anti mouse cd4  (Thermo Fisher)


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    Structured Review

    Thermo Fisher anti mouse cd4
    Th2 and Th1 response to TM in Mina KO mice. Shown are the percentage and absolute number of (A) <t>CD4</t> + IL4 + cells and (B) CD4 + IFNγ + cells and (C) the level of secreted IL4 and IFNγ from mesenteric lymph node cells of Mina KO and WT littermate control mice infected 21 days earlier with 150 embryonated TM eggs. Data are mean ± SD (A: WT n = 8, KO n = 10 each, B: WT and KO: n = 15 and 14 each respectively, C: WT and KO n = 7 each respectively) from 2 independent experiments). Statistical significance was computed by the two-tailed Student’s t-test.
    Anti Mouse Cd4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 7305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 7305 article reviews
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    anti mouse cd4 - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Myc-induced nuclear antigen constrains a latent intestinal epithelial cell-intrinsic anthelmintic pathway"

    Article Title: Myc-induced nuclear antigen constrains a latent intestinal epithelial cell-intrinsic anthelmintic pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211244

    Th2 and Th1 response to TM in Mina KO mice. Shown are the percentage and absolute number of (A) CD4 + IL4 + cells and (B) CD4 + IFNγ + cells and (C) the level of secreted IL4 and IFNγ from mesenteric lymph node cells of Mina KO and WT littermate control mice infected 21 days earlier with 150 embryonated TM eggs. Data are mean ± SD (A: WT n = 8, KO n = 10 each, B: WT and KO: n = 15 and 14 each respectively, C: WT and KO n = 7 each respectively) from 2 independent experiments). Statistical significance was computed by the two-tailed Student’s t-test.
    Figure Legend Snippet: Th2 and Th1 response to TM in Mina KO mice. Shown are the percentage and absolute number of (A) CD4 + IL4 + cells and (B) CD4 + IFNγ + cells and (C) the level of secreted IL4 and IFNγ from mesenteric lymph node cells of Mina KO and WT littermate control mice infected 21 days earlier with 150 embryonated TM eggs. Data are mean ± SD (A: WT n = 8, KO n = 10 each, B: WT and KO: n = 15 and 14 each respectively, C: WT and KO n = 7 each respectively) from 2 independent experiments). Statistical significance was computed by the two-tailed Student’s t-test.

    Techniques Used: Mouse Assay, Infection, Two Tailed Test

    2) Product Images from "Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors"

    Article Title: Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors

    Journal: Science Advances

    doi: 10.1126/sciadv.aau3333

    COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P
    Figure Legend Snippet: COR388 target engagement and dose-dependent effects on brain P. gingivalis , Aβ 1–42 , and TNFα in mice. ( A ) COR553 fluorescent activity probe for Kgp. ( B ) COR553 labeling of Kgp in P. gingivalis W83 strain and no labeling in mutant deficient in Kgp (ΔKgp). ( C ) W83 lysates labeled with COR553. Left lane, before immunodepletion; middle lane, after immunodepletion with anti-Kgp–conjugated beads; right lane, after elution from anti-Kgp–conjugated beads. ( D ) W83 strain titrated and labeled with COR553 to determine the limit of bacterial detection. See Results for details. ( E ) Oral plaque samples from human subjects (CB1-5) with periodontal disease were incubated ex vivo with COR553 probe with or without preincubation with COR388. COR553 probe and CAB102 detected Kgp strongly in three subjects (CB1, CB4, and CB5) and weakly in one subject (CB3). COR388 preincubation blocked COR553 probe binding to Kgp. ( F ) qPCR analysis of plaque samples using hmuY gene–specific primers identified P. gingivalis DNA in samples. ( G ) qPCR analysis of saliva samples. The bar graphs in (F) and (G) show the means and SEMs of three replicates. ( H ) COR388 treatment of W83 culture in defined growth medium reduced growth similarly to a Kgp-deficient strain (ΔKgp) over 43 hours. ( I ) Resistance developed rapidly to moxifloxacin but not COR388 with repeat passaging of bacterial culture. ( J to L ) Efficacy of COR388 at three oral doses of 3, 10, and 30 mg/kg twice daily in treating an established P. gingivalis brain infection in mice. Reduction of brain tissue levels of P. gingivalis (J), Aβ 1–42 (K), and TNFα (L). The bar graphs show the means with SEM error bars. *** P

    Techniques Used: Mouse Assay, Activity Assay, Labeling, Mutagenesis, Incubation, Ex Vivo, Binding Assay, Real-time Polymerase Chain Reaction, Passaging, Infection

    Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P
    Figure Legend Snippet: Small-molecule gingipain inhibitors protect neuronal cells against P. gingivalis – and gingipain-induced toxicity in vitro and in vivo. ( A ) Differentiated SH-SY5Y neuroblastoma cells demonstrate cell aggregation after exposure to RgpB (10 μg/ml), Kgp (10 μg/ml), or both for 24 hours. The nonselective cysteine protease inhibitor iodoacetamide (IAM) blocks the gingipain-induced cell aggregation. ( B ) AlamarBlue viability assay shows that P. gingivalis ( P.g. ) is toxic to SH-SY5Y cells (MOI of 400) and that the small-molecule Kgp inhibitor COR271 and the RgpB inhibitor COR286 provide dose-dependent protection. The broad-spectrum antibiotics moxifloxacin and doxycycline and the γ-secretase inhibitor semagacestat did not inhibit the cytotoxic effect of P. gingivalis . ( C ) Fluoro-Jade C (FJC) staining (green) in pyramidal neurons of the CA1 region of the mouse hippocampus indicates neurodegeneration after stereotactic injection of gingipains. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. ( D ) The total number of FJC-positive cells was determined from serial section through the entire hippocampus. Results demonstrate a significant neuroprotective effect of gingipain inhibitors COR271 + COR286 after acute gingipain exposure in the hippocampus (* P

    Techniques Used: In Vitro, In Vivo, Protease Inhibitor, Viability Assay, Staining, Injection

    RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.
    Figure Legend Snippet: RgpB colocalizes with neurons and pathology in AD hippocampus. ( A ) IHC using RgpB-specific monoclonal antibody 18E6 (representative images from a 63-year-old AD patient). The hippocampus shows abundant intracellular RgpB in the hilus (1), CA3 pyramidal layer (2), granular cell layer (3), and molecular layer (4). High-magnification images from the indicated areas (1 to 4) exhibit a granular staining pattern consistent with P. gingivalis intracellular infection. Scale bars, 200 μm (overview), 50 μm (1), and 10 μm (2 to 4). ( B ) AD hippocampus stained with 18E6 (AD) compared to gingival tissue (gingiva) from a patient with periodontal disease as well as a non-AD control and mouse IgG1 control (IgG1) in an adjacent hippocampal section. Scale bars, 50 μm. ( C ) Immunofluorescent colabeling with CAB101 reveals granular intraneuronal staining for RgpB (arrows) in MAP2-positive neurons in both the granular cell layer (GCL) and the pyramidal cell layer (CA1). Scale bars, 10 μm. ( D ) Dense extracellular RgpB-positive aggregates (arrowheads) were closely associated with astrocytes [glial fibrillary acidic protein (GFAP)]. There was no observed association of RgpB with microglia (IBA1). Scale bars, 10 μm. ( E ) RgpB was associated with paired helical filament Tau (PHF-Tau; arrows). RgpB-positive neurons negative for PHF-Tau (arrowheads) were also seen. Intracellular Aβ was often colocalized with RgpB (arrows). In some Aβ-positive cells, RgpB could not be detected (arrowheads). Scale bars, 10 μm.

    Techniques Used: Immunohistochemistry, Staining, Infection

    Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).
    Figure Legend Snippet: Identification of P. gingivalis –specific protein and DNA in cortex from control and AD patients. ( A ) WB with four different strains of P. gingivalis and CAB102 detection of typical molecular weight bands for Kgp in bacterial lysates. ( B ) IP using brain lysates from nondemented controls (C1 to C6; ages 75, 54, 63, 45, 37, and 102 years, respectively) and AD patients (AD1 to AD3; ages 83, 90, and 80 years, respectively) using CAB102 with subsequent WB reveals the ~50-kDa Kgp catalytic subunit (Kgp cat ), along with higher– and lower–molecular weight Kgp species seen in (A). ( C ) qPCR from DNA isolated from the same brain lysates as the protein samples analyzed in (B) shows a positive signal in nondemented control (C1 to C5) and AD (AD1 to AD3) samples. Sample C6 from the 102-year-old nondemented control patient had no detectable qPCR signal in (C) and very faint bands indicating near absence of Kgp (B) (mean with SEM error bars of repeat qPCR runs).

    Techniques Used: Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

    Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.
    Figure Legend Snippet: Detection of P. gingivalis in CSF and oral biofluids from clinical AD subjects. ( A ) Detection and quantitation of P. gingivalis DNA by qPCR in CSF from subjects with probable AD. ( B ) Detection and quantitation of P. gingivalis DNA by qPCR from matching saliva samples. ( C ) Top: PCR products detecting P. gingivalis from CSF in (A) from all subjects run on agarose gel including negative and positive controls containing a synthetic DNA template. Faint or undetectable PCR products from subjects AD1, AD3, and AD5 were below the limit of quantitation for copy number and not of sufficient quantity for sequence analysis. Bottom: qPCR products from CSF from the same subjects for H. pylori. ( D ) Data table includes age and Mini Mental Status Exam (MMSE) score on subjects and sequence identity of PCR products to P. gingivalis hmuY DNA sequence. Sequence data are included in fig. S4. NS, not sequenced.

    Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P
    Figure Legend Snippet: P. gingivalis invasion of the brain induces an Aβ 1–42 response that is blocked by gingipain inhibition in mice. ( A ) P. gingivalis PCR product in mouse brains after oral infection with P. gingivalis W83, with or without treatment with the Kgp inhibitor COR119, or infection with gingipain knockout strain ΔRgpB or ΔKgp. Lanes 1 to 8 represent individual experimental animals. In the first lane ( P.g. ), P. gingivalis W83 was used as a positive control. ( B ) P. gingivalis W83–infected mice, but not COR119-treated mice or mice infected with gingipain knockouts, had significantly higher Aβ 1–42 levels compared to mock-infected mice (*** P

    Techniques Used: Inhibition, Mouse Assay, Polymerase Chain Reaction, Infection, Knock-Out, Positive Control

    P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.
    Figure Legend Snippet: P. gingivalis and gingipains fragment tau. ( A ) WB analysis of total soluble tau in SH-SY5Y cells infected with increasing concentrations of wild-type (WT) P. gingivalis strain W83 ( P.g. ) and P. gingivalis gingipain-deficient mutants either lacking Kgp activity (KgpΔIg-B) or lacking both Kgp and Rgp activity (ΔK/ΔRAB-A) . Uninfected SH-SY5Y cells (No P.g. ) were used as a negative control. Glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a loading control. Total tau was monitored with the monoclonal antibody Tau-5 at 1, 4, and 8 hours after infection. ( B ) Densitometry analysis of the total tau WB images. ( C ) WB analysis of rtau-441 incubated with purified Kgp and RgpB catalytic domains combined (Gp) at various concentrations for 1 hour at 37°C. The blot was probed with tau monoclonal antibody T46. ( D ) Gingipain cleavage sites in rtau-441 deduced from peptide fragments identified by MS for rtau-441 incubated with 1 or 10 nM gingipains. (a) T46 antibody epitope (red). (b) Tau-5 antibody epitope (red). (c) N-terminal tau fragment. (d) C-terminal tau fragment. (e) Kgp-generated tau fragments containing the VQIVYK sequence. (f) Kgp-generated fragments containing the VQIINK sequence. (g) An RgpB-generated tau fragment. *Cleavage sites identified at 1 nM gingipains.

    Techniques Used: Western Blot, Infection, Activity Assay, Negative Control, Incubation, Purification, Mass Spectrometry, Generated, Sequencing

    3) Product Images from "Phenotypic and Functional Profile of HIV-Inhibitory CD8 T Cells Elicited by Natural Infection and Heterologous Prime/Boost Vaccination ▿Phenotypic and Functional Profile of HIV-Inhibitory CD8 T Cells Elicited by Natural Infection and Heterologous Prime/Boost Vaccination ▿ †"

    Article Title: Phenotypic and Functional Profile of HIV-Inhibitory CD8 T Cells Elicited by Natural Infection and Heterologous Prime/Boost Vaccination ▿Phenotypic and Functional Profile of HIV-Inhibitory CD8 T Cells Elicited by Natural Infection and Heterologous Prime/Boost Vaccination ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.00138-10

    Differentiation state of HIV-specific CD8 + T cells from subjects with antiviral activity. CD8 + T cells from 15 virus controllers, 15 HIV-1 + chronic donors, and 20 vaccinees with positive CD8 + T-cell-mediated antiviral activity were analyzed for expression of the differentiation markers CD45RO, CCR7, and CD28. Cells that produced at least one function in response to HIV-1 peptide stimulation were categorized as early memory (CD45RO − CCR7 + CD28 + ), central memory (CM; CD45RO + CCR7 + CD28 + ), transitional memory (TM; CD45RO + CCR7 − CD28 + ), effector memory (EM; CD45RO + CCR7 − CD28 − ), or terminal effector (TE; CD45RO − CCR7 − CD28 − ). Pie charts show the fraction of HIV-1-specific cells expressing the indicated phenotype.
    Figure Legend Snippet: Differentiation state of HIV-specific CD8 + T cells from subjects with antiviral activity. CD8 + T cells from 15 virus controllers, 15 HIV-1 + chronic donors, and 20 vaccinees with positive CD8 + T-cell-mediated antiviral activity were analyzed for expression of the differentiation markers CD45RO, CCR7, and CD28. Cells that produced at least one function in response to HIV-1 peptide stimulation were categorized as early memory (CD45RO − CCR7 + CD28 + ), central memory (CM; CD45RO + CCR7 + CD28 + ), transitional memory (TM; CD45RO + CCR7 − CD28 + ), effector memory (EM; CD45RO + CCR7 − CD28 − ), or terminal effector (TE; CD45RO − CCR7 − CD28 − ). Pie charts show the fraction of HIV-1-specific cells expressing the indicated phenotype.

    Techniques Used: Activity Assay, Expressing, Produced

    CD107a and MIP-1β expression independently correlate with CD8 + T-cell-mediated virus inhibition. CD8 + cells from 40 vaccinees were examined for coexpression of three different effector functions by flow cytometric analysis. CD8 + T-cell responses are shown following Env (A) and Gag (B) peptide stimulation. Asterisks denote effector functions for which there are statistically significant differences between vaccinees with (red symbols) or without (blue) antiviral activity (*, P
    Figure Legend Snippet: CD107a and MIP-1β expression independently correlate with CD8 + T-cell-mediated virus inhibition. CD8 + cells from 40 vaccinees were examined for coexpression of three different effector functions by flow cytometric analysis. CD8 + T-cell responses are shown following Env (A) and Gag (B) peptide stimulation. Asterisks denote effector functions for which there are statistically significant differences between vaccinees with (red symbols) or without (blue) antiviral activity (*, P

    Techniques Used: Expressing, Inhibition, Flow Cytometry, Activity Assay

    Impact of HLA alleles on CD8 + T-cell-mediated virus inhibition. Cohorts were divided based on the expression of HLA-B alleles associated with virus control in vivo (HLA B*57 and B*27). Increased antiviral activity associated with the presence of controlling alleles for two viruses (CXCR4-tropic NL4.3-LucR.T2A and CCR5 + CXCR4-dualtropic NL-LucR.T2A-WEAU.ecto) are indicated by asterisks ( P
    Figure Legend Snippet: Impact of HLA alleles on CD8 + T-cell-mediated virus inhibition. Cohorts were divided based on the expression of HLA-B alleles associated with virus control in vivo (HLA B*57 and B*27). Increased antiviral activity associated with the presence of controlling alleles for two viruses (CXCR4-tropic NL4.3-LucR.T2A and CCR5 + CXCR4-dualtropic NL-LucR.T2A-WEAU.ecto) are indicated by asterisks ( P

    Techniques Used: Inhibition, Expressing, In Vivo, Activity Assay

    DNA prime/rAd5 boost elicits CD8 + T cells capable of mediating HIV-1 inhibition in vitro . CD8 + T-cell antiviral activity against five Env reporter viruses expressing transmitted/founder envelopes or NL4-3 is shown for 23 seronegative donors, 40 DNA/rAd5 vaccinees, 21 HIV-1-positive chronically infected donors, and 16 HIV-1-positive virus controllers. The inhibition is calculated as the log 10 reduction in virus infection, measured as viral luciferase-produced relative light units (RLU) compared to autologous infectivity controls. A threshold for positive antiviral activity was established as 3 standard deviations above the mean of the seronegative inhibition values. (The dotted line shows a 0.8-log reduction.)
    Figure Legend Snippet: DNA prime/rAd5 boost elicits CD8 + T cells capable of mediating HIV-1 inhibition in vitro . CD8 + T-cell antiviral activity against five Env reporter viruses expressing transmitted/founder envelopes or NL4-3 is shown for 23 seronegative donors, 40 DNA/rAd5 vaccinees, 21 HIV-1-positive chronically infected donors, and 16 HIV-1-positive virus controllers. The inhibition is calculated as the log 10 reduction in virus infection, measured as viral luciferase-produced relative light units (RLU) compared to autologous infectivity controls. A threshold for positive antiviral activity was established as 3 standard deviations above the mean of the seronegative inhibition values. (The dotted line shows a 0.8-log reduction.)

    Techniques Used: Inhibition, In Vitro, Activity Assay, Expressing, Infection, Luciferase, Produced

    Env- and Gag-specific CD8 + T cells in vaccinees correlate with HIV-1 inhibition in vitro . Expression of cytokine and degranulation markers was measured in PBMCs from 40 vaccinees by polychromatic flow cytometry. Total CD8 + T-cell responses for each function are shown following Env (A) and Gag (B) peptide stimulation. Asterisks denote functions for which statistically significant differences between vaccinees with antiviral activity (red symbols) and vaccinees without antiviral activity (blue) ( P
    Figure Legend Snippet: Env- and Gag-specific CD8 + T cells in vaccinees correlate with HIV-1 inhibition in vitro . Expression of cytokine and degranulation markers was measured in PBMCs from 40 vaccinees by polychromatic flow cytometry. Total CD8 + T-cell responses for each function are shown following Env (A) and Gag (B) peptide stimulation. Asterisks denote functions for which statistically significant differences between vaccinees with antiviral activity (red symbols) and vaccinees without antiviral activity (blue) ( P

    Techniques Used: Inhibition, In Vitro, Expressing, Flow Cytometry, Cytometry, Activity Assay

    Effector function in HIV-1-infected subjects with antiviral activity. Expression of cytokine and degranulation markers was measured in 15 virus controllers (A) and 15 chronically infected subjects (B) by flow cytometric analysis. Total CD8 + T-cell responses for each function are shown following Env (blue symbols), Gag (red), or Nef (green) peptide stimulation. Shaded bars indicate interquartile range.
    Figure Legend Snippet: Effector function in HIV-1-infected subjects with antiviral activity. Expression of cytokine and degranulation markers was measured in 15 virus controllers (A) and 15 chronically infected subjects (B) by flow cytometric analysis. Total CD8 + T-cell responses for each function are shown following Env (blue symbols), Gag (red), or Nef (green) peptide stimulation. Shaded bars indicate interquartile range.

    Techniques Used: Infection, Activity Assay, Expressing, Flow Cytometry

    Virus inhibition mediated by subsets of CD8 + T cells. CD8 + T cells from 10 donors positive for antiviral activity (4 vaccinees and 6 viral controllers) were sorted by flow cytometry into four of five possible subsets for each donor (sorts performed in each of three different experiments focused on different subsets). Bars represent the mean ± standard error of the mean for those sorts where sufficient cells were isolated for the assay: naïve, n = 6; early memory (CD45RO − CCR7 − CD27 + ), n = 4; central memory (CD45RO + CD27 + ), n = 9; effector memory (CD45RO + CD27 − ), n = 8; and terminal effector (CD57 + ), n = 10. (A) Total inhibitory activity per culture of 45,000 cells; (B) Inhibitory activity normalized by the fraction of HIV-1-specific T cells (determined by intracellular staining) contained within each subset.
    Figure Legend Snippet: Virus inhibition mediated by subsets of CD8 + T cells. CD8 + T cells from 10 donors positive for antiviral activity (4 vaccinees and 6 viral controllers) were sorted by flow cytometry into four of five possible subsets for each donor (sorts performed in each of three different experiments focused on different subsets). Bars represent the mean ± standard error of the mean for those sorts where sufficient cells were isolated for the assay: naïve, n = 6; early memory (CD45RO − CCR7 − CD27 + ), n = 4; central memory (CD45RO + CD27 + ), n = 9; effector memory (CD45RO + CD27 − ), n = 8; and terminal effector (CD57 + ), n = 10. (A) Total inhibitory activity per culture of 45,000 cells; (B) Inhibitory activity normalized by the fraction of HIV-1-specific T cells (determined by intracellular staining) contained within each subset.

