added. During the culture, the cell layer gradually shrank towards the cylinder, resulting in the formation of ring-shaped cardiac tissue.

    Techniques Used: Labeling

    MCL-labeling of cardiomyocytes. ( a ) Measurement of MCL amount captured by the cells. Isolated cardiomyocytes were suspended in the medium containing various MCL concentrations (0, 25, 50, 100 and 200 pg/cell), and incubated for 1 h at 4 °C. Subsequently, the magnetite amount captured by the cells was measured using the potassium thiocyanate method. ( b ) The effect of MCL-labeling on cell viability. Cardiomyocytes with or without MCL-labeling were seeded into wells of a 96-well plate. After 4-day culture, cell viability was measured using the WST-8 assay. Data are expressed as mean ± SD (n = 3). * P
    Figure Legend Snippet: MCL-labeling of cardiomyocytes. ( a ) Measurement of MCL amount captured by the cells. Isolated cardiomyocytes were suspended in the medium containing various MCL concentrations (0, 25, 50, 100 and 200 pg/cell), and incubated for 1 h at 4 °C. Subsequently, the magnetite amount captured by the cells was measured using the potassium thiocyanate method. ( b ) The effect of MCL-labeling on cell viability. Cardiomyocytes with or without MCL-labeling were seeded into wells of a 96-well plate. After 4-day culture, cell viability was measured using the WST-8 assay. Data are expressed as mean ± SD (n = 3). * P

    Techniques Used: Labeling, Isolation, Incubation

    28) Product Images from "Control of histone H3 phosphorylation by CaMKIIδ in response to haemodynamic cardiac stress"

    Article Title: Control of histone H3 phosphorylation by CaMKIIδ in response to haemodynamic cardiac stress

    Journal: The Journal of Pathology

    doi: 10.1002/path.4489

    14–3–3 recruitment to chromatin is important for transcriptional elongation of GATA-4 and of fetal cardiac genes. (A) ChIP–qPCR performed from mouse heart tissues showing 14–3–3 binding at the ANP and β -MHC promoters in CaMKII δ -WT or CaMKII δ -KO mice 21 days after sham operation or TAC. qPCR reactions were also performed at GAPDH and α-actin promoters that are not responsive to pressure overload hypertrophy: results are expressed as percentage input over signals with IgG antibody and represent average ± SD ( n = 3); p values from Student's t test are indicated. (B) Immunoblotting of cardiomyocytes expressing reduced 14–3–3 (si14–3–3) or normal 14–3–3 (siControl). (C) ChIP–qPCR, showing p-RNAPII S2 at the promoter and internal exon of ANP , β-MHC , GATA-4 , GAPDH and actin in primary neonatal rat cardiomyocytes with siCt or si14–3–3 and stimulated with phenylephrine (PE) for 24 h. (D) ChIP–qPCR assay from mouse heart chromatin, showing p-RNAPII S2 at the ANP , β-MHC , actin and GAPDH exonic region in CaMKII δ -WT or CaMKII δ -KO animals, 21 days after sham operation or TAC. (E) ChIP–qPCR assay, showing p-RNAPII S2 at the ANP , β-MHC , actin and GAPDH promoter regions in CaMKII δ -WT or CaMKII δ -KO animals, 21 days after sham operation or TAC: results are expressed as percentage input over signals with IgG antibody, and represent average ± SD ( n = 3); p values from Student's t -test are indicated
    Figure Legend Snippet: 14–3–3 recruitment to chromatin is important for transcriptional elongation of GATA-4 and of fetal cardiac genes. (A) ChIP–qPCR performed from mouse heart tissues showing 14–3–3 binding at the ANP and β -MHC promoters in CaMKII δ -WT or CaMKII δ -KO mice 21 days after sham operation or TAC. qPCR reactions were also performed at GAPDH and α-actin promoters that are not responsive to pressure overload hypertrophy: results are expressed as percentage input over signals with IgG antibody and represent average ± SD ( n = 3); p values from Student's t test are indicated. (B) Immunoblotting of cardiomyocytes expressing reduced 14–3–3 (si14–3–3) or normal 14–3–3 (siControl). (C) ChIP–qPCR, showing p-RNAPII S2 at the promoter and internal exon of ANP , β-MHC , GATA-4 , GAPDH and actin in primary neonatal rat cardiomyocytes with siCt or si14–3–3 and stimulated with phenylephrine (PE) for 24 h. (D) ChIP–qPCR assay from mouse heart chromatin, showing p-RNAPII S2 at the ANP , β-MHC , actin and GAPDH exonic region in CaMKII δ -WT or CaMKII δ -KO animals, 21 days after sham operation or TAC. (E) ChIP–qPCR assay, showing p-RNAPII S2 at the ANP , β-MHC , actin and GAPDH promoter regions in CaMKII δ -WT or CaMKII δ -KO animals, 21 days after sham operation or TAC: results are expressed as percentage input over signals with IgG antibody, and represent average ± SD ( n = 3); p values from Student's t -test are indicated

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Aqueous Normal-phase Chromatography, Mouse Assay, Expressing

    29) Product Images from "Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity"

    Article Title: Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00545-15

    Reduced extracellular-trap (NET) formation in Hs2st-deficient neutrophils. (A) Human neutrophils were treated with 25 nM PMA for 3 h to allow NET formation, treated with heparan lyase III (5 mU/ml), fixed, and stained with mouse anti-stub heparan sulfate MAb, rabbit anti-myeloperoxidase PAb, and DAPI, followed by appropriate fluorochrome-conjugated secondary antibodies. (B) Human neutrophils were treated with 25 nM PMA for 3 h to induce NET formation and then treated with DNase I (10 U/ml) or heparan lyase I and III (5 mU/ml) for 30 min at 37°C, followed by incubation with GBS for 30 min. Surviving GBS were enumerated by serial plating. Differences between groups were calculated by unpaired t test. **, P
    Figure Legend Snippet: Reduced extracellular-trap (NET) formation in Hs2st-deficient neutrophils. (A) Human neutrophils were treated with 25 nM PMA for 3 h to allow NET formation, treated with heparan lyase III (5 mU/ml), fixed, and stained with mouse anti-stub heparan sulfate MAb, rabbit anti-myeloperoxidase PAb, and DAPI, followed by appropriate fluorochrome-conjugated secondary antibodies. (B) Human neutrophils were treated with 25 nM PMA for 3 h to induce NET formation and then treated with DNase I (10 U/ml) or heparan lyase I and III (5 mU/ml) for 30 min at 37°C, followed by incubation with GBS for 30 min. Surviving GBS were enumerated by serial plating. Differences between groups were calculated by unpaired t test. **, P

    Techniques Used: Staining, Incubation

    30) Product Images from "Structural and functional conservation at the boundaries of the chicken ?-globin domain"

    Article Title: Structural and functional conservation at the boundaries of the chicken ?-globin domain

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.10.2315

    Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).
    Figure Legend Snippet: Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).

    Techniques Used: Derivative Assay

    Fig. 4.  Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with  Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from  Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.
    Figure Legend Snippet: Fig. 4. Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.

    Techniques Used: End Labeling, Migration, Derivative Assay, Mutagenesis

    Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.
    Figure Legend Snippet: Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.

    Techniques Used: Southern Blot, Hybridization, Isolation, Derivative Assay

    Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.
    Figure Legend Snippet: Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.

    Techniques Used: Blocking Assay, Activity Assay, Stable Transfection, Transfection, Construct, Expressing

    31) Product Images from "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans"

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000639

    Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).
    Figure Legend Snippet: Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).

    Techniques Used: Staining, SDS Page, Western Blot, Protein Purification, Incubation, Protease Inhibitor

    Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.
    Figure Legend Snippet: Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.

    Techniques Used:

    32) Product Images from "Remodeling of chromatin structure within the promoter is important for bmp-2-induced fgfr3 expression"

    Article Title: Remodeling of chromatin structure within the promoter is important for bmp-2-induced fgfr3 expression

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp261

    Mapping of the BMP-2-induced DNase I hypersensitivity within the proximal FGFR3 promoter. C3H10T1/2 cells were treated with or without 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with increasing concentrations of DNase I (0, 1, 3, 5 U/ml) and DNA was purified and cleaved with Nco I. After Southern blotting, the filter was hybridized with the 32 P-labeled probe used in the MNase digestion assay. The DNase I-hypersensitive sites are indicated by arrowheads. As a control, purified genomic DNA from C3H10T1/2 cells was digested in vitro with increasing concentrations of DNase I. Size markers were generated as described in ‘Material and Methods’ section. The diagrams at the right of each panel indicate the positions of the transcription start site and the probes used for hybridization (solid bars). The large signal near +1 in the figure on the left is an autoradiograph artifact.
    Figure Legend Snippet: Mapping of the BMP-2-induced DNase I hypersensitivity within the proximal FGFR3 promoter. C3H10T1/2 cells were treated with or without 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with increasing concentrations of DNase I (0, 1, 3, 5 U/ml) and DNA was purified and cleaved with Nco I. After Southern blotting, the filter was hybridized with the 32 P-labeled probe used in the MNase digestion assay. The DNase I-hypersensitive sites are indicated by arrowheads. As a control, purified genomic DNA from C3H10T1/2 cells was digested in vitro with increasing concentrations of DNase I. Size markers were generated as described in ‘Material and Methods’ section. The diagrams at the right of each panel indicate the positions of the transcription start site and the probes used for hybridization (solid bars). The large signal near +1 in the figure on the left is an autoradiograph artifact.

    Techniques Used: Purification, Southern Blot, Labeling, In Vitro, Generated, Hybridization, Autoradiography

    Alteration in the restriction enzyme accessibility at the FGFR3 proximal promoter induced by BMP-2 treatment. ( A ) C3H10T1/2 cells were incubated in the presence or absence of 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with 25–200 U of restriction enzyme/ml, and purified DNA was digested to completion with Nco I. Products were detected by Southern blotting (top panel). A tentative assignment of the nucleosome positions in this region based on nuclease digestion is shown and is aligned with the nuclease-hypersensitive sites. Sites of cutting by DNase I and MNase and restriction enzyme are depicted by solid bars for basal conditions. Because MNase preferentially digests DNA in linker regions between nucleosomes, it is possible to locate nucleosomes within the FGFR3 proximal promoter. Hollow arrow heads indicate increases in the nuclease hypersensitivity upon BMP-2 exposure. ( B ) The relative nuclease sensitivity of restriction enzyme sites was quantitated, and the intensities of the radioactive bands were used to calculate the percentage of DNA digested. All the results are the means of three independent experiments ± standard deviation. * P
    Figure Legend Snippet: Alteration in the restriction enzyme accessibility at the FGFR3 proximal promoter induced by BMP-2 treatment. ( A ) C3H10T1/2 cells were incubated in the presence or absence of 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with 25–200 U of restriction enzyme/ml, and purified DNA was digested to completion with Nco I. Products were detected by Southern blotting (top panel). A tentative assignment of the nucleosome positions in this region based on nuclease digestion is shown and is aligned with the nuclease-hypersensitive sites. Sites of cutting by DNase I and MNase and restriction enzyme are depicted by solid bars for basal conditions. Because MNase preferentially digests DNA in linker regions between nucleosomes, it is possible to locate nucleosomes within the FGFR3 proximal promoter. Hollow arrow heads indicate increases in the nuclease hypersensitivity upon BMP-2 exposure. ( B ) The relative nuclease sensitivity of restriction enzyme sites was quantitated, and the intensities of the radioactive bands were used to calculate the percentage of DNA digested. All the results are the means of three independent experiments ± standard deviation. * P

    Techniques Used: Incubation, Purification, Southern Blot, Standard Deviation

    Inducible DNase I hypersensitivity within the FGFR3 promoter. ( A ) Schematic representation of the probe used to map the DNase-hypersensitive sites within the FGFR3 promoter by the indirect end-labeling technique. ( B ) C3H10T1/2 cells were treated with or without 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with increasing concentrations of DNase I (0, 1, 3, 5 U/ml). DNA was purified and cleaved with Hind III. After Southern blotting, the filter was hybridized with the 32 P-labeled probe. The DNase I-hypersensitive sites are indicated by arrowheads. As a control, purified genomic DNA from C3H10T1/2 cells was digested in vitro with increasing concentrations of DNase I (0, 0.05, 0.1, 0.2 U/ml).
    Figure Legend Snippet: Inducible DNase I hypersensitivity within the FGFR3 promoter. ( A ) Schematic representation of the probe used to map the DNase-hypersensitive sites within the FGFR3 promoter by the indirect end-labeling technique. ( B ) C3H10T1/2 cells were treated with or without 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with increasing concentrations of DNase I (0, 1, 3, 5 U/ml). DNA was purified and cleaved with Hind III. After Southern blotting, the filter was hybridized with the 32 P-labeled probe. The DNase I-hypersensitive sites are indicated by arrowheads. As a control, purified genomic DNA from C3H10T1/2 cells was digested in vitro with increasing concentrations of DNase I (0, 0.05, 0.1, 0.2 U/ml).

    Techniques Used: End Labeling, Purification, Southern Blot, Labeling, In Vitro

    33) Product Images from "Exploring protein interfaces with a general photochemical reagent"

    Article Title: Exploring protein interfaces with a general photochemical reagent

    Journal:

    doi: 10.1110/ps.051960406

    Separation by size-exclusion chromatography of tryptic peptides derived from HEWL labeled with methylene carbene. After the clean-up step described above (see Fig. ), HEWL samples were reduced, carbamidomethylated, and digested with TPCK-trypsin
    Figure Legend Snippet: Separation by size-exclusion chromatography of tryptic peptides derived from HEWL labeled with methylene carbene. After the clean-up step described above (see Fig. ), HEWL samples were reduced, carbamidomethylated, and digested with TPCK-trypsin

    Techniques Used: Size-exclusion Chromatography, Derivative Assay, Labeling

    34) Product Images from "Domain of Dentine Sialoprotein Mediates Proliferation and Differentiation of Human Periodontal Ligament Stem Cells"

    Article Title: Domain of Dentine Sialoprotein Mediates Proliferation and Differentiation of Human Periodontal Ligament Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081655

    Product of DSP protein coated on glass slides. A . Coomassie-stained SDS-PAGE gel for analysis of expression of the recombinant COOH-terminal dentin sialoprotein (rC-DSP) protein. The sample in DL21 cells was grown 3 h with 1 mM IPTG induction after reaching O.D.600 of 0.6. The rC-DSP was purified by the column described in the materials and methods. B . Expression of purified rC-DSP was confirmed by Western blotting analysis using goat polyclonal anti-DSP antibody (Santa Cruz Biotechnology Inc.). D - E . Collagen type I and rC-DSP proteins were coated on glass slides for overnight at 37°C. For scanning electron microscopy, the samples were washed and dried as well as treated with sodium deoxycholate. Then the samples were observed under scanning electron microscope (SEM). Fibril matrix was visible on the collagen type I coated slide ( D ) and rC-DSP coated slide ( E ), but no control slide ( C ). Also, there were different morphologies between rC-DSP and collagen type I.
    Figure Legend Snippet: Product of DSP protein coated on glass slides. A . Coomassie-stained SDS-PAGE gel for analysis of expression of the recombinant COOH-terminal dentin sialoprotein (rC-DSP) protein. The sample in DL21 cells was grown 3 h with 1 mM IPTG induction after reaching O.D.600 of 0.6. The rC-DSP was purified by the column described in the materials and methods. B . Expression of purified rC-DSP was confirmed by Western blotting analysis using goat polyclonal anti-DSP antibody (Santa Cruz Biotechnology Inc.). D - E . Collagen type I and rC-DSP proteins were coated on glass slides for overnight at 37°C. For scanning electron microscopy, the samples were washed and dried as well as treated with sodium deoxycholate. Then the samples were observed under scanning electron microscope (SEM). Fibril matrix was visible on the collagen type I coated slide ( D ) and rC-DSP coated slide ( E ), but no control slide ( C ). Also, there were different morphologies between rC-DSP and collagen type I.

    Techniques Used: Staining, SDS Page, Expressing, Recombinant, Purification, Western Blot, Electron Microscopy, Microscopy

    35) Product Images from "Selective alkylation of T–T mismatched DNA using vinyldiaminotriazine–acridine conjugate"

    Article Title: Selective alkylation of T–T mismatched DNA using vinyldiaminotriazine–acridine conjugate

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1278

    HPLC profiles of the enzymatic hydrolysis products. ( A ) HPLC analysis after enzymatic hydrolysis of ODN 5 (top) and ODN 6 (bottom). The enzymatic hydrolysis was performed with ODN 5 or ODN 6 (100 μM), alkaline phosphatase (0.03 U/μl) and phosphodiesterase I (0.03 U/μl) in alkaline phosphatase buffer at 37°C for 1 h. ( B ) Co-injection of dT*1 and dT*2.
    Figure Legend Snippet: HPLC profiles of the enzymatic hydrolysis products. ( A ) HPLC analysis after enzymatic hydrolysis of ODN 5 (top) and ODN 6 (bottom). The enzymatic hydrolysis was performed with ODN 5 or ODN 6 (100 μM), alkaline phosphatase (0.03 U/μl) and phosphodiesterase I (0.03 U/μl) in alkaline phosphatase buffer at 37°C for 1 h. ( B ) Co-injection of dT*1 and dT*2.