    Techniques Used: Inhibition, Activity Assay, Flow Cytometry, Cytometry, Isolation, Staining

    4) Product Images from "Accurate sequential detection of primary tumor and metastatic lymphatics using a temperature-induced phase transition nanoparticulate system"

    Article Title: Accurate sequential detection of primary tumor and metastatic lymphatics using a temperature-induced phase transition nanoparticulate system

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S63720

    Characteristics of DTX/FPR-675 Pluronic NPs. Notes: ( A ) Size changes and ( B ) zeta potential of DTX/FPR-675 Pluronic NPs prepared with different soybean oil/Tween 80 weight ratios. ( C ) Size distribution and morphology and ( D ) in vitro release behavior of DTX/FPR-675 Pluronic NPs. Data are presented as the mean ± standard deviation (n=4). Abbreviations: DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; w, weight.
    Figure Legend Snippet: Characteristics of DTX/FPR-675 Pluronic NPs. Notes: ( A ) Size changes and ( B ) zeta potential of DTX/FPR-675 Pluronic NPs prepared with different soybean oil/Tween 80 weight ratios. ( C ) Size distribution and morphology and ( D ) in vitro release behavior of DTX/FPR-675 Pluronic NPs. Data are presented as the mean ± standard deviation (n=4). Abbreviations: DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; w, weight.

    Techniques Used: In Vitro, Standard Deviation, Fluorescence, Imaging

    Schematic and expected behavior of DTX/FPR-675 Pluronic NPs. Notes: ( A ) Schematic of DTX/FPR-675 Pluronic NPs according to temperature-induced phase transition and ( B ) expected behavior of NPs in blood vessels and lymphatic vessels. Abbreviations: DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; NIR, near-infrared; PEO-PPO-PEO, poly(ethylene oxide)-poly (propylene oxide)-poly(ethylene oxide).
    Figure Legend Snippet: Schematic and expected behavior of DTX/FPR-675 Pluronic NPs. Notes: ( A ) Schematic of DTX/FPR-675 Pluronic NPs according to temperature-induced phase transition and ( B ) expected behavior of NPs in blood vessels and lymphatic vessels. Abbreviations: DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; NIR, near-infrared; PEO-PPO-PEO, poly(ethylene oxide)-poly (propylene oxide)-poly(ethylene oxide).

    Techniques Used: Sublimation, Fluorescence, Imaging

    In vivo real-time and excised lymph node near-infrared fluorescence imaging of tumor-bearing mice injected with DTX/FPR-675 Pluronic NPs for lymph node imaging. Notes: In vivo whole body near-infrared fluorescence images of mice (n=4) 60 minutes post-injection of ( A ) free FPR-675 (upper) and DTX/FPR-675 Pluronic NPs (lower). Comparison of near-infrared fluorescence intensity in ( B ) an excised superficial cervical (right) and a brachial (left) node and ( C ) near-infrared fluorescence images using DTX/FPR-675 Pluronic NPs. ( D ) Representative ex vivo fluorescence images of dissected lymph nodes (superficial cervical, brachial, and inguinal). Abbreviations: DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; IV, intravenous.
    Figure Legend Snippet: In vivo real-time and excised lymph node near-infrared fluorescence imaging of tumor-bearing mice injected with DTX/FPR-675 Pluronic NPs for lymph node imaging. Notes: In vivo whole body near-infrared fluorescence images of mice (n=4) 60 minutes post-injection of ( A ) free FPR-675 (upper) and DTX/FPR-675 Pluronic NPs (lower). Comparison of near-infrared fluorescence intensity in ( B ) an excised superficial cervical (right) and a brachial (left) node and ( C ) near-infrared fluorescence images using DTX/FPR-675 Pluronic NPs. ( D ) Representative ex vivo fluorescence images of dissected lymph nodes (superficial cervical, brachial, and inguinal). Abbreviations: DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; IV, intravenous.

    Techniques Used: In Vivo, Fluorescence, Imaging, Mouse Assay, Injection, Ex Vivo

    Cytotoxicity and in vitro cellular uptake of the NPs in various cells. Notes: ( A ) In vitro cytotoxicity at various concentrations of the DTX/FPR-675 Pluronic NPs from 0.1 g/mL to 1,000 g/mL and ( B ) cellular behavior of the DTX/FPR-675 Pluronic NPs using normal cells (BAECs), macrophages (Raw264.7), and tumor (SCC-7) cells. Data are presented as the mean ± standard deviation (n=6). Abbreviations: BAECs, bovine aortic endothelial cells; DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; TRITC, tetramethylrhodamine; DAPI, 4’,6-diamidino-2-phenylindole.
    Figure Legend Snippet: Cytotoxicity and in vitro cellular uptake of the NPs in various cells. Notes: ( A ) In vitro cytotoxicity at various concentrations of the DTX/FPR-675 Pluronic NPs from 0.1 g/mL to 1,000 g/mL and ( B ) cellular behavior of the DTX/FPR-675 Pluronic NPs using normal cells (BAECs), macrophages (Raw264.7), and tumor (SCC-7) cells. Data are presented as the mean ± standard deviation (n=6). Abbreviations: BAECs, bovine aortic endothelial cells; DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; TRITC, tetramethylrhodamine; DAPI, 4’,6-diamidino-2-phenylindole.

    Techniques Used: In Vitro, Standard Deviation, Fluorescence, Imaging

    In vivo and ex vivo near-infrared fluorescence images of a lymph node with DTX/FPR-675 Pluronic NPs. Notes: ( A ) In vivo real-time near-infrared fluorescence images and ( B ) fluorescence quantification data for a localized lymph node in a mouse (n=4) within 60 minutes of being intradermally injected with DTX/FPR-675 Pluronic NPs (10 μL, 5 mg/mL DTX) in the right forepaw pad. The filter set has Cy5.5 emission (700 nm). ( C ) Fluorescence microscopic images of a dissected brachial lymph node from a non-tumor-bearing mouse (upper) and a tumor-bearing mouse (lower) with DTX/FPR-675 Pluronic NPs 60 minutes post-injection. Abbreviations: DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; TRITC, tetramethylrhodamine.
    Figure Legend Snippet: In vivo and ex vivo near-infrared fluorescence images of a lymph node with DTX/FPR-675 Pluronic NPs. Notes: ( A ) In vivo real-time near-infrared fluorescence images and ( B ) fluorescence quantification data for a localized lymph node in a mouse (n=4) within 60 minutes of being intradermally injected with DTX/FPR-675 Pluronic NPs (10 μL, 5 mg/mL DTX) in the right forepaw pad. The filter set has Cy5.5 emission (700 nm). ( C ) Fluorescence microscopic images of a dissected brachial lymph node from a non-tumor-bearing mouse (upper) and a tumor-bearing mouse (lower) with DTX/FPR-675 Pluronic NPs 60 minutes post-injection. Abbreviations: DTX, docetaxel; NPs, nanoparticles; FPR-675, Flamma™ fluorescence molecular imaging dye; TRITC, tetramethylrhodamine.

    Techniques Used: In Vivo, Ex Vivo, Fluorescence, Injection, Imaging

    5) Product Images from "Caspase-1 Independent Viral Clearance and Adaptive Immunity Against Mucosal Respiratory Syncytial Virus Infection"

    Article Title: Caspase-1 Independent Viral Clearance and Adaptive Immunity Against Mucosal Respiratory Syncytial Virus Infection

    Journal: Immune Network

    doi: 10.4110/in.2015.15.2.73

    The upregulation of CD86 and proinflammatory cytokines, but not IL-1β, are not impaired in caspase-1 deficient BMDCs and BMMs. BMDCs and BMMs were differentiated from the bone marrow of wild type and caspase-1 -/- mice and stimulated with RSV (moi=1, 5), 2.5 µg/ml CpG 2216 , and 100 ng/ml LPS. After 20 hr, IL-1β, IL-6, and IL-12p40 in supernatants from BMDCs (A~C) and BMMs (D~F) were measured by ELISA. CD86 levels from BMDCs (G) and BMMs (H) were analyzed by flow cytometry (red line=caspase-1 +/- ; blue line=caspase-1 -/- ). Data are representative of two independent experiments. Error bars indicate SD.
    Figure Legend Snippet: The upregulation of CD86 and proinflammatory cytokines, but not IL-1β, are not impaired in caspase-1 deficient BMDCs and BMMs. BMDCs and BMMs were differentiated from the bone marrow of wild type and caspase-1 -/- mice and stimulated with RSV (moi=1, 5), 2.5 µg/ml CpG 2216 , and 100 ng/ml LPS. After 20 hr, IL-1β, IL-6, and IL-12p40 in supernatants from BMDCs (A~C) and BMMs (D~F) were measured by ELISA. CD86 levels from BMDCs (G) and BMMs (H) were analyzed by flow cytometry (red line=caspase-1 +/- ; blue line=caspase-1 -/- ). Data are representative of two independent experiments. Error bars indicate SD.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    6) Product Images from "SOCS1 is essential for regulatory T cell functions by preventing loss of Foxp3 expression as well as IFN-? and IL-17A production"

    Article Title: SOCS1 is essential for regulatory T cell functions by preventing loss of Foxp3 expression as well as IFN-? and IL-17A production

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20110428

    exFoxp3 cells instruct differentiation of naive T cells to Th1 and Th17 in vitro. (A) 10 5 Socs1 +/+ , Ifnγ −/− Socs1 +/+ , or Ifnγ −/− Socs1 −/− T reg cells/well were stimulated with anti-CD3/anti-CD28 beads, 10 ng/ml IL-2, and the indicated cytokines, and IL-17A was measured by ELISA. Data are representative of five independent experiments (***, P
    Figure Legend Snippet: exFoxp3 cells instruct differentiation of naive T cells to Th1 and Th17 in vitro. (A) 10 5 Socs1 +/+ , Ifnγ −/− Socs1 +/+ , or Ifnγ −/− Socs1 −/− T reg cells/well were stimulated with anti-CD3/anti-CD28 beads, 10 ng/ml IL-2, and the indicated cytokines, and IL-17A was measured by ELISA. Data are representative of five independent experiments (***, P

    Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay

    IFN-γ suppresses CNS2 methylation and Foxp3 loss in SOCS1-deficient T reg cells. (A) Expression profiles of CD62L and CD44 on CD3 + CD4 + CD25 bright cells of the LN cells from Ifnγ −/− Socs1 +/+ and Ifnγ −/− Socs1 −/− mice. (B–D) 2 × 10 5 T reg cells from Ifnγ −/− Socs1 +/+ mice or Ifnγ −/− Socs1 −/− mice were transferred into Rag2 −/− mice. 6 wk after transfer, recipient mice were analyzed. (B) Body weight changes. (C) Flow cytometric analysis of Foxp3 expression on CD3 + CD4 + T cells from the LN in Rag2 −/− mice (percentage of Foxp3 + indicated in bar graph). (D) CD25 and intracellular CTLA4 expression on CD3 + CD4 + Foxp3 + cells and Foxp3 − cells from the LN in Rag2 −/− mice (right) compared with those on CD3 + CD4 + CD25 bright cells before transfer (left; mean fluorescent intensity [MFI] indicated in bar graph). Data are representative of three independent experiments (*, P
    Figure Legend Snippet: IFN-γ suppresses CNS2 methylation and Foxp3 loss in SOCS1-deficient T reg cells. (A) Expression profiles of CD62L and CD44 on CD3 + CD4 + CD25 bright cells of the LN cells from Ifnγ −/− Socs1 +/+ and Ifnγ −/− Socs1 −/− mice. (B–D) 2 × 10 5 T reg cells from Ifnγ −/− Socs1 +/+ mice or Ifnγ −/− Socs1 −/− mice were transferred into Rag2 −/− mice. 6 wk after transfer, recipient mice were analyzed. (B) Body weight changes. (C) Flow cytometric analysis of Foxp3 expression on CD3 + CD4 + T cells from the LN in Rag2 −/− mice (percentage of Foxp3 + indicated in bar graph). (D) CD25 and intracellular CTLA4 expression on CD3 + CD4 + Foxp3 + cells and Foxp3 − cells from the LN in Rag2 −/− mice (right) compared with those on CD3 + CD4 + CD25 bright cells before transfer (left; mean fluorescent intensity [MFI] indicated in bar graph). Data are representative of three independent experiments (*, P

    Techniques Used: Methylation, Expressing, Mouse Assay, Flow Cytometry

    Ifnγ −/− Socs1 −/− T reg cells fail to suppress colitis in vivo. (A and B) 2 × 10 5 Ly5.2 Ifnγ −/− Socs1 +/+ or Ly5.2 Ifnγ −/− Socs1 −/− T reg cells were cotransferred with 4 × 10 5 Ly5.1 naive T cells into Rag2 −/− mice. 4 wk after transfer, recipient Rag2 −/− mice were analyzed. (A) Body weight changes. (B) Flow cytometric analysis of Foxp3 expression on Ly5.2 + CD3 + CD4 + T cells from the LN in Rag2 −/− mice (percentage of Foxp3 + indicated in bar graph). (C) Flow cytometric analysis of IFN-γ and IL-17A on Ly5.1 + CD3 + CD4 + cells (top) and Ly5.2 + CD3 + CD4 + T cells (middle) from the LN in recipient mice (percent positivity, bottom). (A–C) Data are representative of three independent experiments (*, P
    Figure Legend Snippet: Ifnγ −/− Socs1 −/− T reg cells fail to suppress colitis in vivo. (A and B) 2 × 10 5 Ly5.2 Ifnγ −/− Socs1 +/+ or Ly5.2 Ifnγ −/− Socs1 −/− T reg cells were cotransferred with 4 × 10 5 Ly5.1 naive T cells into Rag2 −/− mice. 4 wk after transfer, recipient Rag2 −/− mice were analyzed. (A) Body weight changes. (B) Flow cytometric analysis of Foxp3 expression on Ly5.2 + CD3 + CD4 + T cells from the LN in Rag2 −/− mice (percentage of Foxp3 + indicated in bar graph). (C) Flow cytometric analysis of IFN-γ and IL-17A on Ly5.1 + CD3 + CD4 + cells (top) and Ly5.2 + CD3 + CD4 + T cells (middle) from the LN in recipient mice (percent positivity, bottom). (A–C) Data are representative of three independent experiments (*, P

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

    7) Product Images from "A Spontaneous Mutation of the Rat Themis Gene Leads to Impaired Function of Regulatory T Cells Linked to Inflammatory Bowel Disease"

    Article Title: A Spontaneous Mutation of the Rat Themis Gene Leads to Impaired Function of Regulatory T Cells Linked to Inflammatory Bowel Disease

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002461

    Impaired suppressive function of BN m Treg is involved in the development of intestinal lesions. (A) Absolute numbers of Foxp3+ CD4+ T cells in thymus, spleen and mLN from BN (n = 7) and BN m (n = 13) rats. Data are representative of three independent experiments. (B) Suppressive activity of thymic CD25 bright CD4+ SP cells (top panel) and peripheral CD25 bright CD4+ T cells (bottom panel) from BN or BN m rats was assessed in co-culture experiments with CFSE-labeled naive LEW CD4 T cells as effector cells. Proliferation was assessed by CFSE dilution (percentages indicate the proportion of CFSE low cells). Data are representative of three independent experiments. (C, D) Disease frequency (C) and duodenum microscopic scores (D) in 12 week-old BN m rats injected with PBS (white column or symbols; n = 21) or with 4.10 6 BN CD25 bright CD4+ T cells (grey columns or symbols; n = 12) at 4 weeks of age. (E) Cytokine protein expression in duodenum from control BN m rats with microscopic intestinal scores (white columns; n = 8) and from BN m rats transferred with CD25 bright CD4+ T cells (grey columns; n = 12).
    Figure Legend Snippet: Impaired suppressive function of BN m Treg is involved in the development of intestinal lesions. (A) Absolute numbers of Foxp3+ CD4+ T cells in thymus, spleen and mLN from BN (n = 7) and BN m (n = 13) rats. Data are representative of three independent experiments. (B) Suppressive activity of thymic CD25 bright CD4+ SP cells (top panel) and peripheral CD25 bright CD4+ T cells (bottom panel) from BN or BN m rats was assessed in co-culture experiments with CFSE-labeled naive LEW CD4 T cells as effector cells. Proliferation was assessed by CFSE dilution (percentages indicate the proportion of CFSE low cells). Data are representative of three independent experiments. (C, D) Disease frequency (C) and duodenum microscopic scores (D) in 12 week-old BN m rats injected with PBS (white column or symbols; n = 21) or with 4.10 6 BN CD25 bright CD4+ T cells (grey columns or symbols; n = 12) at 4 weeks of age. (E) Cytokine protein expression in duodenum from control BN m rats with microscopic intestinal scores (white columns; n = 8) and from BN m rats transferred with CD25 bright CD4+ T cells (grey columns; n = 12).

    Techniques Used: Activity Assay, Co-Culture Assay, Labeling, Injection, Expressing

    BN m rats carry a disrupted Themis gene. (A) Genome scan for loci controlling the percentage of CD4 T cells in the blood of 44 (BN m ×DA)×BN m rats. Horizontal lines represent genome-wide significance thresholds of 5% (significant) and 0.1% (highly significant) as determined by permutation tests. (B) Percentages of CD4 T cells in 44 (BN m ×DA)×BN m backcross rats classified according to their genotypes at the microsatellite marker of chromosome 1 nearest to the QTL. In each group, horizontal bars represent the mean values. nn: homozygous BN (n = 23); nd: heterozygous BN-DA (n = 21). (C) Fine mapping of the BN m mutation in 28 (BN m ×DA) F2 or (BN m ×DA)×BN m backcross rats, among which 16 showed normal proportions of CD4 T lymphocytes (Unaffected) and 12 showed CD4 T cell lymphopenia (Affected). The position of each microsatellite marker on chromosome 1 is indicated in megabases (Mb). White: homozygous BN m ; black: heterozygous BN m /DA. Rec: number of rats characterized by a given recombination. The physical map of the critical 1.5 Mb interval containing 6 genes is shown underneath. (D) Relative mRNA expression of the 6 genes in BN m (n = 4) and BN (n = 4) thymocytes. Data are representative of two independent experiments. (E) Electrophoregram, nucleic acid sequences and corresponding amino acids of the BN and BN m Themis gene in the region surrounding the 4 nucleotide insertion corresponding to the BN m mutation (boxed). (F) Immunoblot analysis of Themis and β-actin in thymocytes from BN, BN m , LEW and DA rats, C57BL/6 mice and in human PBMC using anti-Themis antibodies specific either for the mouse C-terminal (left panel) or the human N-terminal (right panel) portion of the protein.
    Figure Legend Snippet: BN m rats carry a disrupted Themis gene. (A) Genome scan for loci controlling the percentage of CD4 T cells in the blood of 44 (BN m ×DA)×BN m rats. Horizontal lines represent genome-wide significance thresholds of 5% (significant) and 0.1% (highly significant) as determined by permutation tests. (B) Percentages of CD4 T cells in 44 (BN m ×DA)×BN m backcross rats classified according to their genotypes at the microsatellite marker of chromosome 1 nearest to the QTL. In each group, horizontal bars represent the mean values. nn: homozygous BN (n = 23); nd: heterozygous BN-DA (n = 21). (C) Fine mapping of the BN m mutation in 28 (BN m ×DA) F2 or (BN m ×DA)×BN m backcross rats, among which 16 showed normal proportions of CD4 T lymphocytes (Unaffected) and 12 showed CD4 T cell lymphopenia (Affected). The position of each microsatellite marker on chromosome 1 is indicated in megabases (Mb). White: homozygous BN m ; black: heterozygous BN m /DA. Rec: number of rats characterized by a given recombination. The physical map of the critical 1.5 Mb interval containing 6 genes is shown underneath. (D) Relative mRNA expression of the 6 genes in BN m (n = 4) and BN (n = 4) thymocytes. Data are representative of two independent experiments. (E) Electrophoregram, nucleic acid sequences and corresponding amino acids of the BN and BN m Themis gene in the region surrounding the 4 nucleotide insertion corresponding to the BN m mutation (boxed). (F) Immunoblot analysis of Themis and β-actin in thymocytes from BN, BN m , LEW and DA rats, C57BL/6 mice and in human PBMC using anti-Themis antibodies specific either for the mouse C-terminal (left panel) or the human N-terminal (right panel) portion of the protein.