    Techniques Used: High Performance Liquid Chromatography, Injection

    36) 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

    37) Product Images from "Anticancer activity of paclitaxel-loaded keratin nanoparticles in two-dimensional and perfused three-dimensional breast cancer models"

    Article Title: Anticancer activity of paclitaxel-loaded keratin nanoparticles in two-dimensional and perfused three-dimensional breast cancer models

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S159942

    Antiproliferative activity of PTX in a free form, HSA-NPs-PTX, and KER-NPs-PTX on MCF-7 and MDA MB 231 cell lines in 2D model. Notes: Cell viability was evaluated 72 h after exposure to increasing concentrations of PTX (0.00002, 0.02, and 5 µg/mL) by APH assay. Statistical significance versus untreated cells (100%, represented by a dotted line): ** P
    Figure Legend Snippet: Antiproliferative activity of PTX in a free form, HSA-NPs-PTX, and KER-NPs-PTX on MCF-7 and MDA MB 231 cell lines in 2D model. Notes: Cell viability was evaluated 72 h after exposure to increasing concentrations of PTX (0.00002, 0.02, and 5 µg/mL) by APH assay. Statistical significance versus untreated cells (100%, represented by a dotted line): ** P

    Techniques Used: Activity Assay, Multiple Displacement Amplification

    BAX and BCL-2 gene expression analyses in MCF-7 and MDA MB 231 cells in p3D cultures following 24 h treatments. Notes: Cells were incubated for 24 h with PTX in a free form, HSA-NPs-PTX, or KER-NPs-PTX (PTX, 5 µg/mL). GAPDH was used as reference gene to normalize data. The effects of each treatment were compared with gene expression detectable in untreated cells (=1) as indicated by the dotted line. Statistically significant difference versus untreated cells: * P
    Figure Legend Snippet: BAX and BCL-2 gene expression analyses in MCF-7 and MDA MB 231 cells in p3D cultures following 24 h treatments. Notes: Cells were incubated for 24 h with PTX in a free form, HSA-NPs-PTX, or KER-NPs-PTX (PTX, 5 µg/mL). GAPDH was used as reference gene to normalize data. The effects of each treatment were compared with gene expression detectable in untreated cells (=1) as indicated by the dotted line. Statistically significant difference versus untreated cells: * P

    Techniques Used: Expressing, Multiple Displacement Amplification, Incubation

    Representative H E staining of scaffold sections from p3D MCF-7 and MDA MB 231 cultures after treatments. Notes: MCF-7 (upper panels) and MDA MB 231 (lower panels) cells were initially cultured for 7 and 4 days, respectively, and then left untreated ( A ) or treated for 48 h with PTX in a free form ( B ), HSA-NPs-PTX ( C ), and KER-NPs-PTX ( D ) (PTX, 5 µg/mL). Scale bar: 100 µm. Abbreviations: H E, hematoxylin and eosin; HSA-NPs-PTX, PTX loaded in albumin nanoparticles; KER-NPs-PTX, PTX loaded in keratin nanoparticles; p3D, three-dimensional model with perfused bioreactor; PTX, paclitaxel.
    Figure Legend Snippet: Representative H E staining of scaffold sections from p3D MCF-7 and MDA MB 231 cultures after treatments. Notes: MCF-7 (upper panels) and MDA MB 231 (lower panels) cells were initially cultured for 7 and 4 days, respectively, and then left untreated ( A ) or treated for 48 h with PTX in a free form ( B ), HSA-NPs-PTX ( C ), and KER-NPs-PTX ( D ) (PTX, 5 µg/mL). Scale bar: 100 µm. Abbreviations: H E, hematoxylin and eosin; HSA-NPs-PTX, PTX loaded in albumin nanoparticles; KER-NPs-PTX, PTX loaded in keratin nanoparticles; p3D, three-dimensional model with perfused bioreactor; PTX, paclitaxel.

    Techniques Used: Staining, Multiple Displacement Amplification, Cell Culture

    BAX and BCL-2 gene expression analyses in MCF-7 and MDA MB 231 cells cultured in 2D model upon 12 h treatment. Notes: Cells were incubated for 12 h with PTX in a free form, HSA-NPs-PTX, or KER-NPs-PTX (PTX, 5 µg/mL). GAPDH was used as reference gene to normalize data. Effects of each treatment on gene expression levels were compared with those detectable in untreated cells (n=1) as indicated by the dotted line. Statistically significant difference versus untreated cells: * P
    Figure Legend Snippet: BAX and BCL-2 gene expression analyses in MCF-7 and MDA MB 231 cells cultured in 2D model upon 12 h treatment. Notes: Cells were incubated for 12 h with PTX in a free form, HSA-NPs-PTX, or KER-NPs-PTX (PTX, 5 µg/mL). GAPDH was used as reference gene to normalize data. Effects of each treatment on gene expression levels were compared with those detectable in untreated cells (n=1) as indicated by the dotted line. Statistically significant difference versus untreated cells: * P

    Techniques Used: Expressing, Multiple Displacement Amplification, Cell Culture, Incubation

    Effects of PTX in a free form or vehiculated by HSA-NPs or by KER-NPs on cell death in p3D cultures. Notes: MCF-7 and MDA MB 231 cells were exposed to PTX in a free form, HSA-NPs-PTX, and KER-NPs-PTX (PTX, 5 µg/mL) for 24 and 48 h. Apoptosis assays were carried out by flow cytometry, following APC-Annexin V and PI staining. Representative dot plots of MCF-7 ( A ) and MDA MB 231 ( B ) cell lines after each treatment. Percentages of early apoptotic cells (positive to APC-Annexin V and negative to PI) and late apoptotic cells (positive to APC-Annexin V and PI) were evaluated 24 and 48 h after treatment ( C ). Statistical significance versus untreated cells: * P
    Figure Legend Snippet: Effects of PTX in a free form or vehiculated by HSA-NPs or by KER-NPs on cell death in p3D cultures. Notes: MCF-7 and MDA MB 231 cells were exposed to PTX in a free form, HSA-NPs-PTX, and KER-NPs-PTX (PTX, 5 µg/mL) for 24 and 48 h. Apoptosis assays were carried out by flow cytometry, following APC-Annexin V and PI staining. Representative dot plots of MCF-7 ( A ) and MDA MB 231 ( B ) cell lines after each treatment. Percentages of early apoptotic cells (positive to APC-Annexin V and negative to PI) and late apoptotic cells (positive to APC-Annexin V and PI) were evaluated 24 and 48 h after treatment ( C ). Statistical significance versus untreated cells: * P

    Techniques Used: Multiple Displacement Amplification, Flow Cytometry, Cytometry, Staining

    KER-NPs-PTX analysis. Notes: Transmission electron microscopy images of fixed KER-NPs-PTX at two magnifications: scale bar 2 µm ( A ) and scale bar 200 nm ( B ). Morphological analysis showed particles spherical in shape, smooth surface, and an average dry diameter of ~80 nm. Stability of KER-NPs-PTX in physiological conditions ( C ). Fluorescence spectra of KER-NPs-PTX at different PTX loading percentages ( D ). Abbreviations: KER-NPs-PTX, PTX loaded in keratin nanoparticles; PDI, polydispersity index; PTX, paclitaxel.
    Figure Legend Snippet: KER-NPs-PTX analysis. Notes: Transmission electron microscopy images of fixed KER-NPs-PTX at two magnifications: scale bar 2 µm ( A ) and scale bar 200 nm ( B ). Morphological analysis showed particles spherical in shape, smooth surface, and an average dry diameter of ~80 nm. Stability of KER-NPs-PTX in physiological conditions ( C ). Fluorescence spectra of KER-NPs-PTX at different PTX loading percentages ( D ). Abbreviations: KER-NPs-PTX, PTX loaded in keratin nanoparticles; PDI, polydispersity index; PTX, paclitaxel.

    Techniques Used: Transmission Assay, Electron Microscopy, Fluorescence

    Profiles and mechanisms of PTX release from KER-NPs in vitro. Notes: Profile of PTX release from KER-NPs ( A ). Correlation coefficient values ( R 2 ) used to determine the best fitting model among zero-order, first-order, and Higuchi modes ( B ). Abbreviations: KER-NPs, keratin nanoparticles; PTX, paclitaxel.
    Figure Legend Snippet: Profiles and mechanisms of PTX release from KER-NPs in vitro. Notes: Profile of PTX release from KER-NPs ( A ). Correlation coefficient values ( R 2 ) used to determine the best fitting model among zero-order, first-order, and Higuchi modes ( B ). Abbreviations: KER-NPs, keratin nanoparticles; PTX, paclitaxel.

    Techniques Used: In Vitro

    KER-NPs-PTX preparation. Notes: ( A ) KER-NPs-PTX preparation by aggregation method, starting from a solution of pure keratin powder. ( B ) Correlation between PTX loading and KER-NPs-PTX size. Abbreviations: KER-NPs-PTX, PTX loaded in keratin nanoparticles; PDI, polydispersity index; PTX, paclitaxel; RT, room temperature.
    Figure Legend Snippet: KER-NPs-PTX preparation. Notes: ( A ) KER-NPs-PTX preparation by aggregation method, starting from a solution of pure keratin powder. ( B ) Correlation between PTX loading and KER-NPs-PTX size. Abbreviations: KER-NPs-PTX, PTX loaded in keratin nanoparticles; PDI, polydispersity index; PTX, paclitaxel; RT, room temperature.

    Techniques Used:

    Immunofluorescence staining of CC3 in MCF-7 (upper panels) and MDA MB 231 (lower panels) scaffold sections. Notes: Images show DAPI (blue) and CC3 (green) staining in MCF-7 ( A ) and MDA MB 231 ( B ) p3D scaffold sections upon treatment with PTX in a free form (b), HSA-NPs-PTX (c), and KER-NPs-PTX (d) (PTX, 5 µg/mL) for 48 h or untreated (a). Scale bar: 100 µm. Abbreviations: CC3, cleaved caspase 3; DAPI, 4′,6-diamidino-2-phenylindole; HSA-NPs-PTX, PTX loaded in albumin nanoparticles; KER-NPs-PTX, PTX loaded in keratin nanoparticles; p3D, three-dimensional model with perfused bioreactor; PTX, paclitaxel.
    Figure Legend Snippet: Immunofluorescence staining of CC3 in MCF-7 (upper panels) and MDA MB 231 (lower panels) scaffold sections. Notes: Images show DAPI (blue) and CC3 (green) staining in MCF-7 ( A ) and MDA MB 231 ( B ) p3D scaffold sections upon treatment with PTX in a free form (b), HSA-NPs-PTX (c), and KER-NPs-PTX (d) (PTX, 5 µg/mL) for 48 h or untreated (a). Scale bar: 100 µm. Abbreviations: CC3, cleaved caspase 3; DAPI, 4′,6-diamidino-2-phenylindole; HSA-NPs-PTX, PTX loaded in albumin nanoparticles; KER-NPs-PTX, PTX loaded in keratin nanoparticles; p3D, three-dimensional model with perfused bioreactor; PTX, paclitaxel.

    Techniques Used: Immunofluorescence, Staining, Multiple Displacement Amplification

    Induction of apoptosis in tumor cells cultured in 2D by PTX in a free form, HSA-NPs-PTX, or KER-NPs-PTX in 2D model. Notes: MCF-7 and MDA MB 231 cells were exposed to PTX, HSA-NPs-PTX, and KER-NPs-PTX (PTX, 5 µg/mL) for 24 and 48 h. Apoptosis assays were carried out by flow cytometry, following APC-Annexin V and PI staining. Representative dot plots of MCF-7 ( A ) and MDA MB 231 ( B ) cell lines after each treatment. Percentage of early apoptotic cells (positive to APC-Annexin V and negative to PI) and late apoptotic cells (positive to APC-Annexin V and PI) was evaluated 24 and 48 h after treatment ( C ). Statistical significance versus untreated cells: * P
    Figure Legend Snippet: Induction of apoptosis in tumor cells cultured in 2D by PTX in a free form, HSA-NPs-PTX, or KER-NPs-PTX in 2D model. Notes: MCF-7 and MDA MB 231 cells were exposed to PTX, HSA-NPs-PTX, and KER-NPs-PTX (PTX, 5 µg/mL) for 24 and 48 h. Apoptosis assays were carried out by flow cytometry, following APC-Annexin V and PI staining. Representative dot plots of MCF-7 ( A ) and MDA MB 231 ( B ) cell lines after each treatment. Percentage of early apoptotic cells (positive to APC-Annexin V and negative to PI) and late apoptotic cells (positive to APC-Annexin V and PI) was evaluated 24 and 48 h after treatment ( C ). Statistical significance versus untreated cells: * P

    Techniques Used: Cell Culture, Multiple Displacement Amplification, Flow Cytometry, Cytometry, Staining

    38) Product Images from "Fusion-Deficient Insertion Mutants of Herpes Simplex Virus Type 1 Glycoprotein B Adopt the Trimeric Postfusion Conformation ▿"

    Article Title: Fusion-Deficient Insertion Mutants of Herpes Simplex Virus Type 1 Glycoprotein B Adopt the Trimeric Postfusion Conformation ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01791-09

    Limited proteolysis reveals a new chymotrypsin site in the E187i5 mutant. Wild-type gB730 and five insertion mutants were digested with chymotrypsin at decreasing mass ratios of substrate to protease, indicated above each lane. ND, nondigested. For each gel in this figure, molecular mass markers are indicated on the left in kilodaltons. (A) Digestion pattern of wild-type gB730, the I671i5 mutant, and the E187i5 mutant. (B) Digestion pattern of the T665i5 mutant. (C) Digestion pattern of the V667i5 and L673i5 mutants. Asterisks indicate proteolytic products that are seen in the wild-type gB730 and all of the mutants. Arrows in the E187i5 digestion pattern indicate new bands absent from the digests of the wild-type gB730 and the rest of the mutants. Weak bands corresponding to chymotrypsin autoproteolysis products are observed at 15 and 10 kDa in some of the digests at 25:1 and 10:1 substrate/protease ratios.
    Figure Legend Snippet: Limited proteolysis reveals a new chymotrypsin site in the E187i5 mutant. Wild-type gB730 and five insertion mutants were digested with chymotrypsin at decreasing mass ratios of substrate to protease, indicated above each lane. ND, nondigested. For each gel in this figure, molecular mass markers are indicated on the left in kilodaltons. (A) Digestion pattern of wild-type gB730, the I671i5 mutant, and the E187i5 mutant. (B) Digestion pattern of the T665i5 mutant. (C) Digestion pattern of the V667i5 and L673i5 mutants. Asterisks indicate proteolytic products that are seen in the wild-type gB730 and all of the mutants. Arrows in the E187i5 digestion pattern indicate new bands absent from the digests of the wild-type gB730 and the rest of the mutants. Weak bands corresponding to chymotrypsin autoproteolysis products are observed at 15 and 10 kDa in some of the digests at 25:1 and 10:1 substrate/protease ratios.

    Techniques Used: Mutagenesis

    39) Product Images from "Casein kinase I ?/? phosphorylates topoisomerase II? at serine-1106 and modulates DNA cleavage activity"

    Article Title: Casein kinase I ?/? phosphorylates topoisomerase II? at serine-1106 and modulates DNA cleavage activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn934

    CKI inhibitors, CKI-7 and IC261, decrease phosphorylation of the topo IIα peptides containing Ser-1106. HL-60 cells were labeled with [ 32 P] orthophosphoric acid for 3 h at 37 ° C in the absence or presence of CKI inhibitors, CKI-7 (200 μM) or IC261 (10 μM). Lysates of these cells were immunoprecipitated with topo IIα-specific antibodies. The immunoprecipitated protein was subjected to SDS–PAGE and transferred to nitrocellulose membrane. Two-thirds of the topo IIα band was cleaved with CNBr and one-third of the band was proteolysed with trypsin. ( A ) CNBr fragments of topo IIα from cells treated in the absence or presence of CKI-7 or IC261 were separated by SDS–PAGE. ( B ) Phosphopeptide maps of tryptic digests of topo IIα from cells treated in the absence (Control) or presence of CKI-7 or IC261. The phosphochromatography buffer, n -butanol/pyridine/acetic acid/deionized water (5/3.3/1/4, v/v), was used for resolving peptides in the second dimension. Arrows indicate the position of Ser-1106 containing peptides.
    Figure Legend Snippet: CKI inhibitors, CKI-7 and IC261, decrease phosphorylation of the topo IIα peptides containing Ser-1106. HL-60 cells were labeled with [ 32 P] orthophosphoric acid for 3 h at 37 ° C in the absence or presence of CKI inhibitors, CKI-7 (200 μM) or IC261 (10 μM). Lysates of these cells were immunoprecipitated with topo IIα-specific antibodies. The immunoprecipitated protein was subjected to SDS–PAGE and transferred to nitrocellulose membrane. Two-thirds of the topo IIα band was cleaved with CNBr and one-third of the band was proteolysed with trypsin. ( A ) CNBr fragments of topo IIα from cells treated in the absence or presence of CKI-7 or IC261 were separated by SDS–PAGE. ( B ) Phosphopeptide maps of tryptic digests of topo IIα from cells treated in the absence (Control) or presence of CKI-7 or IC261. The phosphochromatography buffer, n -butanol/pyridine/acetic acid/deionized water (5/3.3/1/4, v/v), was used for resolving peptides in the second dimension. Arrows indicate the position of Ser-1106 containing peptides.

    Techniques Used: Labeling, Immunoprecipitation, SDS Page

    Peptides containing phosphorylated Ser-1106 are not detected by LC-MS analysis of topo IIα from si-CKIδ/ɛ transfected HCT-116 cells. HCT-116 cells were transfected with scrambled si-RNA or si-CKIδ/ɛ. Topo IIα protein present in these cells was purified by immunoprecipitation and SDS–PAGE. The stained topo IIα band in the gel was digested with trypsin and the peptides were analyzed by LC–MS as described in Materials and methods section. ( A ) Total ion chromatograms (upper panel) and trypsin autolysis marker at m/z 422 (lower panel) of topo IIα tryptic digests from cells transfected with scrambled si-RNA or si-CKIδ/ɛ. ( B ) SRM chromatograms for the doubly charged peptide ion containing phosphorylated Ser-1106 at m/z 1116 (upper panel) and CID spectrum of doubly charged peptide ion (lower panel) containing phosphorylated Ser-1106 in topo IIα, detected only in samples treated with scrambled si-RNA. ( C ) SRM chromatograms for the triply charged peptide ion containing phosphorylated Ser-1106 at m/z 744 (upper panel) and characteristic H 3 PO 4 neutral loss for the m/z 744 (+3) phosphorylated peptide (middle panel). CID spectrum of triply charged peptide ion (lower panel) containing phosphorylated Ser-1106 in topo IIα was detected only in samples treated with scrambled si-RNA. Characteristic loss of H 3 PO 4 is seen in both CID spectra. Detected b (N-terminal) and y (C-terminal) fragment ions are labeled in the spectra.
    Figure Legend Snippet: Peptides containing phosphorylated Ser-1106 are not detected by LC-MS analysis of topo IIα from si-CKIδ/ɛ transfected HCT-116 cells. HCT-116 cells were transfected with scrambled si-RNA or si-CKIδ/ɛ. Topo IIα protein present in these cells was purified by immunoprecipitation and SDS–PAGE. The stained topo IIα band in the gel was digested with trypsin and the peptides were analyzed by LC–MS as described in Materials and methods section. ( A ) Total ion chromatograms (upper panel) and trypsin autolysis marker at m/z 422 (lower panel) of topo IIα tryptic digests from cells transfected with scrambled si-RNA or si-CKIδ/ɛ. ( B ) SRM chromatograms for the doubly charged peptide ion containing phosphorylated Ser-1106 at m/z 1116 (upper panel) and CID spectrum of doubly charged peptide ion (lower panel) containing phosphorylated Ser-1106 in topo IIα, detected only in samples treated with scrambled si-RNA. ( C ) SRM chromatograms for the triply charged peptide ion containing phosphorylated Ser-1106 at m/z 744 (upper panel) and characteristic H 3 PO 4 neutral loss for the m/z 744 (+3) phosphorylated peptide (middle panel). CID spectrum of triply charged peptide ion (lower panel) containing phosphorylated Ser-1106 in topo IIα was detected only in samples treated with scrambled si-RNA. Characteristic loss of H 3 PO 4 is seen in both CID spectra. Detected b (N-terminal) and y (C-terminal) fragment ions are labeled in the spectra.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Transfection, Purification, Immunoprecipitation, SDS Page, Staining, Marker, Labeling