    Techniques Used: Genome Wide, Marker, Mutagenesis, Expressing, Mouse Assay

    BN m rats exhibit a T cell autonomous lymphopenia restricted to CD4 T cells. (A) Percentage of TCRαβ positive cells in PBMC from BN (n = 15, black bars), BN m (n = 9, white bars) and (BN×BN m ) F1 (n = 5, grey bars) rats. (B) CD4 and CD8 expression in BN and BN m spleen T cells; numbers indicate the cell percentages in the outlined area. (C) Absolute numbers of BN (n = 4) and BNm (n = 9) T cells, CD4+ and CD8+ T cells, and B cells in spleen. Data in B and C are representative of five independent experiments. (D) CD4 and CD8 expression in BN and BN m thymocytes; numbers indicate cell percentages in the outlined area. (E) Absolute numbers of BN (n = 5) and BN m (n = 5) in thymocyte subsets (DN: double negative; DP: double positive; SP: simple positive). Data in D and E are representative of six independent experiments. (F) Absolute numbers of lymphocytes in spleen of lethally irradiated (LEW×BN) F1 rats reconstituted with BN (n = 6) or BN m (n = 8) T cell-depleted bone marrow cells. Data are representative of three independent experiments. (G) Absolute numbers of donor CD4 SP and CD8 SP cells in thymus of sub-lethally irradiated (LEW×BN) F1 rats 15 days after intrathymic injection of DN thymocytes from BN (n = 4) and BN m (n = 4) rats. (BN: black columns, BN m : white columns).
    Figure Legend Snippet: BN m rats exhibit a T cell autonomous lymphopenia restricted to CD4 T cells. (A) Percentage of TCRαβ positive cells in PBMC from BN (n = 15, black bars), BN m (n = 9, white bars) and (BN×BN m ) F1 (n = 5, grey bars) rats. (B) CD4 and CD8 expression in BN and BN m spleen T cells; numbers indicate the cell percentages in the outlined area. (C) Absolute numbers of BN (n = 4) and BNm (n = 9) T cells, CD4+ and CD8+ T cells, and B cells in spleen. Data in B and C are representative of five independent experiments. (D) CD4 and CD8 expression in BN and BN m thymocytes; numbers indicate cell percentages in the outlined area. (E) Absolute numbers of BN (n = 5) and BN m (n = 5) in thymocyte subsets (DN: double negative; DP: double positive; SP: simple positive). Data in D and E are representative of six independent experiments. (F) Absolute numbers of lymphocytes in spleen of lethally irradiated (LEW×BN) F1 rats reconstituted with BN (n = 6) or BN m (n = 8) T cell-depleted bone marrow cells. Data are representative of three independent experiments. (G) Absolute numbers of donor CD4 SP and CD8 SP cells in thymus of sub-lethally irradiated (LEW×BN) F1 rats 15 days after intrathymic injection of DN thymocytes from BN (n = 4) and BN m (n = 4) rats. (BN: black columns, BN m : white columns).

    Techniques Used: Expressing, Irradiation, Injection

    BN m rats exhibit spontaneous T cell activation and skewed cytokine production. (A) Proportions of OX40 high , CD62L low , CD45RC low and CD25 high among CD4 T cells from spleens of BN (n = 4) and BN m (n = 8) rats. (B) Proliferation was assessed by [3H] Thymidine uptake in total CD4 T cells isolated from BN (n = 4) and BN m (n = 4) spleens after stimulation for 48 hours with anti-TCR mAb and increasing concentrations of anti-CD28 mAb (left panel). Cytokine production by CD4 T cells stimulated with anti-TCR mAb and 0.2 µg/ml of anti-CD28 mAb for 48 hours (right panels). (C) Proliferation (left panel) and cytokine production (right panels) by naive CD62L+ CD4 T cells isolated from BN (n = 4) and BN m (n = 4) spleens and stimulated as described above. Data are representative of three independent experiments. (BN: black columns, BN m : white columns).
    Figure Legend Snippet: BN m rats exhibit spontaneous T cell activation and skewed cytokine production. (A) Proportions of OX40 high , CD62L low , CD45RC low and CD25 high among CD4 T cells from spleens of BN (n = 4) and BN m (n = 8) rats. (B) Proliferation was assessed by [3H] Thymidine uptake in total CD4 T cells isolated from BN (n = 4) and BN m (n = 4) spleens after stimulation for 48 hours with anti-TCR mAb and increasing concentrations of anti-CD28 mAb (left panel). Cytokine production by CD4 T cells stimulated with anti-TCR mAb and 0.2 µg/ml of anti-CD28 mAb for 48 hours (right panels). (C) Proliferation (left panel) and cytokine production (right panels) by naive CD62L+ CD4 T cells isolated from BN (n = 4) and BN m (n = 4) spleens and stimulated as described above. Data are representative of three independent experiments. (BN: black columns, BN m : white columns).

    Techniques Used: Activation Assay, Isolation

    8) Product Images from "INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells"

    Article Title: INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells

    Journal: Diabetologia

    doi: 10.1007/s00125-011-2379-y

    INS-GFP + cells can be viably isolated and recultured. a Schematic depicting the experimental outline of sorting and reculture experiments. b , c Cells sorted on the basis of INS-GFP expression display a wide spectrum of GFP fluorescence intensities ( c ; scale bar, 50 μm). After reaggregation, INS-GFP + cells form tight E-cadherin + (E-CAD) insulin producing aggregates (IPAs) that continue to express glucagon (GCG). Scale bar, 10 μm. d Bright field (BF)–GFP overlay images of IPAs cultured in APEL medium supplemented with 100 ng/ml of the indicated growth factors for 7 days. Note the significant growth of the GFP − population and diminishing proportion of INS-GFP + cells present within these aggregates. Scale bar, 50 μm. e Flow cytometric analysis of differentiating cultures of INS GFP/w hESCs labelled with BrdU for 24 h at the indicated time points. Note that GFP expression is not observed before day 15. At day 20, the majority of INS-GFP + cells had not incorporated BrdU. The percentage of cells within each quadrant is indicated. FSC, forward scatter
    Figure Legend Snippet: INS-GFP + cells can be viably isolated and recultured. a Schematic depicting the experimental outline of sorting and reculture experiments. b , c Cells sorted on the basis of INS-GFP expression display a wide spectrum of GFP fluorescence intensities ( c ; scale bar, 50 μm). After reaggregation, INS-GFP + cells form tight E-cadherin + (E-CAD) insulin producing aggregates (IPAs) that continue to express glucagon (GCG). Scale bar, 10 μm. d Bright field (BF)–GFP overlay images of IPAs cultured in APEL medium supplemented with 100 ng/ml of the indicated growth factors for 7 days. Note the significant growth of the GFP − population and diminishing proportion of INS-GFP + cells present within these aggregates. Scale bar, 50 μm. e Flow cytometric analysis of differentiating cultures of INS GFP/w hESCs labelled with BrdU for 24 h at the indicated time points. Note that GFP expression is not observed before day 15. At day 20, the majority of INS-GFP + cells had not incorporated BrdU. The percentage of cells within each quadrant is indicated. FSC, forward scatter

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

    9) Product Images from "Identification of a Novel Gene Product That Promotes Survival of Mycobacterium smegmatis in Macrophages"

    Article Title: Identification of a Novel Gene Product That Promotes Survival of Mycobacterium smegmatis in Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031788

    NF-κB activation in macrophages infected with WT M. smegmatis or Myco132. RAW264.7 mouse macrophage cells stably expressing the NF-κB-dependent ELAM-luciferase reporter construct were exposed to 10 5 cfu formaldehyde-killed wild-type M. smegmatis or Myco132 for 3.5 hours. Mean relative stimulation of luciferase activity for three biological replicates per strain ± SD for a representative experiment from 3 experiments, each performed in triplicate, is shown. Significant differences were determined by Student's t-test and are indicated by * ( p
    Figure Legend Snippet: NF-κB activation in macrophages infected with WT M. smegmatis or Myco132. RAW264.7 mouse macrophage cells stably expressing the NF-κB-dependent ELAM-luciferase reporter construct were exposed to 10 5 cfu formaldehyde-killed wild-type M. smegmatis or Myco132 for 3.5 hours. Mean relative stimulation of luciferase activity for three biological replicates per strain ± SD for a representative experiment from 3 experiments, each performed in triplicate, is shown. Significant differences were determined by Student's t-test and are indicated by * ( p

    Techniques Used: Activation Assay, Infection, Stable Transfection, Expressing, Luciferase, Construct, Activity Assay

    10) Product Images from "Induction of Apoptosis and T Helper 2 (Th2) Responses Correlates with Peptide Affinity for the Major Histocompatibility Complex in Self-reactive T Cell Receptor Transgenic Mice"

    Article Title: Induction of Apoptosis and T Helper 2 (Th2) Responses Correlates with Peptide Affinity for the Major Histocompatibility Complex in Self-reactive T Cell Receptor Transgenic Mice

    Journal: The Journal of Experimental Medicine

    doi:

    CD4 + T cells from MBP-specific TCR transgenic mice express less membrane TNF-α and LT-αβ than do those from HA-specific TCR transgenic mice. Expression of CD69, Fas, FasL, mTNF, and LT-αβ on splenocytes from either the MBP-specific TCR transgenic line cultured with 50 μM Ac1-11 ( Ac1-11 ) or the hemagglutinin-specific TCR transgenic line ( HA ) cultured in vitro with 0.2 μg/ml HA peptide 126-138 for 48 h. Live cells from the 48-h cultures were collected on a Ficoll gradient and washed two times. Cells were then stained with a biotinylated antibody to CD69, a phycoerythrin-conjugated antibody to Fas, or Fas/Fc, TNFR/Fc, or LT-βR/Fc fusion proteins as described in Materials and Methods. Histograms display only large, blasting cells as determined by forward scatter. For CD69 and Fas, dashed lines in plots indicating levels of CD69 or Fas, respectively, of naive cells from PBS-injected MBP-specific TCR transgenic mice. For FasL, mTNF, and LT-αβ expression, dashed lines indicate fluorescence intensity of cells incubated with only the biotinylated secondary antibody and streptavidin-conjugated Texas red as a negative control.
    Figure Legend Snippet: CD4 + T cells from MBP-specific TCR transgenic mice express less membrane TNF-α and LT-αβ than do those from HA-specific TCR transgenic mice. Expression of CD69, Fas, FasL, mTNF, and LT-αβ on splenocytes from either the MBP-specific TCR transgenic line cultured with 50 μM Ac1-11 ( Ac1-11 ) or the hemagglutinin-specific TCR transgenic line ( HA ) cultured in vitro with 0.2 μg/ml HA peptide 126-138 for 48 h. Live cells from the 48-h cultures were collected on a Ficoll gradient and washed two times. Cells were then stained with a biotinylated antibody to CD69, a phycoerythrin-conjugated antibody to Fas, or Fas/Fc, TNFR/Fc, or LT-βR/Fc fusion proteins as described in Materials and Methods. Histograms display only large, blasting cells as determined by forward scatter. For CD69 and Fas, dashed lines in plots indicating levels of CD69 or Fas, respectively, of naive cells from PBS-injected MBP-specific TCR transgenic mice. For FasL, mTNF, and LT-αβ expression, dashed lines indicate fluorescence intensity of cells incubated with only the biotinylated secondary antibody and streptavidin-conjugated Texas red as a negative control.

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Cell Culture, In Vitro, Staining, Injection, Fluorescence, Incubation, Negative Control

    The effect of the addition of anti–class II antibody (10-3.6) on in vitro differentiation of naive TCR transgenic mice T cells by 50, 5, and 0.5 μM Ac1-11[4Y]. Black bars represent responses of T cells cultured with 12.5 μg/ml 10-3.6 and Ac1-11[4Y], shaded bars those of T cells cultured with 3.125 μg/ml 10-3.6 and Ac111[4Y], and cross-hatched bars those of T cells cultured with Ac1-11[4Y] alone. Sorted naive T cells were cultured in 24-well plates with irradiated splenocytes as APCs with various amounts of 10-3.6 and Ac1-11[4Y]. IL-2 production in the primary stimulation was determined on culture supernatant 48 h after initial stimulation with Ac1-11[4Y] and 10-3.6. After a total of 7 d, viable cells were then washed and cultured with 10 μM Ac1-11[4Y] or medium only in 96-well round-bottom plates at 5 × 10 4 cells/well and 5 × 10 5 APCs/well for 48 h. IFN-γ and IL-4 production in the secondary stimulation was determined as described. ( A ) IL-2 production by naive T cells in the primary stimulation is decreased by addition of 10-3.6. ( B ) IFN-γ production by T cells in the secondary stimulation does not change significantly by addition of 10-3.6. ( C ) IL-4 production by T cells in the secondary stimulation increases 2–20-fold, depending on the initial concentration of Ac1-11[4Y].
    Figure Legend Snippet: The effect of the addition of anti–class II antibody (10-3.6) on in vitro differentiation of naive TCR transgenic mice T cells by 50, 5, and 0.5 μM Ac1-11[4Y]. Black bars represent responses of T cells cultured with 12.5 μg/ml 10-3.6 and Ac1-11[4Y], shaded bars those of T cells cultured with 3.125 μg/ml 10-3.6 and Ac111[4Y], and cross-hatched bars those of T cells cultured with Ac1-11[4Y] alone. Sorted naive T cells were cultured in 24-well plates with irradiated splenocytes as APCs with various amounts of 10-3.6 and Ac1-11[4Y]. IL-2 production in the primary stimulation was determined on culture supernatant 48 h after initial stimulation with Ac1-11[4Y] and 10-3.6. After a total of 7 d, viable cells were then washed and cultured with 10 μM Ac1-11[4Y] or medium only in 96-well round-bottom plates at 5 × 10 4 cells/well and 5 × 10 5 APCs/well for 48 h. IFN-γ and IL-4 production in the secondary stimulation was determined as described. ( A ) IL-2 production by naive T cells in the primary stimulation is decreased by addition of 10-3.6. ( B ) IFN-γ production by T cells in the secondary stimulation does not change significantly by addition of 10-3.6. ( C ) IL-4 production by T cells in the secondary stimulation increases 2–20-fold, depending on the initial concentration of Ac1-11[4Y].

    Techniques Used: In Vitro, Transgenic Assay, Mouse Assay, Cell Culture, Irradiation, Concentration Assay

    The induction of Th1 and Th2 type cells in vitro. Lymph node cells from an MBP-specific TCR transgenic mouse were stained with CD8, B220, Mac-1, CD69, and CD44, and the negative cells were collected by flow cytometry as naive, CD4 + T cells (reanalysis of cells by flow cytometry indicated a population > 98% CD4 + ). These cells were cultured in vitro at a concentration of 10 5 CD4 + cells and 10 6 irradiated nontransgenic syngeneic splenocytes per well in 24-well plates with varying concentrations of Ac1-11, Ac1-11[4A], or Ac1-11[4Y]. 10 d later, cells were washed several times and restimulated at a concentration of 5 × 10 4 T cells plus 3 × 10 5 irradiated splenocytes per well in 96-well round-bottom plates with 10 μM Ac1-11, Ac111[4A], or Ac1-11[4Y]. Supernatants were collected at 48 h, and cytokines were detected by ELISA. Black bars, cytokine response to Ac1-11 in secondary challenge; shaded bars, cytokine response to Ac1-11[4A] in secondary challenge; cross-hatched bars, cytokine response to Ac1-11[4Y] in secondary challenge. In general, Ac1-11 induced Th1 type responses regardless of primary concentration, Ac1-11[4A] induced Th2 type responses at high concentrations and Th1 type responses at low concentrations, and Ac1-11[4Y] induced primarily Th2 type responses, except at the lowest two doses. nd, not determined. Previous experiments indicated that too few live cells were recovered at the doses for which cytokine levels were not determined to test a secondary response.
    Figure Legend Snippet: The induction of Th1 and Th2 type cells in vitro. Lymph node cells from an MBP-specific TCR transgenic mouse were stained with CD8, B220, Mac-1, CD69, and CD44, and the negative cells were collected by flow cytometry as naive, CD4 + T cells (reanalysis of cells by flow cytometry indicated a population > 98% CD4 + ). These cells were cultured in vitro at a concentration of 10 5 CD4 + cells and 10 6 irradiated nontransgenic syngeneic splenocytes per well in 24-well plates with varying concentrations of Ac1-11, Ac1-11[4A], or Ac1-11[4Y]. 10 d later, cells were washed several times and restimulated at a concentration of 5 × 10 4 T cells plus 3 × 10 5 irradiated splenocytes per well in 96-well round-bottom plates with 10 μM Ac1-11, Ac111[4A], or Ac1-11[4Y]. Supernatants were collected at 48 h, and cytokines were detected by ELISA. Black bars, cytokine response to Ac1-11 in secondary challenge; shaded bars, cytokine response to Ac1-11[4A] in secondary challenge; cross-hatched bars, cytokine response to Ac1-11[4Y] in secondary challenge. In general, Ac1-11 induced Th1 type responses regardless of primary concentration, Ac1-11[4A] induced Th2 type responses at high concentrations and Th1 type responses at low concentrations, and Ac1-11[4Y] induced primarily Th2 type responses, except at the lowest two doses. nd, not determined. Previous experiments indicated that too few live cells were recovered at the doses for which cytokine levels were not determined to test a secondary response.

    Techniques Used: In Vitro, Transgenic Assay, Staining, Flow Cytometry, Cytometry, Cell Culture, Concentration Assay, Irradiation, Enzyme-linked Immunosorbent Assay

    The effect of anti–B7-2 on the in vivo expression of B7-1 and B7-2 and the development of Th2 responses in vivo after injection of Ac111[4Y]. TCR transgenic mice were injected intraperitoneally with either PBS or 100 μg of anti–B7-2 antibody GL-1 on days −1 and 0. On day 0, mice were injected intravenously with either PBS or 2.4 mg of Ac1-11[4Y], and mice were killed on day 1. ( A ) The expression of B7-1 and B7-2 on B cells and macrophages was determined as described in Materials and Methods. Solid lines, expression of B7-1 or B7-2 on cells from mice injected intravenously with PBS; dashed lines, expression on cells from mice injected with Ac1-11[4Y]. B7-1 was upregulated on macrophages from Ac1-11[4Y]– injected mice treated intraperitoneally with either PBS or anti–B7-2, while B7-2 was upregulated on B cells from Ac1-11[4Y]–injected mice given PBS intraperitoneally, but not on cells from Ac1-11[4Y]–injected mice given anti–B7-2 intraperitoneally. ( B ). The cytokine profile of splenocytes in response to 10 μM Ac1-11, Ac1-11[4A], or Ac1-11[4Y] was determined by ELISA as described. Black bars, cytokine response to Ac1-11 in secondary challenge; shaded bars, cytokine response to Ac1-11[4A] in secondary challenge; cross-hatched bars, cytokine response to Ac1-11[4Y] in secondary challenge. IL-2 production was similar between mice given PBS or Ac1-11[4Y] intravenously, regardless of antibody treatment. IFN-γ production was increased approximately twofold in mice given both Ac1-11[4Y] and anti–B7-2 over that of cells from mice given only Ac1-11[4Y], while IL-4 and IL-10 production by cells from mice given both Ac1-11[4Y] and anti–B7-2 was reduced by about two- to fourfold relative to production by cells from mice given Ac111[4Y] only.
    Figure Legend Snippet: The effect of anti–B7-2 on the in vivo expression of B7-1 and B7-2 and the development of Th2 responses in vivo after injection of Ac111[4Y]. TCR transgenic mice were injected intraperitoneally with either PBS or 100 μg of anti–B7-2 antibody GL-1 on days −1 and 0. On day 0, mice were injected intravenously with either PBS or 2.4 mg of Ac1-11[4Y], and mice were killed on day 1. ( A ) The expression of B7-1 and B7-2 on B cells and macrophages was determined as described in Materials and Methods. Solid lines, expression of B7-1 or B7-2 on cells from mice injected intravenously with PBS; dashed lines, expression on cells from mice injected with Ac1-11[4Y]. B7-1 was upregulated on macrophages from Ac1-11[4Y]– injected mice treated intraperitoneally with either PBS or anti–B7-2, while B7-2 was upregulated on B cells from Ac1-11[4Y]–injected mice given PBS intraperitoneally, but not on cells from Ac1-11[4Y]–injected mice given anti–B7-2 intraperitoneally. ( B ). The cytokine profile of splenocytes in response to 10 μM Ac1-11, Ac1-11[4A], or Ac1-11[4Y] was determined by ELISA as described. Black bars, cytokine response to Ac1-11 in secondary challenge; shaded bars, cytokine response to Ac1-11[4A] in secondary challenge; cross-hatched bars, cytokine response to Ac1-11[4Y] in secondary challenge. IL-2 production was similar between mice given PBS or Ac1-11[4Y] intravenously, regardless of antibody treatment. IFN-γ production was increased approximately twofold in mice given both Ac1-11[4Y] and anti–B7-2 over that of cells from mice given only Ac1-11[4Y], while IL-4 and IL-10 production by cells from mice given both Ac1-11[4Y] and anti–B7-2 was reduced by about two- to fourfold relative to production by cells from mice given Ac111[4Y] only.

    Techniques Used: In Vivo, Expressing, Injection, Transgenic Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    No net change in the numbers of CD4 + T cells in the periphery is observed after peptide injection. The numbers of CD4 + and CD8 + cells from lymph nodes and spleen from TCR transgenic mice injected with PBS, Ac1-11, Ac1-11[4A], or Ac1-11[4Y] are shown 1, 2, and 6 d after a single administration of 2.4 mg of peptide. Black bars represent the numbers of CD4 + T cells in the spleen, dark cross-hatched bars represent the numbers of CD4 + T cells in lymph nodes, shaded bars represent the numbers of CD8 + T cells in the spleen, and light cross-hatched bars represent the numbers of CD8 + T cells in the lymph nodes. Results are pooled from three experiments for days 1 and 6, and two experiments for day 2. In one experiment from day 3, CD4 + T cells had increased in number by 1.5–2-fold in peptide-injected mice (data not shown).
    Figure Legend Snippet: No net change in the numbers of CD4 + T cells in the periphery is observed after peptide injection. The numbers of CD4 + and CD8 + cells from lymph nodes and spleen from TCR transgenic mice injected with PBS, Ac1-11, Ac1-11[4A], or Ac1-11[4Y] are shown 1, 2, and 6 d after a single administration of 2.4 mg of peptide. Black bars represent the numbers of CD4 + T cells in the spleen, dark cross-hatched bars represent the numbers of CD4 + T cells in lymph nodes, shaded bars represent the numbers of CD8 + T cells in the spleen, and light cross-hatched bars represent the numbers of CD8 + T cells in the lymph nodes. Results are pooled from three experiments for days 1 and 6, and two experiments for day 2. In one experiment from day 3, CD4 + T cells had increased in number by 1.5–2-fold in peptide-injected mice (data not shown).

    Techniques Used: Injection, Transgenic Assay, Mouse Assay

    Soluble peptide induces apoptosis in lymph nodes. Sections from brachial or inguinal lymph nodes from Ac1-11–specific TCR transgenic mice removed 24 h after intravenous injection with ( A ) PBS, ( B ) 2.4 mg of Ac1-11, ( C ) 2.4 mg of Ac1-11[4A], and ( D ) 2.4 mg of Ac1-11[4Y]. Apoptotic cells were visualized using the TUNEL reaction as described in Materials and Methods section and appear as dark spots.
    Figure Legend Snippet: Soluble peptide induces apoptosis in lymph nodes. Sections from brachial or inguinal lymph nodes from Ac1-11–specific TCR transgenic mice removed 24 h after intravenous injection with ( A ) PBS, ( B ) 2.4 mg of Ac1-11, ( C ) 2.4 mg of Ac1-11[4A], and ( D ) 2.4 mg of Ac1-11[4Y]. Apoptotic cells were visualized using the TUNEL reaction as described in Materials and Methods section and appear as dark spots.

    Techniques Used: Transgenic Assay, Mouse Assay, Injection, TUNEL Assay

    Thymic architecture of MBP-specific TCR transgenic mice and a normal nontransgenic littermate, as illustrated by ERTR5, an antibody that is specific for medullary epithelium. ( A ) Thymus from a MBPspecific TCR transgenic. ( B ) Thymus from a nontransgenic littermate. Thymi were from the MBP-specific TCR transgenic line that has a CD4/ CD8 ratio of ∼5:1 and a less pronounced thymic defect than the line with the higher CD4/CD8 ratio. Mice were (PL/J × B10.PL)F1. c, cortex; m, medulla.
    Figure Legend Snippet: Thymic architecture of MBP-specific TCR transgenic mice and a normal nontransgenic littermate, as illustrated by ERTR5, an antibody that is specific for medullary epithelium. ( A ) Thymus from a MBPspecific TCR transgenic. ( B ) Thymus from a nontransgenic littermate. Thymi were from the MBP-specific TCR transgenic line that has a CD4/ CD8 ratio of ∼5:1 and a less pronounced thymic defect than the line with the higher CD4/CD8 ratio. Mice were (PL/J × B10.PL)F1. c, cortex; m, medulla.

    Techniques Used: Transgenic Assay, Mouse Assay

    11) Product Images from "Killing of Trypanosomatid Parasites by a Modified Bovine Host Defense Peptide, BMAP-18"

    Article Title: Killing of Trypanosomatid Parasites by a Modified Bovine Host Defense Peptide, BMAP-18

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0000373

    Inhibition of parasite growth by BMAP-27 and BMAP-18 peptides. MIC assays were performed on trypanosomes and Leishmania using alamarBlue as described in the Materials and Methods . Panel A: Comparison of full length and truncated host defense peptides. T. b. brucei 427.01 BSF (black lines), PCF (gray lines) and Sodalis glossinidius (green lines) were tested for sensitivity to BMAP-27 (solid lines) and BMAP-18 (dashed lines). Percent inhibition is shown as a function of peptide concentration. Panel B: Growth inhibition of a variety of kinetoplastid parasites by BMAP-18. T. b. brucei 427.01 PCF (black line), T. b. brucei TSW196 PCF (magenta line), T. b. rhodesiense ViTat 1.1 PCF (solid green line), T. b. gambiense U2 PCF (red line), T. congolense IL3000 PCF (orange line), T. danilewskyi TrCa (dashed green line), L. donovani LD3 (dashed blue line). The results shown are representative of more than 10 individual experiments (Panel A) or 4 individual experiments (Panel B) performed over a one year period.
    Figure Legend Snippet: Inhibition of parasite growth by BMAP-27 and BMAP-18 peptides. MIC assays were performed on trypanosomes and Leishmania using alamarBlue as described in the Materials and Methods . Panel A: Comparison of full length and truncated host defense peptides. T. b. brucei 427.01 BSF (black lines), PCF (gray lines) and Sodalis glossinidius (green lines) were tested for sensitivity to BMAP-27 (solid lines) and BMAP-18 (dashed lines). Percent inhibition is shown as a function of peptide concentration. Panel B: Growth inhibition of a variety of kinetoplastid parasites by BMAP-18. T. b. brucei 427.01 PCF (black line), T. b. brucei TSW196 PCF (magenta line), T. b. rhodesiense ViTat 1.1 PCF (solid green line), T. b. gambiense U2 PCF (red line), T. congolense IL3000 PCF (orange line), T. danilewskyi TrCa (dashed green line), L. donovani LD3 (dashed blue line). The results shown are representative of more than 10 individual experiments (Panel A) or 4 individual experiments (Panel B) performed over a one year period.

    Techniques Used: Inhibition, Concentration Assay

    12) Product Images from "Retroviral transduction of interferon-? cDNA into a nonimmunogenic murinefibrosarcoma: generation of T cells in draining lymph nodes capable of treating established parental metastatic tumor"

    Article Title: Retroviral transduction of interferon-? cDNA into a nonimmunogenic murinefibrosarcoma: generation of T cells in draining lymph nodes capable of treating established parental metastatic tumor

    Journal: Cancer immunology, immunotherapy : CII

    doi:

    In vivo growth of interferon-γ (IFNγ)-transduced tumor clones of MCA-102.24JK.Mice were injected subcutaneously with 1 × 10 6 syngeneic tumor cells. Tumor size was measured every 5 days. Each group represents at least six mice. Mean ± SE values are shown. (●) NV, non-transduced tumor; (□) Neo , tumor expressing the Neo R gene only; △ IFNγ , IFNγ-gene-modified clone
    Figure Legend Snippet: In vivo growth of interferon-γ (IFNγ)-transduced tumor clones of MCA-102.24JK.Mice were injected subcutaneously with 1 × 10 6 syngeneic tumor cells. Tumor size was measured every 5 days. Each group represents at least six mice. Mean ± SE values are shown. (●) NV, non-transduced tumor; (□) Neo , tumor expressing the Neo R gene only; △ IFNγ , IFNγ-gene-modified clone

    Techniques Used: In Vivo, Clone Assay, Mouse Assay, Injection, Expressing, Modification

    13) Product Images from "Molecular Landscape of Anti-Drug Antibodies Reveals the Mechanism of the Immune Response Following Treatment with TNFα Antagonists"

    Article Title: Molecular Landscape of Anti-Drug Antibodies Reveals the Mechanism of the Immune Response Following Treatment with TNFα Antagonists

    Journal: bioRxiv

    doi: 10.1101/509489

    Configuration of the assay for determining the neutralization index of ADA in patient sera and competitive ELISA between ADA and rhTNFα. (A) The newly developed mAb-F(ab’) 2 based bio-immunoassay configuration (left) and the modified configuration where mAb-F(ab’) 2 binding site is blocked by saturating the assay with rhTNFα (right). (B) Competitive effect of rhTNFα on ADA binding to IFX-F(ab’) 2 . ELISA plate was coated with 5μg/ml of IFX-F(ab’) 2 . ADA standard was diluted 3-fold in blocking solution supplemented with 5nM rhTNFα. ADA diluted 3-fold in blocking solution without the presence of rhTNFα served as a control.
    Figure Legend Snippet: Configuration of the assay for determining the neutralization index of ADA in patient sera and competitive ELISA between ADA and rhTNFα. (A) The newly developed mAb-F(ab’) 2 based bio-immunoassay configuration (left) and the modified configuration where mAb-F(ab’) 2 binding site is blocked by saturating the assay with rhTNFα (right). (B) Competitive effect of rhTNFα on ADA binding to IFX-F(ab’) 2 . ELISA plate was coated with 5μg/ml of IFX-F(ab’) 2 . ADA standard was diluted 3-fold in blocking solution supplemented with 5nM rhTNFα. ADA diluted 3-fold in blocking solution without the presence of rhTNFα served as a control.

    Techniques Used: Neutralization, Competitive ELISA, Modification, Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

    14) Product Images from "Transient “rest” induces functional reinvigoration and epigenetic remodeling in exhausted CAR-T cells"

    Article Title: Transient “rest” induces functional reinvigoration and epigenetic remodeling in exhausted CAR-T cells

    Journal: bioRxiv

    doi: 10.1101/2020.01.26.920496

    A GD2-targeting CAR fused to an ecDHFR destabilizing domain exhibits rapid ON/OFF kinetics and drug-dependent, analog control of expression and function in vivo . Related to Figure 1 . A) CAR-T cells were stained with 1A7 anti-CAR idiotype antibody and analyzed via flow cytometry. Dose-dependent control (left) and ON/OFF kinetics (middle, right) were demonstrated via trimethoprim (TMP). CAR MFI was used to generate non-linear dose-response curves. Error bars represent mean ± SEM of 3 individual donors. B) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice. On days 5-7 post-engraftment, mice were dosed with vehicle (water, CAR OFF) or 200mg/kg trimethoprim (TMP, CAR ON) once daily. On day 7 post-engraftment, 1×10 7 GD2.28 ζ .ecDHFR CAR-T cells were infused. 24 hours after CAR-T cell infusion, peripheral blood and spleens were harvested for flow cytometry analyses. Contour plots demonstrate a TMP-dependent increase in co-expression of CAR and the activation marker CD69 in CAR ON mice compared CAR OFF mice. Representative mouse from n=7 total mice from 2 independent experiments. C) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice at day 0 and 2×10 6 GD2.28 ζ .ecDHFR CAR-T cells expanded in vitro in the absence of TMP for 15 days were infused IV on day 7 post-engraftment. Mice were dosed 6 days per week with vehicle (CAR OFF) or 200mg/kg TMP (CAR ON). Bioluminescence imaging of the tumor indicates TMP-dependent anti-tumor activity in CAR ON mice compared to CAR OFF mice (n=5 mice/group). Representative experiment (also shown in Figure 1F-G ) from 3 individual experiments.
    Figure Legend Snippet: A GD2-targeting CAR fused to an ecDHFR destabilizing domain exhibits rapid ON/OFF kinetics and drug-dependent, analog control of expression and function in vivo . Related to Figure 1 . A) CAR-T cells were stained with 1A7 anti-CAR idiotype antibody and analyzed via flow cytometry. Dose-dependent control (left) and ON/OFF kinetics (middle, right) were demonstrated via trimethoprim (TMP). CAR MFI was used to generate non-linear dose-response curves. Error bars represent mean ± SEM of 3 individual donors. B) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice. On days 5-7 post-engraftment, mice were dosed with vehicle (water, CAR OFF) or 200mg/kg trimethoprim (TMP, CAR ON) once daily. On day 7 post-engraftment, 1×10 7 GD2.28 ζ .ecDHFR CAR-T cells were infused. 24 hours after CAR-T cell infusion, peripheral blood and spleens were harvested for flow cytometry analyses. Contour plots demonstrate a TMP-dependent increase in co-expression of CAR and the activation marker CD69 in CAR ON mice compared CAR OFF mice. Representative mouse from n=7 total mice from 2 independent experiments. C) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice at day 0 and 2×10 6 GD2.28 ζ .ecDHFR CAR-T cells expanded in vitro in the absence of TMP for 15 days were infused IV on day 7 post-engraftment. Mice were dosed 6 days per week with vehicle (CAR OFF) or 200mg/kg TMP (CAR ON). Bioluminescence imaging of the tumor indicates TMP-dependent anti-tumor activity in CAR ON mice compared to CAR OFF mice (n=5 mice/group). Representative experiment (also shown in Figure 1F-G ) from 3 individual experiments.

    Techniques Used: Expressing, In Vivo, Staining, Flow Cytometry, Mouse Assay, Activation Assay, Marker, In Vitro, Imaging, Activity Assay

    Transient rest reinvigorates exhausted CAR-T cells and improves anti-tumor function. A) CAR expression was normalized across all HA.28z.FKBP-expressing cells ( Figure 4A ) by adding shield-1 to cultures approximately 16 hours prior to in vitro tumor challenge. B) D15 incucyte assay shows that transient rest and α PD-1 enhances cytotoxicity in response to 143B-GL osteosarcoma (1:8 E:T, normalized to t=0) compared to exhausted Always ON controls. Error bars represent mean ± SD of 3 triplicate wells from one representative donor (n=3 individual donors). C) D15 co-culture assay with Nalm6-GD2 leukemia demonstrates that transient rest, but not α PD-1, augments IL-2 and IFN γ secretion. Error bars represent mean ± SEM of 4-6 individual donors. D-E) Intracellular cytokine staining and flow cytometry analyses of CD8+ CAR+ T cells demonstrate that rest reduces the frequency of non-responsive cells and increases the frequency of polyfunctional cells. SPICE analysis from 1 representative donor was conducted in (D). Error bars in (E) represent mean ± SEM of 3 individual donors. F-G) CAR-T cells were crosslinked with immobilized 1A7 anti-CAR idiotype antibody for 24 hours. (F) Non-linear dose-response curves demonstrate that T cell rest, but not PD-1 blockade, augments IL-2 and IFN γ secretion in response to both low and high idiotype densities. Error bars represent mean ± SD of 3 triplicate wells from one representative donor (n=3 individual donors). (G) IL-2 secretion in response to low density (1.25 μ g/mL) anti-CAR idiotype was normalized to secretion levels from Always OFF cells. Error bars represent mean ± SEM of 3 individual donors. H-I) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice on day 0 and 1-2×10 6 HA.28 ζ .ecDHFR CAR-T cells expanded for 15 days in vitro (as depicted in Figure 4A ) and were infused IV on day 7 post-engraftment. Mice were dosed with 200mg/kg TMP 6 days/week. (I) Bioluminescent imaging on day 30 post-engraftment demonstrates augmented control of tumor growth in Always OFF and rested conditions compared to Always ON cells. Error bars represent mean ± SEM of 3-5 mice from 1 representative experiment (n=3 independent experiments). Statistics were calculated using one or two-way ANOVA and Dunnett’s multiple comparisons test. *, p
    Figure Legend Snippet: Transient rest reinvigorates exhausted CAR-T cells and improves anti-tumor function. A) CAR expression was normalized across all HA.28z.FKBP-expressing cells ( Figure 4A ) by adding shield-1 to cultures approximately 16 hours prior to in vitro tumor challenge. B) D15 incucyte assay shows that transient rest and α PD-1 enhances cytotoxicity in response to 143B-GL osteosarcoma (1:8 E:T, normalized to t=0) compared to exhausted Always ON controls. Error bars represent mean ± SD of 3 triplicate wells from one representative donor (n=3 individual donors). C) D15 co-culture assay with Nalm6-GD2 leukemia demonstrates that transient rest, but not α PD-1, augments IL-2 and IFN γ secretion. Error bars represent mean ± SEM of 4-6 individual donors. D-E) Intracellular cytokine staining and flow cytometry analyses of CD8+ CAR+ T cells demonstrate that rest reduces the frequency of non-responsive cells and increases the frequency of polyfunctional cells. SPICE analysis from 1 representative donor was conducted in (D). Error bars in (E) represent mean ± SEM of 3 individual donors. F-G) CAR-T cells were crosslinked with immobilized 1A7 anti-CAR idiotype antibody for 24 hours. (F) Non-linear dose-response curves demonstrate that T cell rest, but not PD-1 blockade, augments IL-2 and IFN γ secretion in response to both low and high idiotype densities. Error bars represent mean ± SD of 3 triplicate wells from one representative donor (n=3 individual donors). (G) IL-2 secretion in response to low density (1.25 μ g/mL) anti-CAR idiotype was normalized to secretion levels from Always OFF cells. Error bars represent mean ± SEM of 3 individual donors. H-I) 1×10 6 Nalm6-GD2 leukemia cells were engrafted in mice on day 0 and 1-2×10 6 HA.28 ζ .ecDHFR CAR-T cells expanded for 15 days in vitro (as depicted in Figure 4A ) and were infused IV on day 7 post-engraftment. Mice were dosed with 200mg/kg TMP 6 days/week. (I) Bioluminescent imaging on day 30 post-engraftment demonstrates augmented control of tumor growth in Always OFF and rested conditions compared to Always ON cells. Error bars represent mean ± SEM of 3-5 mice from 1 representative experiment (n=3 independent experiments). Statistics were calculated using one or two-way ANOVA and Dunnett’s multiple comparisons test. *, p

    Techniques Used: Expressing, In Vitro, Co-culture Assay, Staining, Flow Cytometry, Mouse Assay, Imaging

    15) Product Images from "Mitophagy contributes to alpha-tocopheryl succinate toxicity in GSNOR-deficient hepatocellular carcinoma"

    Article Title: Mitophagy contributes to alpha-tocopheryl succinate toxicity in GSNOR-deficient hepatocellular carcinoma

    Journal: bioRxiv

    doi: 10.1101/867846

    Mitophagy impairment contributes to GSNOR-deficient hepatocellular carcinoma sensitivity to αTOS. (A) Western blot analysis of GSNOR and Parkin in HUH7 cells transiently downregulating GSNOR (siGSNOR) and/or Parkin (siParkin). Vinculin was used as loading control. (B) Cell viability fluorescent assay performed in HUH7 cells singly or doubly transfected with siGSNOR or siParkin RNAs, and treated with 40 μM αTOS. Dead cells were stained with propidium iodide (red), whereas living cells were stained with calcein-AM (green). DMSO was selected as a control. (C) Cell death evaluation as ratio between dead and total cells and expressed as mean ± SEM of n = 5 different fields of n=3 independent experiments. ** p
    Figure Legend Snippet: Mitophagy impairment contributes to GSNOR-deficient hepatocellular carcinoma sensitivity to αTOS. (A) Western blot analysis of GSNOR and Parkin in HUH7 cells transiently downregulating GSNOR (siGSNOR) and/or Parkin (siParkin). Vinculin was used as loading control. (B) Cell viability fluorescent assay performed in HUH7 cells singly or doubly transfected with siGSNOR or siParkin RNAs, and treated with 40 μM αTOS. Dead cells were stained with propidium iodide (red), whereas living cells were stained with calcein-AM (green). DMSO was selected as a control. (C) Cell death evaluation as ratio between dead and total cells and expressed as mean ± SEM of n = 5 different fields of n=3 independent experiments. ** p

    Techniques Used: Western Blot, Fluorescence, Transfection, Staining

    αTOS alters mitochondria structure and induces autophagosome formation. Transmission electron microscopy images of shScr and shGSNOR HepG2 treated for 24 h with 40 μM αTOS or vehicle (DMSO). Magnification of selected fields (yellow dotted squares) are shown on the right panels. Mitochondria are highlighted in light brown and double-membraned structures are highlighted in light blue. Scale bar 5μm
    Figure Legend Snippet: αTOS alters mitochondria structure and induces autophagosome formation. Transmission electron microscopy images of shScr and shGSNOR HepG2 treated for 24 h with 40 μM αTOS or vehicle (DMSO). Magnification of selected fields (yellow dotted squares) are shown on the right panels. Mitochondria are highlighted in light brown and double-membraned structures are highlighted in light blue. Scale bar 5μm

    Techniques Used: Transmission Assay, Electron Microscopy

    GSNOR-deficient hepatocellular carcinoma cells are insensitive to α TOS-induced mitophagy. (A) Mitophagy evaluation by fluorescence confocal microscopy of siScr, siGSNOR and siParkin HUH7 cells treated for 24 h with 40 μM αTOS and incubated with chloroquine (CQ) for 4 h to block mitochondrial degradation within autophagolysosomes. Mitochondria were labeled with an antibody against TOM20 (red), whereas autophagosomes were detected by using an anti-LC3 antibody (green). Hoechst 33342 (blue) was used to visualize nuclei. 3 central z-stacks (0.3 µm size) were merged in the microscopy pictures while > 6 stacks were used for the 3D-rendering of TOM20 and LC3 signals (right panels). (B) Number of mitochondria colocalizing with LC3-positive puncta in CQ ± αTOS-treated cells calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. * p
    Figure Legend Snippet: GSNOR-deficient hepatocellular carcinoma cells are insensitive to α TOS-induced mitophagy. (A) Mitophagy evaluation by fluorescence confocal microscopy of siScr, siGSNOR and siParkin HUH7 cells treated for 24 h with 40 μM αTOS and incubated with chloroquine (CQ) for 4 h to block mitochondrial degradation within autophagolysosomes. Mitochondria were labeled with an antibody against TOM20 (red), whereas autophagosomes were detected by using an anti-LC3 antibody (green). Hoechst 33342 (blue) was used to visualize nuclei. 3 central z-stacks (0.3 µm size) were merged in the microscopy pictures while > 6 stacks were used for the 3D-rendering of TOM20 and LC3 signals (right panels). (B) Number of mitochondria colocalizing with LC3-positive puncta in CQ ± αTOS-treated cells calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. * p

    Techniques Used: Fluorescence, Confocal Microscopy, Incubation, Blocking Assay, Labeling, Microscopy, Software