    Down-regulation of CKIδ or CK1ɛ with targeted si-RNAs in HCT-116 cells decreases phosphorylation of the topo IIα peptide containing Ser-1106. HCT-116 cells were transfected with scrambled si-RNA, si-CKIδ or si-CKIɛ as described in Materials and methods section. A small aliquot of the cells was lysed in RIPA buffer and subjected to western blot analysis to determine down-regulation of CKIδ and CKIɛ ( A ). The remaining cells were labeled with [ 32 P] orthophosphoric acid for 3 h, lysed in RIPA buffer and the cell lysates were immunoprecipitated with topo IIα-specific antibodies. The immunoprecipitated topo IIα protein was subjected to SDS–PAGE and transferred to nitrocellulose membrane. The stained topo IIα band was excised, proteolysed with trypsin and the labeled tryptic peptides analyzed by 2D-phosphopeptide mapping ( B , C ). The isobutyric acid buffer (isobutyric acid/ n -butanol/pyridine/acetic acid/deionized water) (32.9/1/2.5/1.5/14.7, v/v) was used for resolving the peptides in the second dimension. The arrow indicates the position of Ser-1106 containing peptide.
    Figure Legend Snippet: Down-regulation of CKIδ or CK1ɛ with targeted si-RNAs in HCT-116 cells decreases phosphorylation of the topo IIα peptide containing Ser-1106. HCT-116 cells were transfected with scrambled si-RNA, si-CKIδ or si-CKIɛ as described in Materials and methods section. A small aliquot of the cells was lysed in RIPA buffer and subjected to western blot analysis to determine down-regulation of CKIδ and CKIɛ ( A ). The remaining cells were labeled with [ 32 P] orthophosphoric acid for 3 h, lysed in RIPA buffer and the cell lysates were immunoprecipitated with topo IIα-specific antibodies. The immunoprecipitated topo IIα protein was subjected to SDS–PAGE and transferred to nitrocellulose membrane. The stained topo IIα band was excised, proteolysed with trypsin and the labeled tryptic peptides analyzed by 2D-phosphopeptide mapping ( B , C ). The isobutyric acid buffer (isobutyric acid/ n -butanol/pyridine/acetic acid/deionized water) (32.9/1/2.5/1.5/14.7, v/v) was used for resolving the peptides in the second dimension. The arrow indicates the position of Ser-1106 containing peptide.

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

    Phosphorylation of the topo IIα peptide containing Ser-1106 is reduced in HCT-116 cells transfected with si-CKIδ/ɛ, but not in nocadazole treated cells arrested in the G 2 +M phase of the cell cycle. HCT-116 cells were transfected with scrambled si-RNA or si-CKIδ/ɛ as described in Materials and methods section, or treated for 16 h in the absence or presence of 200 nM nocadazole. For cells transfected with scrambled si-RNA or si-CKIδ/ɛ a small aliquot was lysed in RIPA buffer and subjected to western blot analysis to determine down-regulation of CKIδ and CK1ɛ ( A ). The remaining transfected cells ( B ), and cells treated in the absence or presence of nocadazole ( C ) were labeled with [ 32 P] orthophosphoric acid for 3 h. At the end of the incubation, cells were lysed in RIPA buffer and cell lysates were immunoprecipitated with topo IIα-specific antibodies. The immunoprecipitated topo IIα protein was subjected to SDS–PAGE and transferred to nitrocellulose membrane. The topo IIα band was excised, proteolysed with trypsin and the labeled tryptic peptides analyzed by 2D-phosphopeptide mapping. The isobutyric acid buffer (isobutyric acid/ n -butanol/pyridine/acetic acid/deionized water) (32.9/1/2.5/1.5/14.7, v/v) was used for resolving the peptides in the second dimension. The arrow indicates the position of Ser-1106 containing peptide.
    Figure Legend Snippet: Phosphorylation of the topo IIα peptide containing Ser-1106 is reduced in HCT-116 cells transfected with si-CKIδ/ɛ, but not in nocadazole treated cells arrested in the G 2 +M phase of the cell cycle. HCT-116 cells were transfected with scrambled si-RNA or si-CKIδ/ɛ as described in Materials and methods section, or treated for 16 h in the absence or presence of 200 nM nocadazole. For cells transfected with scrambled si-RNA or si-CKIδ/ɛ a small aliquot was lysed in RIPA buffer and subjected to western blot analysis to determine down-regulation of CKIδ and CK1ɛ ( A ). The remaining transfected cells ( B ), and cells treated in the absence or presence of nocadazole ( C ) were labeled with [ 32 P] orthophosphoric acid for 3 h. At the end of the incubation, cells were lysed in RIPA buffer and cell lysates were immunoprecipitated with topo IIα-specific antibodies. The immunoprecipitated topo IIα protein was subjected to SDS–PAGE and transferred to nitrocellulose membrane. The topo IIα band was excised, proteolysed with trypsin and the labeled tryptic peptides analyzed by 2D-phosphopeptide mapping. The isobutyric acid buffer (isobutyric acid/ n -butanol/pyridine/acetic acid/deionized water) (32.9/1/2.5/1.5/14.7, v/v) was used for resolving the peptides in the second dimension. The arrow indicates the position of Ser-1106 containing peptide.

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

    Phosphorylation of Ser-1106 in human topo IIα expressing HRR25Δ S. cerevisae cells is reduced, but can be enhanced following transformation of the cells with human CKIɛ. WT W303 cells or HRR25Δ isolate isogenic to W303 cells transformed with human topo IIα ( A ), or topo IIα expressing HRR25Δ W303 cells transformed with vector control or human CKIɛ cDNA ( B ) were labeled with [ 32 P] orthophosphoric acid for 1 h as described in Materials and methods section. Topo IIα from cell lysates prepared in Y-PER lysis buffer was purified by Ni 2+ -nitrilotriacetic acid or immunoprecipitated with topo IIα antibody. Purified topo IIα was subjected to SDS–PAGE and transferred to nitrocellulose membrane. The stained topo IIα band was excised, proteolysed with trypsin and the labeled tryptic peptides analyzed by 2D-phosphopeptide mapping. The phosphochromatography buffer, n -butanol/pyridine/acetic acid/deionized water (5/3.3/1/4, v/v), was used for separating the peptides in the second dimension (A) and the isobutyric acid buffer (isobutyric acid/ n -butanol/pyridine/acetic acid/deionized water) (32.9/1/2.5/1.5/14.7, v/v) was used for resolving the peptides in the second dimension (B). The arrow indicates the position of Ser-1106 containing peptide.
    Figure Legend Snippet: Phosphorylation of Ser-1106 in human topo IIα expressing HRR25Δ S. cerevisae cells is reduced, but can be enhanced following transformation of the cells with human CKIɛ. WT W303 cells or HRR25Δ isolate isogenic to W303 cells transformed with human topo IIα ( A ), or topo IIα expressing HRR25Δ W303 cells transformed with vector control or human CKIɛ cDNA ( B ) were labeled with [ 32 P] orthophosphoric acid for 1 h as described in Materials and methods section. Topo IIα from cell lysates prepared in Y-PER lysis buffer was purified by Ni 2+ -nitrilotriacetic acid or immunoprecipitated with topo IIα antibody. Purified topo IIα was subjected to SDS–PAGE and transferred to nitrocellulose membrane. The stained topo IIα band was excised, proteolysed with trypsin and the labeled tryptic peptides analyzed by 2D-phosphopeptide mapping. The phosphochromatography buffer, n -butanol/pyridine/acetic acid/deionized water (5/3.3/1/4, v/v), was used for separating the peptides in the second dimension (A) and the isobutyric acid buffer (isobutyric acid/ n -butanol/pyridine/acetic acid/deionized water) (32.9/1/2.5/1.5/14.7, v/v) was used for resolving the peptides in the second dimension (B). The arrow indicates the position of Ser-1106 containing peptide.