    Working model of mitophagy-dependent α TOS toxicity in GSNOR-deficient cancer cells. Nitric Oxide (NO) is a signaling molecule physiologically produced by a class of nitric oxide synthases (NOS). The denitrosylase S- nitrosoglutathione reductase (GSNOR), by reducing the nitrosylated form of glutathione (GSH), S- nitrosoglutathione (GSNO), indirectly controls the extent of S- nitrosylated proteins (PSNO) (Upper left). GSNOR downregulation, a condition occurring during aging and in some cancer types, results in the accumulation of PSNOs (Upper right). αTOS, by targeting complex II of the mitochondrial electron transport chain, induces mitochondrial damage that, if not neutralized, could lead to cell death. In GSNOR-proficient cells, the selective recognition and degradation of mitochondria (mitophagy) is fully working and ensures a correct clearance of αTOS-damaged mitochondria (Center left). Damaged mitochondria are labeled by means of polyubiquitination of mitochondrial membrane-associated proteins ( e.g. Mitofusin 2, Mfn2; Voltage-dependent anion channel, VDAC), carried out by E3 protein ubiquitin-ligases, among which Parkin represents one of the best characterized examples. Upon damage, PINK1 recruits Parkin onto mitochondria, this representing the starting point for ubiquitin-based labeling of damaged organelles. These are finally recognized by adaptor proteins ( e.g. p62 and LC3); surrounded by a double membrane; engulfed, and degraded by fusing with a lysosome. Removal of defective mitochondria by mitophagy has pro-survival effects and contributes to chemoresistance (Bottom left). On the other hand, when GSNOR is mutated or loss, Parkin is inactivated by S- nitrosylation, becoming inactive and unable to be recruited in the proximity of damaged mitochondria, a condition similar to Parkin-deficient cells. In these conditions, αTOS treatment results in the accumulation of defective mitochondria, finally leading to cell death (Bottom right).
    Figure Legend Snippet: Working model of mitophagy-dependent α TOS toxicity in GSNOR-deficient cancer cells. Nitric Oxide (NO) is a signaling molecule physiologically produced by a class of nitric oxide synthases (NOS). The denitrosylase S- nitrosoglutathione reductase (GSNOR), by reducing the nitrosylated form of glutathione (GSH), S- nitrosoglutathione (GSNO), indirectly controls the extent of S- nitrosylated proteins (PSNO) (Upper left). GSNOR downregulation, a condition occurring during aging and in some cancer types, results in the accumulation of PSNOs (Upper right). αTOS, by targeting complex II of the mitochondrial electron transport chain, induces mitochondrial damage that, if not neutralized, could lead to cell death. In GSNOR-proficient cells, the selective recognition and degradation of mitochondria (mitophagy) is fully working and ensures a correct clearance of αTOS-damaged mitochondria (Center left). Damaged mitochondria are labeled by means of polyubiquitination of mitochondrial membrane-associated proteins ( e.g. Mitofusin 2, Mfn2; Voltage-dependent anion channel, VDAC), carried out by E3 protein ubiquitin-ligases, among which Parkin represents one of the best characterized examples. Upon damage, PINK1 recruits Parkin onto mitochondria, this representing the starting point for ubiquitin-based labeling of damaged organelles. These are finally recognized by adaptor proteins ( e.g. p62 and LC3); surrounded by a double membrane; engulfed, and degraded by fusing with a lysosome. Removal of defective mitochondria by mitophagy has pro-survival effects and contributes to chemoresistance (Bottom left). On the other hand, when GSNOR is mutated or loss, Parkin is inactivated by S- nitrosylation, becoming inactive and unable to be recruited in the proximity of damaged mitochondria, a condition similar to Parkin-deficient cells. In these conditions, αTOS treatment results in the accumulation of defective mitochondria, finally leading to cell death (Bottom right).

    Techniques Used: Produced, Labeling

    16) Product Images from "The α-synuclein hereditary mutation E46K unlocks a more stable, pathogenic fibril structure"

    Article Title: The α-synuclein hereditary mutation E46K unlocks a more stable, pathogenic fibril structure

    Journal: bioRxiv

    doi: 10.1101/868869

    Biochemical analysis of E46K vs. wild-type fibrils a) Fibrils of E46K and wild-type α-syn were sonicated and transfected into HEK293T α-syn-A53T-YFP biosensor cells, and aggregation is measured by the normally soluble and diffuse intracellular α-syn-A53T-YFP forming discrete puncta, which are then quantified through fluorescent image analysis 24 . At all concentrations but 20 nM, E46K fibrils are significantly more powerful seeds than wild-type fibrils. Error bars represent the standard deviation of four technical replicates. b) In order to assay the toxicity of E46K and wild-type fibrils, we treated PC12 cells with sonicated fibrils and measured mitochondrial activity with an MTT assay 26 , 27 . E46K fibrils require a lower concentration to significantly impair mitochondrial activity compared to wild-type fibrils. Error bars represent the standard deviation of 7-14 technical replicates. **** = p-value ≤ 0.0001. ** = p-value ≤ 0.01. ns = p-value > 0.05. P-values were calculated using an unpaired, two-tailed t-test with a 95% CI.
    Figure Legend Snippet: Biochemical analysis of E46K vs. wild-type fibrils a) Fibrils of E46K and wild-type α-syn were sonicated and transfected into HEK293T α-syn-A53T-YFP biosensor cells, and aggregation is measured by the normally soluble and diffuse intracellular α-syn-A53T-YFP forming discrete puncta, which are then quantified through fluorescent image analysis 24 . At all concentrations but 20 nM, E46K fibrils are significantly more powerful seeds than wild-type fibrils. Error bars represent the standard deviation of four technical replicates. b) In order to assay the toxicity of E46K and wild-type fibrils, we treated PC12 cells with sonicated fibrils and measured mitochondrial activity with an MTT assay 26 , 27 . E46K fibrils require a lower concentration to significantly impair mitochondrial activity compared to wild-type fibrils. Error bars represent the standard deviation of 7-14 technical replicates. **** = p-value ≤ 0.0001. ** = p-value ≤ 0.01. ns = p-value > 0.05. P-values were calculated using an unpaired, two-tailed t-test with a 95% CI.

    Techniques Used: Sonication, Transfection, Standard Deviation, Activity Assay, MTT Assay, Concentration Assay, Two Tailed Test

    17) Product Images from "Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies"

    Article Title: Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00862-15

    Treatment with caspofungin leads to a compensatory increase in chitin content in A. fumigatus . The wild-type strain was grown at 37°C for 12 h in RPMI 1640 in the presence and absence of 2 μg/ml caspofungin. (A) CFW-stained fluorescent images; scale bars are 10 μm. (B) Cell wall chitin assays were performed three times on three biologically independent samples (average ± standard deviation [SD], n = 9). The asterisk indicates significant difference ( P
    Figure Legend Snippet: Treatment with caspofungin leads to a compensatory increase in chitin content in A. fumigatus . The wild-type strain was grown at 37°C for 12 h in RPMI 1640 in the presence and absence of 2 μg/ml caspofungin. (A) CFW-stained fluorescent images; scale bars are 10 μm. (B) Cell wall chitin assays were performed three times on three biologically independent samples (average ± standard deviation [SD], n = 9). The asterisk indicates significant difference ( P

    Techniques Used: Staining, Standard Deviation

    Intrahyphal growth of A. fumigatus corresponds to areas of increased chitin content in response to caspofungin treatment. A. fumigatus spores were trapped in a microfluidics chamber and grown in Sabdex broth plus 2% glucose for 2 h. Cells were then treated with 32 μg/ml caspofungin for 6 h, followed by combination treatment with 32 μg/ml caspofungin and 64 μg/ml nikkomycin Z for a further 6 h. Yellow arrowheads indicate intrahyphal hyphae. DIC (top) and CFW fluorescent images (bottom). Scale bars = 10 μm.
    Figure Legend Snippet: Intrahyphal growth of A. fumigatus corresponds to areas of increased chitin content in response to caspofungin treatment. A. fumigatus spores were trapped in a microfluidics chamber and grown in Sabdex broth plus 2% glucose for 2 h. Cells were then treated with 32 μg/ml caspofungin for 6 h, followed by combination treatment with 32 μg/ml caspofungin and 64 μg/ml nikkomycin Z for a further 6 h. Yellow arrowheads indicate intrahyphal hyphae. DIC (top) and CFW fluorescent images (bottom). Scale bars = 10 μm.

    Techniques Used:

    Pregrowing A. fumigatus in CaCl 2 and CFW reduces susceptibility to caspofungin on solid medium. Plate dilution sensitivity tests of the A. fumigatus wild-type strain (H237) and various Δ Afchs mutants on RPMI 1640 agar supplemented with 4 μg/ml caspofungin. Rows marked with an asterisk indicate pregrowth of the inoculum in Sabdex broth containing 200 mM CaCl 2 and 100 μg/ml CFW. Plates were incubated for 24 h at 37°C. Spore numbers per spot are 5,000, 500, 50, and 5 spores, from left to right.
    Figure Legend Snippet: Pregrowing A. fumigatus in CaCl 2 and CFW reduces susceptibility to caspofungin on solid medium. Plate dilution sensitivity tests of the A. fumigatus wild-type strain (H237) and various Δ Afchs mutants on RPMI 1640 agar supplemented with 4 μg/ml caspofungin. Rows marked with an asterisk indicate pregrowth of the inoculum in Sabdex broth containing 200 mM CaCl 2 and 100 μg/ml CFW. Plates were incubated for 24 h at 37°C. Spore numbers per spot are 5,000, 500, 50, and 5 spores, from left to right.

    Techniques Used: Incubation

    Treatment with CaCl 2 and CFW increases A. fumigatus cell wall chitin. The A. fumigatus wild-type strain (H237) was grown at 37°C for 8 h in Sabouraud dextrose broth alone and supplemented with 200 mM CaCl 2 or 100 μg/ml CFW or a combination. (A) Cell wall chitin assays were performed three times on three biologically independent samples (average ± SD, n = 9). An asterisk indicates significant difference ( P
    Figure Legend Snippet: Treatment with CaCl 2 and CFW increases A. fumigatus cell wall chitin. The A. fumigatus wild-type strain (H237) was grown at 37°C for 8 h in Sabouraud dextrose broth alone and supplemented with 200 mM CaCl 2 or 100 μg/ml CFW or a combination. (A) Cell wall chitin assays were performed three times on three biologically independent samples (average ± SD, n = 9). An asterisk indicates significant difference ( P

    Techniques Used:

    Morphological changes in A. fumigatus in response to caspofungin. A. fumigatus spores were trapped in a microfluidics chamber and grown in Sabouraud plus 2% glucose for 6 h in the absence (A) or presence (B) of CaCl 2 and CFW. Then all cells were treated with 32 μg/ml caspofungin for another 6 h and grown in fresh medium for 8 h. Yellow arrowheads indicate intrahyphal hyphae. White arrowheads indicate newly formed septum. Scale bars = 20 μm.
    Figure Legend Snippet: Morphological changes in A. fumigatus in response to caspofungin. A. fumigatus spores were trapped in a microfluidics chamber and grown in Sabouraud plus 2% glucose for 6 h in the absence (A) or presence (B) of CaCl 2 and CFW. Then all cells were treated with 32 μg/ml caspofungin for another 6 h and grown in fresh medium for 8 h. Yellow arrowheads indicate intrahyphal hyphae. White arrowheads indicate newly formed septum. Scale bars = 20 μm.

    Techniques Used:

    Treatment with CaCl 2 and CFW leads to an increase in chitin synthesis in Δ Afchs mutants of A. fumigatus . CFW-stained fluorescent images of the wild-type strain and Δ Afchs mutants in Sabdex broth alone (A) or supplemented with 200 mM CaCl 2 and 100 μg/ml CFW (B) after 12 h at 37°C. Scale bars = 10 μm.
    Figure Legend Snippet: Treatment with CaCl 2 and CFW leads to an increase in chitin synthesis in Δ Afchs mutants of A. fumigatus . CFW-stained fluorescent images of the wild-type strain and Δ Afchs mutants in Sabdex broth alone (A) or supplemented with 200 mM CaCl 2 and 100 μg/ml CFW (B) after 12 h at 37°C. Scale bars = 10 μm.

    Techniques Used: Staining

    Disruption of AfCHSG leads to hypersensitivity to caspofungin. (A) Plate dilution sensitivity tests of the A. fumigatus wild-type strain on RPMI 1640 agar alone (i) or supplemented with 2 μg/ml caspofungin (ii), 2 μg/ml nikkomycin Z (iii), or a combination (iv). (B) The Δ Afchs mutants were grown on RPMI 1640 agar with or without 2 μg/ml caspofungin. Plates were incubated for 48 h at 37°C. Spore numbers per spot are 5,000, 500, 50, and 5 spores, from left to right. (C) Dry weights of wild-type and Δ Afchs mutants were determined after 24 h growth in RPMI 1640 broth alone or supplemented with 2 μg/ml caspofungin. The wild-type strain was also treated with 2 μg/ml nikkomycin Z alone and in combination with caspofungin. Error bars are SD ( n = 3, from three independent experiments). Asterisks indicate significant differences ( P
    Figure Legend Snippet: Disruption of AfCHSG leads to hypersensitivity to caspofungin. (A) Plate dilution sensitivity tests of the A. fumigatus wild-type strain on RPMI 1640 agar alone (i) or supplemented with 2 μg/ml caspofungin (ii), 2 μg/ml nikkomycin Z (iii), or a combination (iv). (B) The Δ Afchs mutants were grown on RPMI 1640 agar with or without 2 μg/ml caspofungin. Plates were incubated for 48 h at 37°C. Spore numbers per spot are 5,000, 500, 50, and 5 spores, from left to right. (C) Dry weights of wild-type and Δ Afchs mutants were determined after 24 h growth in RPMI 1640 broth alone or supplemented with 2 μg/ml caspofungin. The wild-type strain was also treated with 2 μg/ml nikkomycin Z alone and in combination with caspofungin. Error bars are SD ( n = 3, from three independent experiments). Asterisks indicate significant differences ( P

    Techniques Used: Incubation

    18) Product Images from "Ex Vivo Response to Histone Deacetylase (HDAC) Inhibitors of the HIV Long Terminal Repeat (LTR) Derived from HIV-Infected Patients on Antiretroviral Therapy"

    Article Title: Ex Vivo Response to Histone Deacetylase (HDAC) Inhibitors of the HIV Long Terminal Repeat (LTR) Derived from HIV-Infected Patients on Antiretroviral Therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113341

    Ex vivo response of patient-derived HIV LTRs to HDACi in A. SVG and B. HeLa cell lines. (I) Each cell line was incubated with different concentrations of HDACi for 24 hr and toxicity was measured by the MTS assay. The cytotoxic concentration 50 (CC 50 ) for each drug is shown. (II) SVG or HeLa cells were transiently transfected with the wild-type NL4-3 LTR- pCEP4 and treated with various concentrations of HDACi for 24 hr. Co-transfection with Tat (4 ng) or incubation with PMA (20 nM) were used as positive controls. The activity of the LTR was measured as the fold change in luciferase compared to the untreated sample. # indicates doses of individual HDACi that were closest to the CC 50 and induced the largest fold change increase in luciferase activity. (III) Luciferase expression following transfection of pCEP4 containing LTR sequences isolated from total memory CD4 + T-cells prior to or after cART and treated with the optimal dose of HDACi. (C) Comparison of the luciferase expression in HeLa (red) and SVG (blue) following transfection of pCEP4 containing LTR sequences from (B III) and treated with the optimal dose of HDACi. Boxes represent the median, 25th and 75th percentiles and error bars the 10th and 90th percentiles. Ns = not statistically significant.
    Figure Legend Snippet: Ex vivo response of patient-derived HIV LTRs to HDACi in A. SVG and B. HeLa cell lines. (I) Each cell line was incubated with different concentrations of HDACi for 24 hr and toxicity was measured by the MTS assay. The cytotoxic concentration 50 (CC 50 ) for each drug is shown. (II) SVG or HeLa cells were transiently transfected with the wild-type NL4-3 LTR- pCEP4 and treated with various concentrations of HDACi for 24 hr. Co-transfection with Tat (4 ng) or incubation with PMA (20 nM) were used as positive controls. The activity of the LTR was measured as the fold change in luciferase compared to the untreated sample. # indicates doses of individual HDACi that were closest to the CC 50 and induced the largest fold change increase in luciferase activity. (III) Luciferase expression following transfection of pCEP4 containing LTR sequences isolated from total memory CD4 + T-cells prior to or after cART and treated with the optimal dose of HDACi. (C) Comparison of the luciferase expression in HeLa (red) and SVG (blue) following transfection of pCEP4 containing LTR sequences from (B III) and treated with the optimal dose of HDACi. Boxes represent the median, 25th and 75th percentiles and error bars the 10th and 90th percentiles. Ns = not statistically significant.

    Techniques Used: Ex Vivo, Derivative Assay, Incubation, MTS Assay, Concentration Assay, Transfection, Cotransfection, Activity Assay, Luciferase, Expressing, Isolation

    Vorinostat synergises with Tat and not PMA to increase transcription of the HIV LTR. SVG cells were transiently transfected with pCEP4 plasmid containing either NL4-3 or patient-derived HIV LTRs and luciferase activity was quantified following treatment with vorinostat (5 µM) and co-transfection with Tat using (A) non-mutated or (B) hypermutated ( P
    Figure Legend Snippet: Vorinostat synergises with Tat and not PMA to increase transcription of the HIV LTR. SVG cells were transiently transfected with pCEP4 plasmid containing either NL4-3 or patient-derived HIV LTRs and luciferase activity was quantified following treatment with vorinostat (5 µM) and co-transfection with Tat using (A) non-mutated or (B) hypermutated ( P

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

    Cloning of patient-derived HIV LTRs into pCEP4. (A) Total memory T cells from HIV-infected individuals were isolated from blood collected prior to or after receiving cART. The integrated HIV LTRs from these cells were isolated by triple nested Alu-LTR PCR. (B)(I) The plasmid pCEP4 was digested with Sal I to remove the entire PCMV promoter region and the SV40 poly A sequences. (II) DNA sequence of the Δ-57/-4 HIV LTR and the luciferase gene was generated by digestion of the Δ-57/-4 LTR pGL3-Basic vector [20] and ligated into pCEP4. (III) Patient-derived HIV LTRs were cloned into the Δ-57/-4 LTR-pCEP4 vector using the Acc 6 5 I and Hind III sites. (IV) Patient LTR pCEP4 was transfected into SVG and HeLa cell lines; the activity of various HDACi on LTR transcription was measured by quantification of luciferase activity.
    Figure Legend Snippet: Cloning of patient-derived HIV LTRs into pCEP4. (A) Total memory T cells from HIV-infected individuals were isolated from blood collected prior to or after receiving cART. The integrated HIV LTRs from these cells were isolated by triple nested Alu-LTR PCR. (B)(I) The plasmid pCEP4 was digested with Sal I to remove the entire PCMV promoter region and the SV40 poly A sequences. (II) DNA sequence of the Δ-57/-4 HIV LTR and the luciferase gene was generated by digestion of the Δ-57/-4 LTR pGL3-Basic vector [20] and ligated into pCEP4. (III) Patient-derived HIV LTRs were cloned into the Δ-57/-4 LTR-pCEP4 vector using the Acc 6 5 I and Hind III sites. (IV) Patient LTR pCEP4 was transfected into SVG and HeLa cell lines; the activity of various HDACi on LTR transcription was measured by quantification of luciferase activity.

    Techniques Used: Clone Assay, Derivative Assay, Infection, Isolation, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Luciferase, Generated, Transfection, Activity Assay

    Phylogenetic analyses of DNA sequences derived from integrated virus in CD4 + memory T-cells. Phylogenetic trees were constructed using a neighbour-joining method with sequences from nucleotide 6 to 548 of the LTR derived from memory CD4 + T-cells prior to the initiation of cART ( open symbols ), after at least 18 months of cART ( closed symbols ) in four participants and the consensus sequence from NL4-3 (square symbol). Arrows indicate clones selected at random for cloning into pCEP4. Scale-bars indicate genetic distance (e.g., 0.01 = 1% genetic distance). Bootstrap values of > 75 are shown on branches. All hypermutated clones ( P
    Figure Legend Snippet: Phylogenetic analyses of DNA sequences derived from integrated virus in CD4 + memory T-cells. Phylogenetic trees were constructed using a neighbour-joining method with sequences from nucleotide 6 to 548 of the LTR derived from memory CD4 + T-cells prior to the initiation of cART ( open symbols ), after at least 18 months of cART ( closed symbols ) in four participants and the consensus sequence from NL4-3 (square symbol). Arrows indicate clones selected at random for cloning into pCEP4. Scale-bars indicate genetic distance (e.g., 0.01 = 1% genetic distance). Bootstrap values of > 75 are shown on branches. All hypermutated clones ( P

    Techniques Used: Derivative Assay, Construct, Sequencing, Clone Assay

    19) Product Images from "Subconjunctivally applied naïve Tregs support corneal graft survival in baby rats"

    Article Title: Subconjunctivally applied naïve Tregs support corneal graft survival in baby rats

    Journal: Molecular Vision

    doi:

    Inhibitory potential of Tregs in a T cell stimulation assay. T cells were stimulated with CD3 and CD28 antibodies and Tregs of 3- and 10-week old rats were added in a co-culture experiment. Division of cells was traced with CFSE and ratio of divided to undivided cells was determined. Treg of both cohorts showed inhibitory potential (*p
    Figure Legend Snippet: Inhibitory potential of Tregs in a T cell stimulation assay. T cells were stimulated with CD3 and CD28 antibodies and Tregs of 3- and 10-week old rats were added in a co-culture experiment. Division of cells was traced with CFSE and ratio of divided to undivided cells was determined. Treg of both cohorts showed inhibitory potential (*p

    Techniques Used: Cell Stimulation, Co-Culture Assay

    20) Product Images from "RGS10 deficiency ameliorates the severity of disease in experimental autoimmune encephalomyelitis"

    Article Title: RGS10 deficiency ameliorates the severity of disease in experimental autoimmune encephalomyelitis

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0491-0

    RGS10-null mice with symptomatic EAE have fewer leukocytes in the CNS. Mononuclear cells from the spinal cord were isolated from MOG 35–55 -immunized WT ( n = 3–4) and RGS10-null ( n = 4) mice at day 15 p.i. as described in the “Methods” section. Cells were then stained with mAbs to surface markers and/or intracellular cytokines and analyzed by flow cytometry. a Plot shows the CD3+ T cells, CD19+ B cells, Ly6G+ neutrophils, and CD11b+ myeloid cells among CD45+ gated cells in the spinal cord. *, p
    Figure Legend Snippet: RGS10-null mice with symptomatic EAE have fewer leukocytes in the CNS. Mononuclear cells from the spinal cord were isolated from MOG 35–55 -immunized WT ( n = 3–4) and RGS10-null ( n = 4) mice at day 15 p.i. as described in the “Methods” section. Cells were then stained with mAbs to surface markers and/or intracellular cytokines and analyzed by flow cytometry. a Plot shows the CD3+ T cells, CD19+ B cells, Ly6G+ neutrophils, and CD11b+ myeloid cells among CD45+ gated cells in the spinal cord. *, p

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

    RGS10-null CD4+ T cells displayed attenuated migration to CXCL12, a ligand for CXCR4. CD4+ T cell migration to CXCL12 was tested in an in vitro transwell chemotaxis assay. a Histogram shows the levels of chemokine receptor CXCR4 staining on CD3+ CD19− CD4+ T lymphocytes from WT and RGS10-null mouse spleens, as determined by flow cytometry. b Percentage of CD4+ T cells from WT or RGS10 null mice ( n = 3) that migrated to the lower chamber containing serum free-medium (VEH), CXCL12 (10 nM) or under control conditions (CXCL12 in both lower and upper chambers) *, p
    Figure Legend Snippet: RGS10-null CD4+ T cells displayed attenuated migration to CXCL12, a ligand for CXCR4. CD4+ T cell migration to CXCL12 was tested in an in vitro transwell chemotaxis assay. a Histogram shows the levels of chemokine receptor CXCR4 staining on CD3+ CD19− CD4+ T lymphocytes from WT and RGS10-null mouse spleens, as determined by flow cytometry. b Percentage of CD4+ T cells from WT or RGS10 null mice ( n = 3) that migrated to the lower chamber containing serum free-medium (VEH), CXCL12 (10 nM) or under control conditions (CXCL12 in both lower and upper chambers) *, p

    Techniques Used: Migration, In Vitro, Chemotaxis Assay, Staining, Flow Cytometry, Cytometry, Mouse Assay

    RGS10-null dendritic cells (DCs) displayed intact antigen presentation capacity. DCs derived from WT and RGS10-null mice were incubated with CD4+ T cells from 2D2 TCR mice for 72 h in the presence of the indicated concentrations of MOG 35–55. As a positive control, CD4+ T cells from 2D2 TCR mice were stimulated in vitro with anti-CD3/CD28 (5 μg/ml) for 72 h. T cell proliferation was measured by MTS incorporation assay ( n = 3 mice per group)
    Figure Legend Snippet: RGS10-null dendritic cells (DCs) displayed intact antigen presentation capacity. DCs derived from WT and RGS10-null mice were incubated with CD4+ T cells from 2D2 TCR mice for 72 h in the presence of the indicated concentrations of MOG 35–55. As a positive control, CD4+ T cells from 2D2 TCR mice were stimulated in vitro with anti-CD3/CD28 (5 μg/ml) for 72 h. T cell proliferation was measured by MTS incorporation assay ( n = 3 mice per group)

    Techniques Used: Derivative Assay, Mouse Assay, Incubation, Positive Control, In Vitro

    RGS10-null naïve CD4+ T cells displayed intact mitogen-mediated activation and polarization into Th1 or Th17. a CD4+ T cells were isolated from spleens of WT and RGS10-null mice. Cells were treated with PMA (20 ng/ml)/ionomycin (1 μM) or anti-CD3/CD28 (5 μg/ml) in vitro for 72 h. Proliferation was measured by MTS incorporation assay and ( b ) cytokine production was measured by multiplexed immunoassays (MSD). *, p
    Figure Legend Snippet: RGS10-null naïve CD4+ T cells displayed intact mitogen-mediated activation and polarization into Th1 or Th17. a CD4+ T cells were isolated from spleens of WT and RGS10-null mice. Cells were treated with PMA (20 ng/ml)/ionomycin (1 μM) or anti-CD3/CD28 (5 μg/ml) in vitro for 72 h. Proliferation was measured by MTS incorporation assay and ( b ) cytokine production was measured by multiplexed immunoassays (MSD). *, p

    Techniques Used: Activation Assay, Isolation, Mouse Assay, In Vitro

    21) Product Images from "Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes"

    Article Title: Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0152583

    ASM secreted during cell injury associates with the PM and is proteolytically modulated. (A) Effect of protease inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca 2+ media containing (green) or not (blue) a protease inhibitor cocktail and incubated at 37°C for the indicated time. As controls, cells not treated with SLO (NT) were incubated with (purple) or without (red) inhibitors. Supernatant samples were placed on ice, protease inhibitors were added and ASM activity was assayed. Similar results were obtained with HeLa cells (not shown). (B) Effect of inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca 2+ -containing media with 100 μM E64, 100 μM pepstatin-A (PEP-A) or no inhibitors, and incubated at 37°C for 30 s. The data represent the mean +/- SD of triplicate assays. * P = 0.039, Student’s t test. (C) Detection of cell-associated ASM during the first seconds after SLO wounding. NRK cells were permeabilized with SLO (100 ng/ml), incubated at 37°C in Ca 2+ -containing media for the indicated time, washed and immunofluorescence was performed with rabbit anti-ASM antibodies, followed by imaging under identical settings. (D) Detection of the active 65 kDa form of ASM in supernatant and membrane-associated fractions of wounded cells. NRK cells were permeabilized or not with SLO (100 ng/ml) with or without 100 μM E64 or 100 μM pepstatin-A for the indicated periods of time, and samples of the supernatant or of material removed from the cell surface by an acid wash were analyzed by Western blot with rabbit anti-ASM antibodies. The data are representative of at least three independent experiments. (E) Detection of the 65 kDa ASM form in the supernatant and membrane-associated fractions of wounded cells depleted or not in cathepsin D (CATD). NRK cells were permeabilized or not with SLO and analyzed as described in (D). The data are representative of at least three independent experiments.
    Figure Legend Snippet: ASM secreted during cell injury associates with the PM and is proteolytically modulated. (A) Effect of protease inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca 2+ media containing (green) or not (blue) a protease inhibitor cocktail and incubated at 37°C for the indicated time. As controls, cells not treated with SLO (NT) were incubated with (purple) or without (red) inhibitors. Supernatant samples were placed on ice, protease inhibitors were added and ASM activity was assayed. Similar results were obtained with HeLa cells (not shown). (B) Effect of inhibitors on ASM activity released from wounded cells. NRK cells were permeabilized with SLO (100 ng/ml) in Ca 2+ -containing media with 100 μM E64, 100 μM pepstatin-A (PEP-A) or no inhibitors, and incubated at 37°C for 30 s. The data represent the mean +/- SD of triplicate assays. * P = 0.039, Student’s t test. (C) Detection of cell-associated ASM during the first seconds after SLO wounding. NRK cells were permeabilized with SLO (100 ng/ml), incubated at 37°C in Ca 2+ -containing media for the indicated time, washed and immunofluorescence was performed with rabbit anti-ASM antibodies, followed by imaging under identical settings. (D) Detection of the active 65 kDa form of ASM in supernatant and membrane-associated fractions of wounded cells. NRK cells were permeabilized or not with SLO (100 ng/ml) with or without 100 μM E64 or 100 μM pepstatin-A for the indicated periods of time, and samples of the supernatant or of material removed from the cell surface by an acid wash were analyzed by Western blot with rabbit anti-ASM antibodies. The data are representative of at least three independent experiments. (E) Detection of the 65 kDa ASM form in the supernatant and membrane-associated fractions of wounded cells depleted or not in cathepsin D (CATD). NRK cells were permeabilized or not with SLO and analyzed as described in (D). The data are representative of at least three independent experiments.

    Techniques Used: Activity Assay, Protease Inhibitor, Incubation, Immunofluorescence, Imaging, Western Blot

    Rapid extracellular proteolysis triggered by SLO wounding is required for PM repair. (A) Cleavage of extracellularly added DQ-BSA during PM wounding and repair. HeLa cells treated or not with 200 ng/ml SLO were incubated with DQ-BSA for 2 min at the indicated temperature, followed by measuring the de-quenching generated fluorescence. The data represent the mean +/- SD of triplicate assays. ** P = 0.00297, Student’s t test. (B) De-quenched DQ-BSA associated with the surface of wounded cells. NRK cells treated or not with 50 ng/ml SLO in the presence or absence of Ca 2+ were incubated with DQ-BSA followed by fluorescence deconvolution imaging (top panel). Bars = 10 μm. The mean cell-associated fluorescence intensity was determined and expressed as fold increase relative to the starting level (bottom panel). The data represent the mean +/- SD of fluorescence intensity values associated with 24–26 individual cells, and are representative of two independent experiments. (C) FACS quantification of PI staining in NRK cells permeabilized with SLO (200 ng/ml) in the presence (red) or absence (blue) of the protease inhibitor alpha-2-macroglobulin (20 μg/ml). The dotted histogram shows the Ca 2+ -free permeabilization control, which determined the gating (dashed line). The inset shows the percentage of cells that excluded PI after 2 min at 37°C. The data represent the mean +/- SD of three independent experiments. (D) Time-lapse live imaging of FM1-43 influx into NRK cells exposed to SLO (350 ng/ml) in the presence or absence of Ca 2+ and the protease inhibitor alpha-2-macroglobulin (20 μg/ml). The data represent the mean +/- SEM of 27–52 cells per condition.
    Figure Legend Snippet: Rapid extracellular proteolysis triggered by SLO wounding is required for PM repair. (A) Cleavage of extracellularly added DQ-BSA during PM wounding and repair. HeLa cells treated or not with 200 ng/ml SLO were incubated with DQ-BSA for 2 min at the indicated temperature, followed by measuring the de-quenching generated fluorescence. The data represent the mean +/- SD of triplicate assays. ** P = 0.00297, Student’s t test. (B) De-quenched DQ-BSA associated with the surface of wounded cells. NRK cells treated or not with 50 ng/ml SLO in the presence or absence of Ca 2+ were incubated with DQ-BSA followed by fluorescence deconvolution imaging (top panel). Bars = 10 μm. The mean cell-associated fluorescence intensity was determined and expressed as fold increase relative to the starting level (bottom panel). The data represent the mean +/- SD of fluorescence intensity values associated with 24–26 individual cells, and are representative of two independent experiments. (C) FACS quantification of PI staining in NRK cells permeabilized with SLO (200 ng/ml) in the presence (red) or absence (blue) of the protease inhibitor alpha-2-macroglobulin (20 μg/ml). The dotted histogram shows the Ca 2+ -free permeabilization control, which determined the gating (dashed line). The inset shows the percentage of cells that excluded PI after 2 min at 37°C. The data represent the mean +/- SD of three independent experiments. (D) Time-lapse live imaging of FM1-43 influx into NRK cells exposed to SLO (350 ng/ml) in the presence or absence of Ca 2+ and the protease inhibitor alpha-2-macroglobulin (20 μg/ml). The data represent the mean +/- SEM of 27–52 cells per condition.

    Techniques Used: Incubation, Generated, Fluorescence, Imaging, FACS, Staining, Protease Inhibitor

    Extracellular proteolysis releases surface proteins and promotes PM repair. (A) Effect of proteinase K treatment on Ca 2+- dependent PM repair. NRK cells pre-treated with increasing concentrations of proteinase K were permeabilized with SLO (150 ng/ml), incubated at 37°C for 5 min, stained with PI and analyzed by FACS. The inset on the right shows one example of PI quantification for cells untreated (blue) or pre-treated with 50 μg/ml proteinase K (red). The dotted histogram shows the Ca 2+ -free permeabilization control, which determined the gating (dashed line). The data are representative of three independent experiments. (B) Effect of proteinase K treatment on SM-induced Ca 2+ -free PM repair. NRK cells treated (red) or not (blue) with 50 μg/ml proteinase K were permeabilized with SLO (15 ng/ml), incubated at 37°C for 5 min in Ca 2+ -free DMEM containing 10 μU/ml SM, stained with PI and analyzed by FACS. The dotted black histogram shows the Ca 2+ -free permeabilization control in the absence of sphingomyelinase; the data are representative of five independent experiments. (C, D) Biotinylated surface proteins released in soluble form during SLO wounding and repair. NRK cells were biotinylated at 4°C and permeabilized or not with SLO (100 ng/ml) in the presence or not of Ca 2+ and containing or not a cocktail of protease inhibitors (Prot. Inh.), 100 μM E64, pepstatin-A (PEP-A) or AEBSF for 30 s, followed by collection of the supernatant, centrifugation at 100,000 g and analysis by Western blot with streptavidin-HRP. A diluted sample of the total cell extract before SLO wounding is shown on the left. The data are representative of two (C) or three (D) independent experiments.
    Figure Legend Snippet: Extracellular proteolysis releases surface proteins and promotes PM repair. (A) Effect of proteinase K treatment on Ca 2+- dependent PM repair. NRK cells pre-treated with increasing concentrations of proteinase K were permeabilized with SLO (150 ng/ml), incubated at 37°C for 5 min, stained with PI and analyzed by FACS. The inset on the right shows one example of PI quantification for cells untreated (blue) or pre-treated with 50 μg/ml proteinase K (red). The dotted histogram shows the Ca 2+ -free permeabilization control, which determined the gating (dashed line). The data are representative of three independent experiments. (B) Effect of proteinase K treatment on SM-induced Ca 2+ -free PM repair. NRK cells treated (red) or not (blue) with 50 μg/ml proteinase K were permeabilized with SLO (15 ng/ml), incubated at 37°C for 5 min in Ca 2+ -free DMEM containing 10 μU/ml SM, stained with PI and analyzed by FACS. The dotted black histogram shows the Ca 2+ -free permeabilization control in the absence of sphingomyelinase; the data are representative of five independent experiments. (C, D) Biotinylated surface proteins released in soluble form during SLO wounding and repair. NRK cells were biotinylated at 4°C and permeabilized or not with SLO (100 ng/ml) in the presence or not of Ca 2+ and containing or not a cocktail of protease inhibitors (Prot. Inh.), 100 μM E64, pepstatin-A (PEP-A) or AEBSF for 30 s, followed by collection of the supernatant, centrifugation at 100,000 g and analysis by Western blot with streptavidin-HRP. A diluted sample of the total cell extract before SLO wounding is shown on the left. The data are representative of two (C) or three (D) independent experiments.

    Techniques Used: Incubation, Staining, FACS, Centrifugation, Western Blot

    Active lysosomal proteases are released with differential kinetics during PM wounding and repair. NRK cells were pre-incubated on ice with SLO (100 ng/ml) followed by addition of Ca 2+ -containing media pre-warmed at 37°C to trigger pore-formation and lysosomal exocytosis. The supernatant was collected at the indicated periods of time and analyzed for activity of the cysteine proteases cathepsins B and L and the aspartyl protease cathepsin D. Results are expressed as percentage of the total activity present in the cells (determined by assaying enzymatic activity in whole cell lysates), and correspond to the mean +/- SD of three independent experiments.
    Figure Legend Snippet: Active lysosomal proteases are released with differential kinetics during PM wounding and repair. NRK cells were pre-incubated on ice with SLO (100 ng/ml) followed by addition of Ca 2+ -containing media pre-warmed at 37°C to trigger pore-formation and lysosomal exocytosis. The supernatant was collected at the indicated periods of time and analyzed for activity of the cysteine proteases cathepsins B and L and the aspartyl protease cathepsin D. Results are expressed as percentage of the total activity present in the cells (determined by assaying enzymatic activity in whole cell lysates), and correspond to the mean +/- SD of three independent experiments.

    Techniques Used: Incubation, Activity Assay

    Protease inhibitors rapidly modulate the repair of SLO wounds. NRK (left column) or HeLa (right column) cells were wounded with SLO (NRK 150 ng/ml, HeLa 250 ng/ml) and PM repair assays were performed in the absence (A, E) or in the presence of the following protease inhibitors: (B, F) E64 (100 μM); (C, G) Pepstatin-A (PEP-A,100 μM); (D, H) AEBSF (100 μM). After a resealing period of 2 min at 37°C the cell population was stained with PI and analyzed by FACS. In (A, E) the green histograms show the FACS profile of not wounded cells, and the dotted black histograms show the Ca 2+ -free permeabilization controls, which determined the gating (dashed line). The data are representative of at least three independent experiments.
    Figure Legend Snippet: Protease inhibitors rapidly modulate the repair of SLO wounds. NRK (left column) or HeLa (right column) cells were wounded with SLO (NRK 150 ng/ml, HeLa 250 ng/ml) and PM repair assays were performed in the absence (A, E) or in the presence of the following protease inhibitors: (B, F) E64 (100 μM); (C, G) Pepstatin-A (PEP-A,100 μM); (D, H) AEBSF (100 μM). After a resealing period of 2 min at 37°C the cell population was stained with PI and analyzed by FACS. In (A, E) the green histograms show the FACS profile of not wounded cells, and the dotted black histograms show the Ca 2+ -free permeabilization controls, which determined the gating (dashed line). The data are representative of at least three independent experiments.

    Techniques Used: Staining, FACS

    22) Product Images from "Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *"

    Article Title: Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.704734

    Affinity purification of recombinant proteins used in this study. A , Coomassie Brilliant Blue-stained SDS-polyacrylamide quality control gels of recombinantly expressed and affinity-purified BMP-7 PD variants and proteins representing the fibrillin-1 N terminus. B ( left ), size exclusion chromatogram of the BMP-7 PD-GF complex after chelating chromatography utilizing the His 6 tag placed at the N terminus of the PD. The chromatogram shows the BMP-7 PD-GF complex mainly eluting in one peak. Right , Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing the purity of the peak fraction. C , Coomassie Brilliant Blue-stained SDS-polyacrylamide gels showing successful separation of the GF from the PD. The separation was performed as described previously ( 20 ). BMP-7 complex was separated into BMP-7 PD (34 kDa) and GF dimer (31 kDa) after dialysis into 8 m urea followed by chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through.
    Figure Legend Snippet: Affinity purification of recombinant proteins used in this study. A , Coomassie Brilliant Blue-stained SDS-polyacrylamide quality control gels of recombinantly expressed and affinity-purified BMP-7 PD variants and proteins representing the fibrillin-1 N terminus. B ( left ), size exclusion chromatogram of the BMP-7 PD-GF complex after chelating chromatography utilizing the His 6 tag placed at the N terminus of the PD. The chromatogram shows the BMP-7 PD-GF complex mainly eluting in one peak. Right , Coomassie Brilliant Blue-stained SDS-polyacrylamide gel showing the purity of the peak fraction. C , Coomassie Brilliant Blue-stained SDS-polyacrylamide gels showing successful separation of the GF from the PD. The separation was performed as described previously ( 20 ). BMP-7 complex was separated into BMP-7 PD (34 kDa) and GF dimer (31 kDa) after dialysis into 8 m urea followed by chelating chromatography, where the PD was bound to the affinity column, and the GF was obtained in the flow-through.

    Techniques Used: Affinity Purification, Recombinant, Staining, Chromatography, Affinity Column, Flow Cytometry

    Interaction studies to identify the fibrillin-1 binding motif within the BMP-7 PD. A , domain structure of the N-terminal fibrillin-1 fragment used in the interaction study. B , sensorgrams of SPR binding studies suggesting that the fibrillin-1 binding domain within the BMP-7 PD resides within residues Gly 74 –Phe 185 . 0–80 n m rF87, containing the N-terminal unique domain, the first three EGF-like domains, the first hybrid motif, and the first two calcium-binding EGF- like domains of fibrillin-1, was injected onto immobilized BMP-7 PD variants. All injections were performed in HBS-EP buffer.
    Figure Legend Snippet: Interaction studies to identify the fibrillin-1 binding motif within the BMP-7 PD. A , domain structure of the N-terminal fibrillin-1 fragment used in the interaction study. B , sensorgrams of SPR binding studies suggesting that the fibrillin-1 binding domain within the BMP-7 PD resides within residues Gly 74 –Phe 185 . 0–80 n m rF87, containing the N-terminal unique domain, the first three EGF-like domains, the first hybrid motif, and the first two calcium-binding EGF- like domains of fibrillin-1, was injected onto immobilized BMP-7 PD variants. All injections were performed in HBS-EP buffer.