    Techniques Used: Expressing, Transformation Assay, Plasmid Preparation, Labeling, Lysis, Purification, Immunoprecipitation, SDS Page, Staining

    Down-regulation of CKIδ and CKIɛ in HCT-116 cells leads to reduced topo IIα–DNA cleavage activity. HCT-116 cells were treated with scrambled si-CKIδ (75 nM) plus si-CKIɛ (75 nM) as described in Materials and methods section and subsequently treated with etoposide for 1 h. An aliquot of the cell lysate was used to determine down regulation of CKIδ and CKIɛ ( A ). Cells labeled with [ 14 C] thymidine were lysed and precipitated with SDS–KCl. The dpm present in the precipitate was determined by liquid scintillation counting ( B ). *Significantly ( P
    Figure Legend Snippet: Down-regulation of CKIδ and CKIɛ in HCT-116 cells leads to reduced topo IIα–DNA cleavage activity. HCT-116 cells were treated with scrambled si-CKIδ (75 nM) plus si-CKIɛ (75 nM) as described in Materials and methods section and subsequently treated with etoposide for 1 h. An aliquot of the cell lysate was used to determine down regulation of CKIδ and CKIɛ ( A ). Cells labeled with [ 14 C] thymidine were lysed and precipitated with SDS–KCl. The dpm present in the precipitate was determined by liquid scintillation counting ( B ). *Significantly ( P

    Techniques Used: Activity Assay, Labeling

    Down-regulation of CKIIα and α′ with targeted si-RNAs does not alter phosphorylation of the topo IIα peptide containing Ser-1106. An aliquot of HCT-116 cells transfected with scrambled si-RNA or si - CKIIα plus si-CKIIα′, as described in Materials and methods section, were lysed in RIPA buffer and subjected to western blot analysis to determine down-regulation of CKIIα and CKIIα′ ( A ). The remaining cells were labeled with [ 32 P] orthophosphoric acid for 3 h. Cell lysates prepared in RIPA buffer were immunoprecipitated with topo IIα-specific antibodies, subjected to SDS–PAGE and transferred to nitrocellulose membrane. The stained topo IIα band was excised, proteolysed with trypsin and the labeled tryptic peptides analyzed by 2D-phosphopeptide mapping ( B ). The isobutyric acid buffer (isobutyric acid/ n -butanol/pyridine/acetic acid/deionized water) (32.9/1/2.5/1.5/14.7, v/v) was used for resolving the peptides in the second dimension. The arrow indicates the position of Ser-1106 containing peptide.
    Figure Legend Snippet: Down-regulation of CKIIα and α′ with targeted si-RNAs does not alter phosphorylation of the topo IIα peptide containing Ser-1106. An aliquot of HCT-116 cells transfected with scrambled si-RNA or si - CKIIα plus si-CKIIα′, as described in Materials and methods section, were lysed in RIPA buffer and subjected to western blot analysis to determine down-regulation of CKIIα and CKIIα′ ( A ). The remaining cells were labeled with [ 32 P] orthophosphoric acid for 3 h. Cell lysates prepared in RIPA buffer were immunoprecipitated with topo IIα-specific antibodies, subjected to SDS–PAGE and transferred to nitrocellulose membrane. The stained topo IIα band was excised, proteolysed with trypsin and the labeled tryptic peptides analyzed by 2D-phosphopeptide mapping ( B ). The isobutyric acid buffer (isobutyric acid/ n -butanol/pyridine/acetic acid/deionized water) (32.9/1/2.5/1.5/14.7, v/v) was used for resolving the peptides in the second dimension. The arrow indicates the position of Ser-1106 containing peptide.

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

    40) Product Images from "Bach1 deficiency reduces severity of osteoarthritis through upregulation of heme oxygenase-1"

    Article Title: Bach1 deficiency reduces severity of osteoarthritis through upregulation of heme oxygenase-1

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-015-0792-1

    Expression of heme oxygenase-1 ( HO-1 ) in articular cartilages from wild-type ( WT ) mice and Bach1 -/- mice. Primary articular chondrocytes were isolated from mouse femoral head cartilage (n = 4 per group) at the age of 1 month. a Real-time PCR assay for Hmox1. b Western blot analysis of HO-1 protein (n = 4). c Evaluation of the phenotype of articular chondrocytes in Bach1 -/- mice. Expression of cartilage-associated genes was determined by real-time PCR assay (n = 4 per group). d Comparison of HO-1 expression in articular cartilage from 22-month-old wild-type mice and Bach1 -/- mice (n = 4 per group). Knee joints were assessed by immunohistochemistry using anti-HO-1 antibody. Original magnification × 10 ( left ; scale bars 200 μm, and × 20 ( right ; scale bars 100 μm). Values in a , c and d were expressed as means ± SD. Statistical analysis was performed with the Mann–Whitney U test; ** P
    Figure Legend Snippet: Expression of heme oxygenase-1 ( HO-1 ) in articular cartilages from wild-type ( WT ) mice and Bach1 -/- mice. Primary articular chondrocytes were isolated from mouse femoral head cartilage (n = 4 per group) at the age of 1 month. a Real-time PCR assay for Hmox1. b Western blot analysis of HO-1 protein (n = 4). c Evaluation of the phenotype of articular chondrocytes in Bach1 -/- mice. Expression of cartilage-associated genes was determined by real-time PCR assay (n = 4 per group). d Comparison of HO-1 expression in articular cartilage from 22-month-old wild-type mice and Bach1 -/- mice (n = 4 per group). Knee joints were assessed by immunohistochemistry using anti-HO-1 antibody. Original magnification × 10 ( left ; scale bars 200 μm, and × 20 ( right ; scale bars 100 μm). Values in a , c and d were expressed as means ± SD. Statistical analysis was performed with the Mann–Whitney U test; ** P

    Techniques Used: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, MANN-WHITNEY

    The involvement of heme oxygenase-1 ( HO-1 ) in changes observed in Bach1 -/- articular chondrocytes. Small interfering HO-1 ( siHO-1 ) or control siRNA (10 uM) was transfected into chondrocytes from wild-type ( WT ) and Bach1 -/- mice at 1 month of age (n = 4 per group). a The expression of superoxide dismutase 2 ( SOD2 ) protein was detected by immunoblot analysis. Values are the mean ± SD. Statistical analysis was performed with the Steel–Dwass test; * P
    Figure Legend Snippet: The involvement of heme oxygenase-1 ( HO-1 ) in changes observed in Bach1 -/- articular chondrocytes. Small interfering HO-1 ( siHO-1 ) or control siRNA (10 uM) was transfected into chondrocytes from wild-type ( WT ) and Bach1 -/- mice at 1 month of age (n = 4 per group). a The expression of superoxide dismutase 2 ( SOD2 ) protein was detected by immunoblot analysis. Values are the mean ± SD. Statistical analysis was performed with the Steel–Dwass test; * P

    Techniques Used: Transfection, Mouse Assay, Expressing

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    Article Snippet: .. Primary Culture of Neonatal Rat Cardiomyocytes Primary neonatal rat cardiomyocytes were isolated using a neonatal cardiomyocyte isolation kit (Worthington Biochemical, Lakewood, NJ, USA) according to the published procedure [ ]. .. Briefly, ventricles from 2–4 day-old Sprague-Dawley rats (Japan SLC, Inc., Hamamatsu, Japan) were incubated for 16–20 h at 4 °C in Hank’s balanced salt solution containing trypsin (50–100 μg/mL), followed by digestion with collagenase (75 U/mL) for 40 min at 37 °C.

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    Article Snippet: .. RNVC were extracted from the hearts of Sprague Dawley rat pups (strain code 001; Charles River) 1–3 days after birth using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corporation). .. The isolated ventricles were incubated in calcium/magnesium-free Hank’s Balanced Salt Solution containing trypsin (50 µg/mL) in vented-cap tubes with slow agitation at 4 °C for 18 h. The trypsin was then inhibited by adding Soybean Trypsin Inhibitor (200 µg/mL), and the ventricles were further digested with collagenase (100 units/mL) at 37 °C for 30 min.

    Article Title: Pro-survival function of MEF2 in cardiomyocytes is enhanced by β-blockers
    Article Snippet: .. Cell culture Primary neonatal rat cardiomyocytes were prepared from 1- to 3-day old Sprague Dawley rats using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp, Lakewood, NJ, USA). .. Briefly, whole hearts were dissociated with trypsin (Promega, Madison, WI, USA) and collagenase (Worthington Biochemical Corp).

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    Article Snippet: .. Cardiac cells of 2–4-day-old WKY rats were isolated using a neonatal cardiomyocyte isolation system (Worthington Biochemical Corp, Lakewood, New Jersey, USA). .. After pre-plating to reduce contaminating non-muscle cells, cardiomyocytes were plated on Bioflex collagen 6-well plates (Flexcell, McKeesport, Pennsylvania, USA) and cultured in DMEM/F-12 (1:1) media supplemented with 10% fetal bovine serum for approximately 1.5 days prior to switching to serum-free medium.

    Cell Culture:

    Article Title: Pro-survival function of MEF2 in cardiomyocytes is enhanced by β-blockers
    Article Snippet: .. Cell culture Primary neonatal rat cardiomyocytes were prepared from 1- to 3-day old Sprague Dawley rats using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp, Lakewood, NJ, USA). .. Briefly, whole hearts were dissociated with trypsin (Promega, Madison, WI, USA) and collagenase (Worthington Biochemical Corp).

    Concentration Assay:

    Article Title: Single Cell Transcriptomics Reconstructs Fate Conversion from Fibroblast to Cardiomyocyte
    Article Snippet: .. Neonatal CMs were isolated using the neonatal cardiomyocytes isolation system (Worthington Biochemical Corporation) except that all enzymes were used at a ¼ of the recommended concentration to increase cell viability. .. After a 1.5 hour pre-plating on uncoated surface to remove attached nonmyocytes, the unattached CMs were collected in TRIzol ( > 80% viability by Trypan blue staining).