    Techniques Used: Binding Assay, SPR Assay, Injection

    Identification of the GF binding motif within the BMP-7 PD. A , solid phase binding ELISA-style assays with immobilized BMP-7 PD truncation variants and GF in solution. B , SPR binding studies of BMP-7 N-terminal PD truncation variants and BMP-7 GF. Top panel , GF binding to the PD was robust to the presence of 1 m urea and pH reduction to 4.5 (full-length PD(30–292) immobilized, GF in solution). Bottom panels , GF was immobilized, and PD variants were injected in solution. C , BMP-7 reconstitution after dialysis of PD variants and GF. Successful reconstitution was monitored in a sandwich ELISA using an anti-His 6 antibody against the C-terminal His 6 tag on the PD as capture and a polyclonal anti-BMP-7 GF antibody as detector. Error bars , S.D. from three independent experiments. D , sequence alignment using ClustalW identifies the 65 PHRP 68 motif ( red ) as conserved within the BMP-5, -6, -7 subgroup of the TGF-β superfamily. Blue , predicted Ile 58 -Leu 59 -Leu 62 -Leu 64 GF binding motif ( 35 ). E , CD spectra of systematic truncation variants between Arg 67 and Pro 69 . Deletion of Pro 65 -His 66 results in a significant increase of α-helical content of 8% when compared with the 65–292 PD variant ( Table 1 ). This increase returned to normal levels upon stepwise N-terminal truncation of the subsequent two residues. The point mutation P65A resulted in a 4% increase of α-helical content ( Table 1 ), whereas H66A or P68A resulted in no or little change. Additional mutation of the subsequent three residues resulted in no additional change in α-helical content in the quadruple mutant variant P65A/H66A/R67A/P68A ( Table 1 ).
    Figure Legend Snippet: Identification of the GF binding motif within the BMP-7 PD. A , solid phase binding ELISA-style assays with immobilized BMP-7 PD truncation variants and GF in solution. B , SPR binding studies of BMP-7 N-terminal PD truncation variants and BMP-7 GF. Top panel , GF binding to the PD was robust to the presence of 1 m urea and pH reduction to 4.5 (full-length PD(30–292) immobilized, GF in solution). Bottom panels , GF was immobilized, and PD variants were injected in solution. C , BMP-7 reconstitution after dialysis of PD variants and GF. Successful reconstitution was monitored in a sandwich ELISA using an anti-His 6 antibody against the C-terminal His 6 tag on the PD as capture and a polyclonal anti-BMP-7 GF antibody as detector. Error bars , S.D. from three independent experiments. D , sequence alignment using ClustalW identifies the 65 PHRP 68 motif ( red ) as conserved within the BMP-5, -6, -7 subgroup of the TGF-β superfamily. Blue , predicted Ile 58 -Leu 59 -Leu 62 -Leu 64 GF binding motif ( 35 ). E , CD spectra of systematic truncation variants between Arg 67 and Pro 69 . Deletion of Pro 65 -His 66 results in a significant increase of α-helical content of 8% when compared with the 65–292 PD variant ( Table 1 ). This increase returned to normal levels upon stepwise N-terminal truncation of the subsequent two residues. The point mutation P65A resulted in a 4% increase of α-helical content ( Table 1 ), whereas H66A or P68A resulted in no or little change. Additional mutation of the subsequent three residues resulted in no additional change in α-helical content in the quadruple mutant variant P65A/H66A/R67A/P68A ( Table 1 ).

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, SPR Assay, Injection, Sandwich ELISA, Sequencing, Variant Assay, Mutagenesis

    SAXS data collected for BMP-7 complex. A , x-ray scattering profile of BMP-7 complex showing intensity as a function of q ( gray triangles ) and Gnom fit to the data ( black line ). Inset , Guinier plot showing R g of 4.8 nm. B , P ( r ) plot showing D max of 16 nm. C , Kratky plot showing profile typical of a folded protein. D , plateau in the Porod-Debye plot indicative of a non-flexible protein. E , ab initio models generated from SAXS data using GASBOR with 2-fold symmetry ( blue ); the averaged model is shown along with three representative models.
    Figure Legend Snippet: SAXS data collected for BMP-7 complex. A , x-ray scattering profile of BMP-7 complex showing intensity as a function of q ( gray triangles ) and Gnom fit to the data ( black line ). Inset , Guinier plot showing R g of 4.8 nm. B , P ( r ) plot showing D max of 16 nm. C , Kratky plot showing profile typical of a folded protein. D , plateau in the Porod-Debye plot indicative of a non-flexible protein. E , ab initio models generated from SAXS data using GASBOR with 2-fold symmetry ( blue ); the averaged model is shown along with three representative models.

    Techniques Used: Generated

    Three-dimensional EM and solution SAXS models of BMP-7 PD-GF complex. Three-dimensional structure of BMP-7 complex was generated using TEM. A ( top ), representative electron micrograph of BMP-7 complex molecules ( scale bar , 100 nm); bottom , 12 images selected from 140 class sum images of 9,000 particles that represent different views of BMP-7 complex ( box size = 29.4 × 29.4 nm). B , class sum images were used to generate a three-dimensional TEM model of BMP-7 complex with 2-fold symmetry using angular reconstitution. C , superimposition of the BMP-9 complex structure ( 33 ) at 20 Å with the determined BMP-7 EM envelope suggests that the angle between the boomerang arms may be wider in BMP-9.
    Figure Legend Snippet: Three-dimensional EM and solution SAXS models of BMP-7 PD-GF complex. Three-dimensional structure of BMP-7 complex was generated using TEM. A ( top ), representative electron micrograph of BMP-7 complex molecules ( scale bar , 100 nm); bottom , 12 images selected from 140 class sum images of 9,000 particles that represent different views of BMP-7 complex ( box size = 29.4 × 29.4 nm). B , class sum images were used to generate a three-dimensional TEM model of BMP-7 complex with 2-fold symmetry using angular reconstitution. C , superimposition of the BMP-9 complex structure ( 33 ) at 20 Å with the determined BMP-7 EM envelope suggests that the angle between the boomerang arms may be wider in BMP-9.

    Techniques Used: Generated, Transmission Electron Microscopy

    The 65 PHRP 68 motif within the N-terminal region of the BMP-7 PD is required for competition with the BMP type II receptor for GF binding. A , scheme of the experimental set-up. Full-length BMP-7 PD (residues 30–292) and the N-terminal truncation variant 65–292 were immobilized, and 100 n m BMP-7 GF was injected in the presence of 0–500 n m BMPRII receptor extracellular domain onto the chip first, followed by a second injection of 100 n m mAb anti-BMP-7 GF antibody to detect bound GF (all injections were in HBS-EP buffer). B , sensorgrams of 100 n m injected mAb anti-BMP-7 GF antibody to detect bound GF. C , increasing amounts of receptor resulted in comparable inhibition of GF binding to both immobilized PD variants, suggesting that the presence of the 65 PHRP 68 motif in 65–292 is responsible for PD competition with the type II receptor for the same binding site on the GF. Error bars , S.D. from three independent experiments. The schematic shows GF ( orange ) and type II receptor ( blue ) binding sites.
    Figure Legend Snippet: The 65 PHRP 68 motif within the N-terminal region of the BMP-7 PD is required for competition with the BMP type II receptor for GF binding. A , scheme of the experimental set-up. Full-length BMP-7 PD (residues 30–292) and the N-terminal truncation variant 65–292 were immobilized, and 100 n m BMP-7 GF was injected in the presence of 0–500 n m BMPRII receptor extracellular domain onto the chip first, followed by a second injection of 100 n m mAb anti-BMP-7 GF antibody to detect bound GF (all injections were in HBS-EP buffer). B , sensorgrams of 100 n m injected mAb anti-BMP-7 GF antibody to detect bound GF. C , increasing amounts of receptor resulted in comparable inhibition of GF binding to both immobilized PD variants, suggesting that the presence of the 65 PHRP 68 motif in 65–292 is responsible for PD competition with the type II receptor for the same binding site on the GF. Error bars , S.D. from three independent experiments. The schematic shows GF ( orange ) and type II receptor ( blue ) binding sites.

    Techniques Used: Binding Assay, Variant Assay, Injection, Chromatin Immunoprecipitation, Inhibition

    Homology models of the BMP-7 complex and model of extracellular control of BMP GF activity via PD interactions with fibrillin-1 microfibrils. A ( top ), in the unbound, bioactive state, the BMP-7 complex adopts an open V-like shape. In this conformation, the PDs are in contact with each other via the first 18 N-terminal residues ( green ). The GF shows an extended, open conformation similar to the TGF-β-1 GF in the SLC ( 34 ), which enables positioning of the α1-helix ( purple rod ) of the PD within a pocket of the GF. The PD contains a 65 PHRP 68 motif ( red hinge ) located between the α1- and α2-helix ( red rod ), which serves as an important “molecular clamp” for maintaining interaction with the GF and is therefore required for proper PD competition with type II receptor binding. In this conformation, the α2-helix is not occupying the type II receptor binding site on the GF. Bottom , upon binding to fibrillin-1, the BMP-7 complex undergoes a conformational change. In this latent, closed conformation, the two PD arms may interact with each other via unmasked C-terminal self-interaction epitopes, which in turn facilitate the ring closure. In the closed ring shape conformation, the α2 occupies the type II receptor binding site, which confers latency to the GF. B , in solution, binding of type II receptors to the GF moiety of the BMP-7 complex results in displacement of the PDs as a dimer. The PDs remain tethered to each other via their N-terminal self-interaction epitopes ( green ). Binding to fibrillin-1 microfibrils ( green ) induces a conformational change within the PD that enables a closed ring-shaped conformation of the BMP-7 complex, rendering the GF latent. Homology models of the BMP-7 complex in its open and closed forms were generated using the structure of the TGF-β-1 SLC ( 34 ) and fitted into the shapes determined by TEM. For the open BMP-7 form, the model is fitted in the electron density map from EM, and for the closed form the model is shown as electron density rendered at 20 Å resolution. Orange , GF dimer; yellow circle , type II receptor binding site; green , N-terminal self-interaction epitope; magenta , α1-helix; red , α2-helix; red , stretch connecting α1- and α2-helix containing the 65 PHRP 68 motif; light blue , C-terminal portion of BMP-7 PD.
    Figure Legend Snippet: Homology models of the BMP-7 complex and model of extracellular control of BMP GF activity via PD interactions with fibrillin-1 microfibrils. A ( top ), in the unbound, bioactive state, the BMP-7 complex adopts an open V-like shape. In this conformation, the PDs are in contact with each other via the first 18 N-terminal residues ( green ). The GF shows an extended, open conformation similar to the TGF-β-1 GF in the SLC ( 34 ), which enables positioning of the α1-helix ( purple rod ) of the PD within a pocket of the GF. The PD contains a 65 PHRP 68 motif ( red hinge ) located between the α1- and α2-helix ( red rod ), which serves as an important “molecular clamp” for maintaining interaction with the GF and is therefore required for proper PD competition with type II receptor binding. In this conformation, the α2-helix is not occupying the type II receptor binding site on the GF. Bottom , upon binding to fibrillin-1, the BMP-7 complex undergoes a conformational change. In this latent, closed conformation, the two PD arms may interact with each other via unmasked C-terminal self-interaction epitopes, which in turn facilitate the ring closure. In the closed ring shape conformation, the α2 occupies the type II receptor binding site, which confers latency to the GF. B , in solution, binding of type II receptors to the GF moiety of the BMP-7 complex results in displacement of the PDs as a dimer. The PDs remain tethered to each other via their N-terminal self-interaction epitopes ( green ). Binding to fibrillin-1 microfibrils ( green ) induces a conformational change within the PD that enables a closed ring-shaped conformation of the BMP-7 complex, rendering the GF latent. Homology models of the BMP-7 complex in its open and closed forms were generated using the structure of the TGF-β-1 SLC ( 34 ) and fitted into the shapes determined by TEM. For the open BMP-7 form, the model is fitted in the electron density map from EM, and for the closed form the model is shown as electron density rendered at 20 Å resolution. Orange , GF dimer; yellow circle , type II receptor binding site; green , N-terminal self-interaction epitope; magenta , α1-helix; red , α2-helix; red , stretch connecting α1- and α2-helix containing the 65 PHRP 68 motif; light blue , C-terminal portion of BMP-7 PD.

    Techniques Used: Activity Assay, Binding Assay, Generated, Transmission Electron Microscopy

    Binding to fibrillin-1 induces a conformational change of the BMP-7 complex, resulting in GF inhibition. A , domain structure of fibrillin-1 and used variants. B , BMP activity assay with BMP-7 complex captured via PD interactions, mAb against the N-terminal His 6 tag, or the N-terminal half of fibrillin-1 (rF11). C2C12 cells were seeded onto immobilized BMP-7 complex, and Id3 expression was measured as a read-out for BMP activity. Shown is dot blotting analysis of stripped BMP-7 complex by comparison with a diluted series of dots containing BMP-7 complex at known concentrations. The schematic depicts the different ways BMP-7 PD-GF complex is presented to the reporter cells. Antibody capture of the N-terminally placed His 6 tag on the PD ( green ) does not affect bioactivity; however, binding of fibrillin-1 within the PD ( blue ) induces a conformational change into a ring shape that confers latency. Orange , GF dimer; yellow circle , type II receptor binding site; green , N-terminal His 6 tag; magenta , α1-helix; red , α2-helix; red , stretch connecting α1- and α2-helix containing the 65 PHRP 68 motif; light blue , C-terminal portion of BMP-7 PD. C , dialyzing the small fibrillin-1 N-terminal fragment FUN to BMP-7 complex resulted in the formation of ring shapes and open intermediates that were absent in the BMP-7 complex-only sample ( Fig. 2 A ). Shown are a representative TEM electron micrograph ( scale bar , 100 nm) and 12 from 100 class averages of 11,000 particles ( box size , 28 × 28 nm). The small fibrillin-1 fragment FUN was not distinguishable from the background.
    Figure Legend Snippet: Binding to fibrillin-1 induces a conformational change of the BMP-7 complex, resulting in GF inhibition. A , domain structure of fibrillin-1 and used variants. B , BMP activity assay with BMP-7 complex captured via PD interactions, mAb against the N-terminal His 6 tag, or the N-terminal half of fibrillin-1 (rF11). C2C12 cells were seeded onto immobilized BMP-7 complex, and Id3 expression was measured as a read-out for BMP activity. Shown is dot blotting analysis of stripped BMP-7 complex by comparison with a diluted series of dots containing BMP-7 complex at known concentrations. The schematic depicts the different ways BMP-7 PD-GF complex is presented to the reporter cells. Antibody capture of the N-terminally placed His 6 tag on the PD ( green ) does not affect bioactivity; however, binding of fibrillin-1 within the PD ( blue ) induces a conformational change into a ring shape that confers latency. Orange , GF dimer; yellow circle , type II receptor binding site; green , N-terminal His 6 tag; magenta , α1-helix; red , α2-helix; red , stretch connecting α1- and α2-helix containing the 65 PHRP 68 motif; light blue , C-terminal portion of BMP-7 PD. C , dialyzing the small fibrillin-1 N-terminal fragment FUN to BMP-7 complex resulted in the formation of ring shapes and open intermediates that were absent in the BMP-7 complex-only sample ( Fig. 2 A ). Shown are a representative TEM electron micrograph ( scale bar , 100 nm) and 12 from 100 class averages of 11,000 particles ( box size , 28 × 28 nm). The small fibrillin-1 fragment FUN was not distinguishable from the background.

    Techniques Used: Binding Assay, Inhibition, Activity Assay, Expressing, Transmission Electron Microscopy

    BMP-7 PD interacts with itself. A , dialysis of BMP-7 complex into 0.25–4 m urea reveals the presence of PD dimers monitored by velocity sedimentation experiments using 5–20% sucrose gradients. Each gradient was collected in 28 fractions (fraction 1 at 5% and fraction 28 at 20% sucrose) and subjected to Western blotting analysis for BMP-7 complex components. Western blots were incubated with anti-BMP-7 PD antibody first, stripped, and subsequently re-incubated with anti-BMP-7 GF antibody. B , SPR sensorgrams of self-interaction studies with BMP-7 full-length PD, 48–292, and 55–292, respectively, of immobilized full-length PD and PD variants representing the C-terminal end. C , BMP-7 complex reconstitution is affected by 30% upon deletion of the N-terminal PD self-interaction site. Error bars , mean ± S.D. from three independent experiments.
    Figure Legend Snippet: BMP-7 PD interacts with itself. A , dialysis of BMP-7 complex into 0.25–4 m urea reveals the presence of PD dimers monitored by velocity sedimentation experiments using 5–20% sucrose gradients. Each gradient was collected in 28 fractions (fraction 1 at 5% and fraction 28 at 20% sucrose) and subjected to Western blotting analysis for BMP-7 complex components. Western blots were incubated with anti-BMP-7 PD antibody first, stripped, and subsequently re-incubated with anti-BMP-7 GF antibody. B , SPR sensorgrams of self-interaction studies with BMP-7 full-length PD, 48–292, and 55–292, respectively, of immobilized full-length PD and PD variants representing the C-terminal end. C , BMP-7 complex reconstitution is affected by 30% upon deletion of the N-terminal PD self-interaction site. Error bars , mean ± S.D. from three independent experiments.

    Techniques Used: Sedimentation, Western Blot, Incubation, SPR Assay

    Secondary structure analysis of BMP-7 PD. A , CD measurements of N-terminal BMP-7 PD truncation variants ( red ) in comparison with full-length BMP-7 PD (30–292, blue ). Bottom panel , middle , truncation of the first 43 residues in a shorter variant covering the first 184 N-terminal residues ( red ) results in significant reduction of the α-helical peak at 209 nm compared with the control ( blue ). Construct 166–217 suggests the existence of a third α-helix within this region. Secondary structure percentage calculated from these CD curves is listed in Tables 1 and 2 . B , secondary structure prediction based on CD measurements shows the position of α-helices ( red ). The position of β-sheets ( blue ) was guided by predicted secondary structure elements (PSIPRED) marked below the sequence ( yellow , α-helical regions; light blue , β-sheets).
    Figure Legend Snippet: Secondary structure analysis of BMP-7 PD. A , CD measurements of N-terminal BMP-7 PD truncation variants ( red ) in comparison with full-length BMP-7 PD (30–292, blue ). Bottom panel , middle , truncation of the first 43 residues in a shorter variant covering the first 184 N-terminal residues ( red ) results in significant reduction of the α-helical peak at 209 nm compared with the control ( blue ). Construct 166–217 suggests the existence of a third α-helix within this region. Secondary structure percentage calculated from these CD curves is listed in Tables 1 and 2 . B , secondary structure prediction based on CD measurements shows the position of α-helices ( red ). The position of β-sheets ( blue ) was guided by predicted secondary structure elements (PSIPRED) marked below the sequence ( yellow , α-helical regions; light blue , β-sheets).