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    Worthington Biochemical hiv rt rnase h
    Antiviral mechanism of ODN sequences. a Schematic representation of reverse transcription of <t>HIV</t> and mechanism of ODN A action. The HIV-1 extended polypurine tract (PPT) is indicated as black box with the RT/RNase H cleavage side depicted in white. ODN A interacts with the highly conserved PPT, mimicking the natural replication intermediate RNA-DNA hybrid, which results in premature activation of reverse transcriptase (RT)/RNase H hydroloysis of the viral RNA genome. b Sequences of the ODNs used. All ODNs form hairpin-like structures with an antisense ( lower ) and passenger ( upper ) strand linked by four thymidines. Watson-Crick bonds are shown as vertical bars . Phosphorothioate-modified nucleotides are shown in bold and marked by a star . The ODN A sequence is complementary to the extended PPT, and ODN Co targets an HIV-1 RNA region outside of the PPT. ODN G serves as a further control, with a similar passenger strand sequence compared to ODN A but a non-complementary HIV-1 PPT sequence. c Sequence of the extended polypurine tract of HIV-1 recognized by the viral RNase H, whose specific cleavage site is indicated by an arrow
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    Vitamin D deficiency does not affect the immunogenicity of dendritic cells or the numbers of regulatory T cells in the airway-draining lymph nodes. Eight week-old male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-containing or -null diets (respectively) were sensitized intraperitoneally with 1 µg ovalbumin <t>(OVA)</t> (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitization, and one week later their airways challenged with aerosolised OVA (1% in saline). Cells were assessed twenty-four h after OVA challenge. In (A), the capacity of CD11c+ cells isolated from the ADLN to process and present antigen to co-cultured CD4+ T cells from DO11.10 mice was determined by [ 3 H]-thymidine incorporation for the final 24 h of 96 h co-culture with 1 µg/ml OVA peptide. Results are shown of a representative of 3 independent experiments (mean±SEM) for CD11c+ cells cultured at various ratios with CD4+ cells. In (B), the FACS gating strategy used to determine the percentage of ClassII+CD11c+CD8− cells in ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (C), the FACS gating strategy used to determine the percentage of CD3+CD4+ T cells and CD3+CD4+CD25+Foxp3+ and CD3+CD4+CD25+Foxp3− cells in the ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (D), the expression of CCL5, CCL11, CCL19, CCL20, CXCL1 and IL-18RA mRNAs in CD11c+ cells isolated from pooled ADLN of ten mice per treatment with results shown from 3 independent experiments (mean+SEM). (*p
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    Vitamin D deficiency does not affect the immunogenicity of dendritic cells or the numbers of regulatory T cells in the airway-draining lymph nodes. Eight week-old male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-containing or -null diets (respectively) were sensitized intraperitoneally with 1 µg ovalbumin <t>(OVA)</t> (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitization, and one week later their airways challenged with aerosolised OVA (1% in saline). Cells were assessed twenty-four h after OVA challenge. In (A), the capacity of CD11c+ cells isolated from the ADLN to process and present antigen to co-cultured CD4+ T cells from DO11.10 mice was determined by [ 3 H]-thymidine incorporation for the final 24 h of 96 h co-culture with 1 µg/ml OVA peptide. Results are shown of a representative of 3 independent experiments (mean±SEM) for CD11c+ cells cultured at various ratios with CD4+ cells. In (B), the FACS gating strategy used to determine the percentage of ClassII+CD11c+CD8− cells in ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (C), the FACS gating strategy used to determine the percentage of CD3+CD4+ T cells and CD3+CD4+CD25+Foxp3+ and CD3+CD4+CD25+Foxp3− cells in the ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (D), the expression of CCL5, CCL11, CCL19, CCL20, CXCL1 and IL-18RA mRNAs in CD11c+ cells isolated from pooled ADLN of ten mice per treatment with results shown from 3 independent experiments (mean+SEM). (*p
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    Vitamin D deficiency does not affect the immunogenicity of dendritic cells or the numbers of regulatory T cells in the airway-draining lymph nodes. Eight week-old male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-containing or -null diets (respectively) were sensitized intraperitoneally with 1 µg ovalbumin <t>(OVA)</t> (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitization, and one week later their airways challenged with aerosolised OVA (1% in saline). Cells were assessed twenty-four h after OVA challenge. In (A), the capacity of CD11c+ cells isolated from the ADLN to process and present antigen to co-cultured CD4+ T cells from DO11.10 mice was determined by [ 3 H]-thymidine incorporation for the final 24 h of 96 h co-culture with 1 µg/ml OVA peptide. Results are shown of a representative of 3 independent experiments (mean±SEM) for CD11c+ cells cultured at various ratios with CD4+ cells. In (B), the FACS gating strategy used to determine the percentage of ClassII+CD11c+CD8− cells in ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (C), the FACS gating strategy used to determine the percentage of CD3+CD4+ T cells and CD3+CD4+CD25+Foxp3+ and CD3+CD4+CD25+Foxp3− cells in the ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (D), the expression of CCL5, CCL11, CCL19, CCL20, CXCL1 and IL-18RA mRNAs in CD11c+ cells isolated from pooled ADLN of ten mice per treatment with results shown from 3 independent experiments (mean+SEM). (*p
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    Antiviral mechanism of ODN sequences. a Schematic representation of reverse transcription of HIV and mechanism of ODN A action. The HIV-1 extended polypurine tract (PPT) is indicated as black box with the RT/RNase H cleavage side depicted in white. ODN A interacts with the highly conserved PPT, mimicking the natural replication intermediate RNA-DNA hybrid, which results in premature activation of reverse transcriptase (RT)/RNase H hydroloysis of the viral RNA genome. b Sequences of the ODNs used. All ODNs form hairpin-like structures with an antisense ( lower ) and passenger ( upper ) strand linked by four thymidines. Watson-Crick bonds are shown as vertical bars . Phosphorothioate-modified nucleotides are shown in bold and marked by a star . The ODN A sequence is complementary to the extended PPT, and ODN Co targets an HIV-1 RNA region outside of the PPT. ODN G serves as a further control, with a similar passenger strand sequence compared to ODN A but a non-complementary HIV-1 PPT sequence. c Sequence of the extended polypurine tract of HIV-1 recognized by the viral RNase H, whose specific cleavage site is indicated by an arrow

    Journal: BMC Infectious Diseases

    Article Title: Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide

    doi: 10.1186/s12879-016-1713-x

    Figure Lengend Snippet: Antiviral mechanism of ODN sequences. a Schematic representation of reverse transcription of HIV and mechanism of ODN A action. The HIV-1 extended polypurine tract (PPT) is indicated as black box with the RT/RNase H cleavage side depicted in white. ODN A interacts with the highly conserved PPT, mimicking the natural replication intermediate RNA-DNA hybrid, which results in premature activation of reverse transcriptase (RT)/RNase H hydroloysis of the viral RNA genome. b Sequences of the ODNs used. All ODNs form hairpin-like structures with an antisense ( lower ) and passenger ( upper ) strand linked by four thymidines. Watson-Crick bonds are shown as vertical bars . Phosphorothioate-modified nucleotides are shown in bold and marked by a star . The ODN A sequence is complementary to the extended PPT, and ODN Co targets an HIV-1 RNA region outside of the PPT. ODN G serves as a further control, with a similar passenger strand sequence compared to ODN A but a non-complementary HIV-1 PPT sequence. c Sequence of the extended polypurine tract of HIV-1 recognized by the viral RNase H, whose specific cleavage site is indicated by an arrow

    Article Snippet: For annealing, purified RNA2 transcripts were mixed with 50 nM of ODNs in hybridization buffer (50 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 0.4 mM spermine hydrochloride, 25 mM Tris-acetate, pH 6.8), heat-treated for 3 min at 90 °C, cooled, and incubated at 37 °C for 30 min. After annealing, samples were incubated with 0.05 units/μl of HIV RT/RNase H (Worthington, USA) for a further 30 min at 37 °C.

    Techniques: Activation Assay, Modification, Sequencing

    ODN A is active in vitro and in cell culture infection assays after long-term storage at 37 °C. a ODN A (8 μM) was stored for the indicated duration in PBS or ( b ) in H 2 O at 37 °C. Samples were analyzed by non-denaturing 10 % PAGE. c ODN A (50 nM), either freshly thawed or stored for the indicated time periods at 37 °C in PBS or H 2 O was hybridized to 50 nM in vitro transcribed γ-32-ATP 5′-labeled PPT-containing RNA in the presence of HIV-1 RT/RNase H. The cleavage products were analyzed by denaturing polyacrylamide/8 M urea gel electrophoresis and are presented schematically on the right . Cleavage sites are indicated by arrowheads and labeled products are shown in black . ODN Co, annealing to sequences downstream of the PPT, served as a control. d Following the experimental procedure shown at the top : Freshly thawed ODN A or ODN Co (250 nM), or ODN A stored for 102 days at 37 °C in PBS were incubated with replication-competent HIV-1 particles (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were infected with the mixtures overnight and HIV-1 p24 antigen in the supernatant was detected at 3–14 days post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was highly significant ( p

    Journal: BMC Infectious Diseases

    Article Title: Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide

    doi: 10.1186/s12879-016-1713-x

    Figure Lengend Snippet: ODN A is active in vitro and in cell culture infection assays after long-term storage at 37 °C. a ODN A (8 μM) was stored for the indicated duration in PBS or ( b ) in H 2 O at 37 °C. Samples were analyzed by non-denaturing 10 % PAGE. c ODN A (50 nM), either freshly thawed or stored for the indicated time periods at 37 °C in PBS or H 2 O was hybridized to 50 nM in vitro transcribed γ-32-ATP 5′-labeled PPT-containing RNA in the presence of HIV-1 RT/RNase H. The cleavage products were analyzed by denaturing polyacrylamide/8 M urea gel electrophoresis and are presented schematically on the right . Cleavage sites are indicated by arrowheads and labeled products are shown in black . ODN Co, annealing to sequences downstream of the PPT, served as a control. d Following the experimental procedure shown at the top : Freshly thawed ODN A or ODN Co (250 nM), or ODN A stored for 102 days at 37 °C in PBS were incubated with replication-competent HIV-1 particles (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were infected with the mixtures overnight and HIV-1 p24 antigen in the supernatant was detected at 3–14 days post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was highly significant ( p

    Article Snippet: For annealing, purified RNA2 transcripts were mixed with 50 nM of ODNs in hybridization buffer (50 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 0.4 mM spermine hydrochloride, 25 mM Tris-acetate, pH 6.8), heat-treated for 3 min at 90 °C, cooled, and incubated at 37 °C for 30 min. After annealing, samples were incubated with 0.05 units/μl of HIV RT/RNase H (Worthington, USA) for a further 30 min at 37 °C.

    Techniques: In Vitro, Cell Culture, Infection, Polyacrylamide Gel Electrophoresis, Labeling, Nucleic Acid Electrophoresis, Incubation, Inhibition

    The lubricant CMC (K-Y Jelly) does not influence the stability and antiviral activity of ODN A. a ODN A (8 μM) was incubated for the indicated days in 25 % K-Y Jelly (in PBS) at 37 °C. The samples were analyzed by 10 % non-denaturing polyacrylamide gel electrophoresis. b Analysis of ODN A-triggered, RNase H-mediated cleavage of PPT RNA in vitro. Assays were performed as described in Fig. 2c . Cleavage sites are indicated by arrowheads and the labeled products are indicated in black . c Following the experimental procedure shown at the top : ODN A pre-incubated in 25 % K-Y Jelly/PBS for 57 days, freshly thawed ODN A, or ODN Co (both 250 nM) were incubated with replication-competent HIV-1 virions (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were subsequently infected overnight. Virus replication was monitored by HIV-1 p24 antigen ELISA of culture supernatants at day 3–14 post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was significant (day 7, p

    Journal: BMC Infectious Diseases

    Article Title: Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide

    doi: 10.1186/s12879-016-1713-x

    Figure Lengend Snippet: The lubricant CMC (K-Y Jelly) does not influence the stability and antiviral activity of ODN A. a ODN A (8 μM) was incubated for the indicated days in 25 % K-Y Jelly (in PBS) at 37 °C. The samples were analyzed by 10 % non-denaturing polyacrylamide gel electrophoresis. b Analysis of ODN A-triggered, RNase H-mediated cleavage of PPT RNA in vitro. Assays were performed as described in Fig. 2c . Cleavage sites are indicated by arrowheads and the labeled products are indicated in black . c Following the experimental procedure shown at the top : ODN A pre-incubated in 25 % K-Y Jelly/PBS for 57 days, freshly thawed ODN A, or ODN Co (both 250 nM) were incubated with replication-competent HIV-1 virions (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were subsequently infected overnight. Virus replication was monitored by HIV-1 p24 antigen ELISA of culture supernatants at day 3–14 post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was significant (day 7, p

    Article Snippet: For annealing, purified RNA2 transcripts were mixed with 50 nM of ODNs in hybridization buffer (50 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 0.4 mM spermine hydrochloride, 25 mM Tris-acetate, pH 6.8), heat-treated for 3 min at 90 °C, cooled, and incubated at 37 °C for 30 min. After annealing, samples were incubated with 0.05 units/μl of HIV RT/RNase H (Worthington, USA) for a further 30 min at 37 °C.

    Techniques: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, In Vitro, Labeling, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay, Inhibition

    Vitamin D deficiency does not affect the immunogenicity of dendritic cells or the numbers of regulatory T cells in the airway-draining lymph nodes. Eight week-old male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-containing or -null diets (respectively) were sensitized intraperitoneally with 1 µg ovalbumin (OVA) (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitization, and one week later their airways challenged with aerosolised OVA (1% in saline). Cells were assessed twenty-four h after OVA challenge. In (A), the capacity of CD11c+ cells isolated from the ADLN to process and present antigen to co-cultured CD4+ T cells from DO11.10 mice was determined by [ 3 H]-thymidine incorporation for the final 24 h of 96 h co-culture with 1 µg/ml OVA peptide. Results are shown of a representative of 3 independent experiments (mean±SEM) for CD11c+ cells cultured at various ratios with CD4+ cells. In (B), the FACS gating strategy used to determine the percentage of ClassII+CD11c+CD8− cells in ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (C), the FACS gating strategy used to determine the percentage of CD3+CD4+ T cells and CD3+CD4+CD25+Foxp3+ and CD3+CD4+CD25+Foxp3− cells in the ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (D), the expression of CCL5, CCL11, CCL19, CCL20, CXCL1 and IL-18RA mRNAs in CD11c+ cells isolated from pooled ADLN of ten mice per treatment with results shown from 3 independent experiments (mean+SEM). (*p

    Journal: PLoS ONE

    Article Title: Reversible Control by Vitamin D of Granulocytes and Bacteria in the Lungs of Mice: An Ovalbumin-Induced Model of Allergic Airway Disease

    doi: 10.1371/journal.pone.0067823

    Figure Lengend Snippet: Vitamin D deficiency does not affect the immunogenicity of dendritic cells or the numbers of regulatory T cells in the airway-draining lymph nodes. Eight week-old male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-containing or -null diets (respectively) were sensitized intraperitoneally with 1 µg ovalbumin (OVA) (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitization, and one week later their airways challenged with aerosolised OVA (1% in saline). Cells were assessed twenty-four h after OVA challenge. In (A), the capacity of CD11c+ cells isolated from the ADLN to process and present antigen to co-cultured CD4+ T cells from DO11.10 mice was determined by [ 3 H]-thymidine incorporation for the final 24 h of 96 h co-culture with 1 µg/ml OVA peptide. Results are shown of a representative of 3 independent experiments (mean±SEM) for CD11c+ cells cultured at various ratios with CD4+ cells. In (B), the FACS gating strategy used to determine the percentage of ClassII+CD11c+CD8− cells in ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (C), the FACS gating strategy used to determine the percentage of CD3+CD4+ T cells and CD3+CD4+CD25+Foxp3+ and CD3+CD4+CD25+Foxp3− cells in the ADLN for 8–9 mice per treatment pooled from 3 independent experiments (mean±SEM). In (D), the expression of CCL5, CCL11, CCL19, CCL20, CXCL1 and IL-18RA mRNAs in CD11c+ cells isolated from pooled ADLN of ten mice per treatment with results shown from 3 independent experiments (mean+SEM). (*p

    Article Snippet: In vitro antigen-presenting cell assay Twenty-four h after OVA challenge, ADLN were removed and digested with collagenase type 4 (1 mg/ml, Worthington Biochemical, Lakewood, NJ) and DNAse (0.1 mg/ml, Sigma) for 30 min at 37°C.

    Techniques: Mouse Assay, Isolation, Cell Culture, Co-Culture Assay, FACS, Expressing

    Vitamin D supplementation suppresses circulating neutrophil and basophil levels in male mice. Male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-replete or null diets (respectively), or vitamin D-deficient offspring switched to a vitamin D-replete diet from 8 weeks of age (VitD− to vitD+, −/+), were sensitised at 12 weeks of age intraperitoneally with 1 µg ovalbumin (OVA) (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitisation, and one week later their airways challenged with aerosolised OVA (1% in saline). Twenty-four h after the OVA challenge, the proportions of eosinophils, neutrophils and basophils of circulating white blood cells (WBC) is shown as mean + SEM for n = 5 mice/treatment for a representative experiment.

    Journal: PLoS ONE

    Article Title: Reversible Control by Vitamin D of Granulocytes and Bacteria in the Lungs of Mice: An Ovalbumin-Induced Model of Allergic Airway Disease

    doi: 10.1371/journal.pone.0067823

    Figure Lengend Snippet: Vitamin D supplementation suppresses circulating neutrophil and basophil levels in male mice. Male and female offspring born to vitamin D-replete or -deficient mothers and maintained on the vitamin D-replete or null diets (respectively), or vitamin D-deficient offspring switched to a vitamin D-replete diet from 8 weeks of age (VitD− to vitD+, −/+), were sensitised at 12 weeks of age intraperitoneally with 1 µg ovalbumin (OVA) (0.2 mg Aluminium hydroxide (Alum)). Mice were boosted with the same OVA/Alum dose two weeks after the initial sensitisation, and one week later their airways challenged with aerosolised OVA (1% in saline). Twenty-four h after the OVA challenge, the proportions of eosinophils, neutrophils and basophils of circulating white blood cells (WBC) is shown as mean + SEM for n = 5 mice/treatment for a representative experiment.

    Article Snippet: In vitro antigen-presenting cell assay Twenty-four h after OVA challenge, ADLN were removed and digested with collagenase type 4 (1 mg/ml, Worthington Biochemical, Lakewood, NJ) and DNAse (0.1 mg/ml, Sigma) for 30 min at 37°C.

    Techniques: Mouse Assay