    Techniques Used: Variant Assay, Construct, Sequencing

    23) Product Images from "CXCR7 stimulates MAPK signaling to regulate hepatocellular carcinoma progression"

    Article Title: CXCR7 stimulates MAPK signaling to regulate hepatocellular carcinoma progression

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.392

    CXCR7 modulates phenotype of HCC cells in vitro . ( a ) Exogenous CXCR7 was expressed in HepG2 cells transfected with the pBabe vector. Both shCXCR7-1 and shCXCR7-2 were used to knockdown CXCR7 in LM3 cells. Parental cells with empty vector (pBabe or pLKO.1) were used as control. The surface expression of CXCR7 were evaluated by FACS analysis, using a phycoerythrin (PE)–anti-CXCR7 monoclonal antibody to detect CXCR7 expression, and a matched PE mouse IgG served as isotype control. ( b ) Western blot analysis comfirmed the expression of CXCR7 in HepG2- and LM3-transfected cells. ( c ) Cell proliferations were examined by CCK-8 experiments for HCC cell lines in response to altered CXCR7 levels with or without CXCL12 stimulation. * denotes significant difference from respective controls (as compared with cells transfected with empty vector); # , significant from nontreatment controls by CXCL12 (100 ng/ml) stimulation ( P
    Figure Legend Snippet: CXCR7 modulates phenotype of HCC cells in vitro . ( a ) Exogenous CXCR7 was expressed in HepG2 cells transfected with the pBabe vector. Both shCXCR7-1 and shCXCR7-2 were used to knockdown CXCR7 in LM3 cells. Parental cells with empty vector (pBabe or pLKO.1) were used as control. The surface expression of CXCR7 were evaluated by FACS analysis, using a phycoerythrin (PE)–anti-CXCR7 monoclonal antibody to detect CXCR7 expression, and a matched PE mouse IgG served as isotype control. ( b ) Western blot analysis comfirmed the expression of CXCR7 in HepG2- and LM3-transfected cells. ( c ) Cell proliferations were examined by CCK-8 experiments for HCC cell lines in response to altered CXCR7 levels with or without CXCL12 stimulation. * denotes significant difference from respective controls (as compared with cells transfected with empty vector); # , significant from nontreatment controls by CXCL12 (100 ng/ml) stimulation ( P

    Techniques Used: In Vitro, Transfection, Plasmid Preparation, Expressing, FACS, Western Blot, CCK-8 Assay

    24) Product Images from "mTORC1-Activated Monocytes Increase Tregs and Inhibit the Immune Response to Bacterial Infections"

    Article Title: mTORC1-Activated Monocytes Increase Tregs and Inhibit the Immune Response to Bacterial Infections

    Journal: Mediators of Inflammation

    doi: 10.1155/2016/7369351

    mTORC1 influences the numbers of Tregs and Th17 and Th1 cells in lymph nodes and spleen. (a) The percentage and number of Tregs increased in TSC1 KO lymph nodes and spleens compared to WT mice (four mice per group, three representative experiments). (b) The percentage of Ki67-positive Tregs increased in TSC1 KO lymph nodes and spleens compared to WT mice (four mice per group, three representative experiments). (c) The percentage and number of Th17 cells (CD4 + IL-17 + ) decreased in lymph nodes and spleens in TSC1 KO mice when compared to WT mice (four mice per group, three representative experiments). (d) The number and percentage of Th1 cells (CD4 + IFN-gamma + ) decreased in the LNs and spleen of TSC1 KO mice compared to those of WT mice (four mice per group, two representative experiments). (e) 2 × 10 5 Tregs (CD4 + CD25 + ) labeled with CFSE (5 μ M; eBioscience) were cocultured with 1 × 10 5 sorted monocytes (CD11b + Ly6G − Ly6C + ) from WT or TSC1 KO mice in DMEM containing IL-2 (100 ng/mL) in 96-well flat-bottomed plates for 6 days. There was no significant difference in proliferation of Tregs cocultured with monocytes obtained from TSC1 KO or WT mice ( n = 3 representative experiments). (f) 2 × 10 5 naïve CD4 + T cells (CD4 + CD44 − CD62 + ) were cocultured with 1 × 10 5 sorted monocytes (CD11b + Ly6G − Ly6C + ) from WT or TSC1 KO mice in DMEM with anti-mouse CD3e (10 μ g/mL, BD Biosciences, USA) and CD28 antibody (10 μ g/mL, BD Biosciences, USA) in 96-well flat-bottomed plates for 6 days. The percentage of Th17 cells of naïve CD4 + T cells (CD4 + CD44 − CD62L + ) cocultured with monocytes obtained from TSC1 KO mice was lower than the WT group at day 6. However, the percentage of Tregs was higher in the TSC1 KO group than in the WT group at day 6 ( n = 3 representative experiments). (g) 2 × 10 5 sorted naïve CD4 + T cells (CD4 + CD44 − CD62L + ) were cocultured with 1 × 10 5 sorted monocytes (CD11b + Ly6G − Ly6C + ) from WT mice in DMEM with or without IL-10 (20 ng/mL, Peprotech, UK). After 6 days, cells were used to estimate Th17 cells using flow cytometry (BD FACSCanto II, USA). ∗ P
    Figure Legend Snippet: mTORC1 influences the numbers of Tregs and Th17 and Th1 cells in lymph nodes and spleen. (a) The percentage and number of Tregs increased in TSC1 KO lymph nodes and spleens compared to WT mice (four mice per group, three representative experiments). (b) The percentage of Ki67-positive Tregs increased in TSC1 KO lymph nodes and spleens compared to WT mice (four mice per group, three representative experiments). (c) The percentage and number of Th17 cells (CD4 + IL-17 + ) decreased in lymph nodes and spleens in TSC1 KO mice when compared to WT mice (four mice per group, three representative experiments). (d) The number and percentage of Th1 cells (CD4 + IFN-gamma + ) decreased in the LNs and spleen of TSC1 KO mice compared to those of WT mice (four mice per group, two representative experiments). (e) 2 × 10 5 Tregs (CD4 + CD25 + ) labeled with CFSE (5 μ M; eBioscience) were cocultured with 1 × 10 5 sorted monocytes (CD11b + Ly6G − Ly6C + ) from WT or TSC1 KO mice in DMEM containing IL-2 (100 ng/mL) in 96-well flat-bottomed plates for 6 days. There was no significant difference in proliferation of Tregs cocultured with monocytes obtained from TSC1 KO or WT mice ( n = 3 representative experiments). (f) 2 × 10 5 naïve CD4 + T cells (CD4 + CD44 − CD62 + ) were cocultured with 1 × 10 5 sorted monocytes (CD11b + Ly6G − Ly6C + ) from WT or TSC1 KO mice in DMEM with anti-mouse CD3e (10 μ g/mL, BD Biosciences, USA) and CD28 antibody (10 μ g/mL, BD Biosciences, USA) in 96-well flat-bottomed plates for 6 days. The percentage of Th17 cells of naïve CD4 + T cells (CD4 + CD44 − CD62L + ) cocultured with monocytes obtained from TSC1 KO mice was lower than the WT group at day 6. However, the percentage of Tregs was higher in the TSC1 KO group than in the WT group at day 6 ( n = 3 representative experiments). (g) 2 × 10 5 sorted naïve CD4 + T cells (CD4 + CD44 − CD62L + ) were cocultured with 1 × 10 5 sorted monocytes (CD11b + Ly6G − Ly6C + ) from WT mice in DMEM with or without IL-10 (20 ng/mL, Peprotech, UK). After 6 days, cells were used to estimate Th17 cells using flow cytometry (BD FACSCanto II, USA). ∗ P

    Techniques Used: Mouse Assay, Labeling, Flow Cytometry, Cytometry

    After E. coli infection, the expression of activators in monocytes decreased and the activation of effector T cells declined in TSC1 KO mice. (a) 5 ∗ 10 7 E. coli (ATCC, USA) were injected into each mouse through the tail vein for infection. Eight hours postinfection, the percentage and number of activated effector CD4+ T cells decreased in LNs and spl of TSC1 KO mice compared to WT mice. (b) 5 ∗ 10 7 E. coli (ATCC, USA) were injected into each mouse through the tail vein for infection. Eight hours postinfection, the expression levels of activators, including CD40, CD80, CD86, MHC-II, and CD14 in monocytes, decrease in TSC1 KO mice than WT mice. (c) 2 × 10 5 naïve CD4 + T cells (CD4 + CD44 − CD62 + ) were cocultured with 1 × 10 5 sorted monocytes (CD11b + Ly6G − Ly6C + ) from WT or TSC1 KO mice in DMEM with anti-mouse CD3e (10 μ g/mL, BD Biosciences, USA) and CD28 antibody (10 μ g/mL, BD Biosciences, USA) in 96-well flat-bottomed plates for 6 days. The proliferation of naïve CD4 + T cells cocultured with TSC1 KO monocytes was lower than cells cocultured with the WT group. ∗ P
    Figure Legend Snippet: After E. coli infection, the expression of activators in monocytes decreased and the activation of effector T cells declined in TSC1 KO mice. (a) 5 ∗ 10 7 E. coli (ATCC, USA) were injected into each mouse through the tail vein for infection. Eight hours postinfection, the percentage and number of activated effector CD4+ T cells decreased in LNs and spl of TSC1 KO mice compared to WT mice. (b) 5 ∗ 10 7 E. coli (ATCC, USA) were injected into each mouse through the tail vein for infection. Eight hours postinfection, the expression levels of activators, including CD40, CD80, CD86, MHC-II, and CD14 in monocytes, decrease in TSC1 KO mice than WT mice. (c) 2 × 10 5 naïve CD4 + T cells (CD4 + CD44 − CD62 + ) were cocultured with 1 × 10 5 sorted monocytes (CD11b + Ly6G − Ly6C + ) from WT or TSC1 KO mice in DMEM with anti-mouse CD3e (10 μ g/mL, BD Biosciences, USA) and CD28 antibody (10 μ g/mL, BD Biosciences, USA) in 96-well flat-bottomed plates for 6 days. The proliferation of naïve CD4 + T cells cocultured with TSC1 KO monocytes was lower than cells cocultured with the WT group. ∗ P

    Techniques Used: Infection, Expressing, Activation Assay, Mouse Assay, Injection

    25) Product Images from "An Id2RFP-Reporter Mouse Redefines Innate Lymphoid Cell Precursor Potentials"

    Article Title: An Id2RFP-Reporter Mouse Redefines Innate Lymphoid Cell Precursor Potentials

    Journal: Immunity

    doi: 10.1016/j.immuni.2019.02.022

    Id2 + Zbtb16 + ILCPs Retain NK-Cell Potential (A) Reconstitution of ILC compartments in mice adoptively transferred with Id2 + Zbtb16 – or Id2 + Zbtb16 + ILCPs (n = 4; error bars represent standard error of the mean). (B) Spleen and liver FACS analysis for ILC1s in mice adoptively transferred with Id2 + Zbtb16 – or Id2 + Zbtb16 + ILCPs. Data are from one experiment representative of two independent experiments. (C) In vitro differentiation of Id2 + Zbtb16 + ILCPs on OP9 or OP9-DL4 cells. Cells were cultured during 15 days with SCF and IL-7 or with IL-33 and/or IL-2 and IL-23. (D) Analysis of perforin and granzyme B expression in NK cells derived from bulk culture of 200 Id2 + Zbtb16 + ILCPs. Cells were cultured during 7 days on OP9 cells with SCF and IL-7 and supplemented for 1 day with IL-12 and IL-15. Data are from one experiment representative of two independent experiments, each including technical triplicates.
    Figure Legend Snippet: Id2 + Zbtb16 + ILCPs Retain NK-Cell Potential (A) Reconstitution of ILC compartments in mice adoptively transferred with Id2 + Zbtb16 – or Id2 + Zbtb16 + ILCPs (n = 4; error bars represent standard error of the mean). (B) Spleen and liver FACS analysis for ILC1s in mice adoptively transferred with Id2 + Zbtb16 – or Id2 + Zbtb16 + ILCPs. Data are from one experiment representative of two independent experiments. (C) In vitro differentiation of Id2 + Zbtb16 + ILCPs on OP9 or OP9-DL4 cells. Cells were cultured during 15 days with SCF and IL-7 or with IL-33 and/or IL-2 and IL-23. (D) Analysis of perforin and granzyme B expression in NK cells derived from bulk culture of 200 Id2 + Zbtb16 + ILCPs. Cells were cultured during 7 days on OP9 cells with SCF and IL-7 and supplemented for 1 day with IL-12 and IL-15. Data are from one experiment representative of two independent experiments, each including technical triplicates.

    Techniques Used: Mouse Assay, FACS, In Vitro, Cell Culture, Expressing, Derivative Assay

    Bcl11b Marks Emergence of an ILC2-Restricted Precursor (A) Flow cytometry characterization of Lin – CD127 + CD25 – ICOS – CD117 + CD135 – α 4 β 7 + cells from Id2 RFP Zbtb16 GFPcre Bcl11b tdTomato mice. (B) Flow cytometry analysis of surface marker and transcription factor expression on Id2 RFP ILCPs according to their expression of Zbtb16 and Bcl11b . (C) Flow cytometry analysis of RORγt and CD27 expression on Id2 RFP ILCPs according to their expression of Zbtb16 and Bcl11b . Data are from one experiment representative of two independent experiments. (D) Flow cytometry analysis for mature ILCs after bulk culture of Id2 + Zbtb16 – Bcl11b – , Id2 + Zbtb16 + Bcl11b – , Id2 + Zbtb16 + Bcl11b + , or Id2 + Zbtb16 – Bcl11b + ILCPs for 7 days on OP9 cells with SCF, IL-7, and IL-33. Data are from one experiment representative of three independent experiments. (E) Percentage of mature ILC subsets among CD45 + cells for (D). (F) In vitro differentiation of single Id2 + Zbtb16 – Bcl11b – , Id2 + Zbtb16 + Bcl11b – , Id2 + Zbtb16 + Bcl11b + , or Id2 + Zbtb16 – Bcl11b + .
    Figure Legend Snippet: Bcl11b Marks Emergence of an ILC2-Restricted Precursor (A) Flow cytometry characterization of Lin – CD127 + CD25 – ICOS – CD117 + CD135 – α 4 β 7 + cells from Id2 RFP Zbtb16 GFPcre Bcl11b tdTomato mice. (B) Flow cytometry analysis of surface marker and transcription factor expression on Id2 RFP ILCPs according to their expression of Zbtb16 and Bcl11b . (C) Flow cytometry analysis of RORγt and CD27 expression on Id2 RFP ILCPs according to their expression of Zbtb16 and Bcl11b . Data are from one experiment representative of two independent experiments. (D) Flow cytometry analysis for mature ILCs after bulk culture of Id2 + Zbtb16 – Bcl11b – , Id2 + Zbtb16 + Bcl11b – , Id2 + Zbtb16 + Bcl11b + , or Id2 + Zbtb16 – Bcl11b + ILCPs for 7 days on OP9 cells with SCF, IL-7, and IL-33. Data are from one experiment representative of three independent experiments. (E) Percentage of mature ILC subsets among CD45 + cells for (D). (F) In vitro differentiation of single Id2 + Zbtb16 – Bcl11b – , Id2 + Zbtb16 + Bcl11b – , Id2 + Zbtb16 + Bcl11b + , or Id2 + Zbtb16 – Bcl11b + .

    Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Marker, Expressing, In Vitro

    Id2 + Zbtb16 + ILCPs Derive from Id2 + Zbtb16 − Cells with Loss of ILC3 Potential (A) Single-cell multiplex qPCR ordered by hierarchical clustering of BM CLPs and Id2 + Zbtb16 – and Id2 + Zbtb16 + ILCPs. (B) RFP and GFP expression on Id2 + Zbtb16 – ILCPs cultured for 48 h on OP9 cells with SCF and IL-7. Data are from one experiment representative of three independent experiments. (C) Flow cytometry analysis for ILC3s after culture of Id2 + Zbtb16 – ILCPs and Id2 + Zbtb16 + ILCPs or 7 days on OP9 or OP9-DL4 cells with SCF and IL-7. Data are from one experiment representative of two independent experiments, each including technical duplicates. (D) Percentage of ILC3s among CD45 + cells after 7 days’ culture of Id2 + Zbtb16 – and Id2 + Zbtb16 + ILCPs on OP9 or OP9-DL4 cells with SCF and IL-7. n = 3 or 4; error bars represent standard error of the mean; ∗ p
    Figure Legend Snippet: Id2 + Zbtb16 + ILCPs Derive from Id2 + Zbtb16 − Cells with Loss of ILC3 Potential (A) Single-cell multiplex qPCR ordered by hierarchical clustering of BM CLPs and Id2 + Zbtb16 – and Id2 + Zbtb16 + ILCPs. (B) RFP and GFP expression on Id2 + Zbtb16 – ILCPs cultured for 48 h on OP9 cells with SCF and IL-7. Data are from one experiment representative of three independent experiments. (C) Flow cytometry analysis for ILC3s after culture of Id2 + Zbtb16 – ILCPs and Id2 + Zbtb16 + ILCPs or 7 days on OP9 or OP9-DL4 cells with SCF and IL-7. Data are from one experiment representative of two independent experiments, each including technical duplicates. (D) Percentage of ILC3s among CD45 + cells after 7 days’ culture of Id2 + Zbtb16 – and Id2 + Zbtb16 + ILCPs on OP9 or OP9-DL4 cells with SCF and IL-7. n = 3 or 4; error bars represent standard error of the mean; ∗ p

    Techniques Used: Multiplex Assay, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Flow Cytometry, Cytometry

    26) Product Images from "Stimulating Respiratory Activity Primes Anaerobically Grown Listeria monocytogenes for Subsequent Intracellular Infections"

    Article Title: Stimulating Respiratory Activity Primes Anaerobically Grown Listeria monocytogenes for Subsequent Intracellular Infections

    Journal: Pathogens

    doi: 10.3390/pathogens7040096

    Fumarate supplementation results in enhanced L. monocytogenes anaerobic respiratory activity. L. monocytogenes was grown overnight (16–20 h) aerobically or anaerobically in BHI at 37 °C with or without supplementation of fumarate (50 mM). Culture optical density was measured at 600 nm ( a ). The pH ( b ) and acetoin concentration ( c ) were determined in culture supernatant. Reduction of tetrazolium salt ( d ) was determined in washed bacterial pellets. Relative intracellular ATP levels ( e ) was quantified using a luciferase-based assay. Averages of triplicates were plotted with error bars representing standard deviation. Data represent at least three independent experiments. Significant differences (*, p
    Figure Legend Snippet: Fumarate supplementation results in enhanced L. monocytogenes anaerobic respiratory activity. L. monocytogenes was grown overnight (16–20 h) aerobically or anaerobically in BHI at 37 °C with or without supplementation of fumarate (50 mM). Culture optical density was measured at 600 nm ( a ). The pH ( b ) and acetoin concentration ( c ) were determined in culture supernatant. Reduction of tetrazolium salt ( d ) was determined in washed bacterial pellets. Relative intracellular ATP levels ( e ) was quantified using a luciferase-based assay. Averages of triplicates were plotted with error bars representing standard deviation. Data represent at least three independent experiments. Significant differences (*, p

    Techniques Used: Activity Assay, Concentration Assay, Luciferase, Standard Deviation

    Modulations of respiratory activity alter production of LLO and actin co-localization. L. monocytogenes was grown overnight at 37 °C in BHI. Culture supernatant was used to quantify LLO activity using a hemolytic assay ( a , b ). Averages of triplicates were plotted with error bars representing standard deviation. Data represent three independent experiments. LLO abundance in culture supernatant was determined using immunoblotting ( c , left ) followed by pixel density analysis ( c , right ). Blot shown is representative of six independent experiments. (rLLO, recombinant LLO control; NS, no supplement; SB, sample buffer; FUM, fumarate-treated L. monocytogenes ; CCCP, CCCP-treated L. monocytogenes .) Averages of pixel density from six blots were plotted with error bars representing standard error of the mean (n = 6). RAW264.7 macrophages were infected with overnight L. monocytogenes for 30 min at a MOI of 10. Actin colocalization was determined at 2 h post infection (hpi). BD, or below detection, indicates a lack of colocalization. Significant differences (*, p
    Figure Legend Snippet: Modulations of respiratory activity alter production of LLO and actin co-localization. L. monocytogenes was grown overnight at 37 °C in BHI. Culture supernatant was used to quantify LLO activity using a hemolytic assay ( a , b ). Averages of triplicates were plotted with error bars representing standard deviation. Data represent three independent experiments. LLO abundance in culture supernatant was determined using immunoblotting ( c , left ) followed by pixel density analysis ( c , right ). Blot shown is representative of six independent experiments. (rLLO, recombinant LLO control; NS, no supplement; SB, sample buffer; FUM, fumarate-treated L. monocytogenes ; CCCP, CCCP-treated L. monocytogenes .) Averages of pixel density from six blots were plotted with error bars representing standard error of the mean (n = 6). RAW264.7 macrophages were infected with overnight L. monocytogenes for 30 min at a MOI of 10. Actin colocalization was determined at 2 h post infection (hpi). BD, or below detection, indicates a lack of colocalization. Significant differences (*, p

    Techniques Used: Activity Assay, Hemolytic Assay, Standard Deviation, Recombinant, Infection

    Inhibition of aerobic respiratory activity with CCCP phenocopies anaerobically grown L. monocytogenes . L. monocytogenes was grown at 37 °C in BHI with or without 1 µM CCCP. Culture optical density was measured at 600 nm. ( a ) pH was measured in culture supernatant. ( b ) While reduction of tetrazolium salts was measured in washed bacterial pellets. ( c ) Bacteria from overnight cultures were used to infect RAW264.7 macrophages for 30 min at an MOI of 10 where intracellular colony forming units (CFU) was determined and compared to the CFU in inoculum. ( d ). Averages of triplicates were plotted with error bars representing standard deviation. Data represent three independent experiments. Significant differences (**, 0.001
    Figure Legend Snippet: Inhibition of aerobic respiratory activity with CCCP phenocopies anaerobically grown L. monocytogenes . L. monocytogenes was grown at 37 °C in BHI with or without 1 µM CCCP. Culture optical density was measured at 600 nm. ( a ) pH was measured in culture supernatant. ( b ) While reduction of tetrazolium salts was measured in washed bacterial pellets. ( c ) Bacteria from overnight cultures were used to infect RAW264.7 macrophages for 30 min at an MOI of 10 where intracellular colony forming units (CFU) was determined and compared to the CFU in inoculum. ( d ). Averages of triplicates were plotted with error bars representing standard deviation. Data represent three independent experiments. Significant differences (**, 0.001

    Techniques Used: Inhibition, Activity Assay, Standard Deviation

    Prior fumarate treatments enhance cell-cell spread. Overnight cultures of L. monocytogenes were washed and used to infect fibroblast monolayer cells for 1 h. After 72 h of incubation, plaque sizes were measured by neutral red staining. Diameters of at least 30 plaques were measured. ( a ) Fumarate (50 mM) or CCCP (1 µM) was supplemented only in the growth of the bacterial inoculum. ( b ) Compared to no fumarate control (NS_NS), fumarate was added only to fibroblasts prior to infection (NS_FUM) or to both the growth of L. monocytogenes and fibroblasts prior to infection (FUM_FUM). No exogenous fumarate was added during infection. Fumarate supplementation in both L. monocytogenes and fibroblasts prior to infection resulted in complete lysis where plaque sizes were not determinable (CL, Complete Lysis). Significant differences (***, p
    Figure Legend Snippet: Prior fumarate treatments enhance cell-cell spread. Overnight cultures of L. monocytogenes were washed and used to infect fibroblast monolayer cells for 1 h. After 72 h of incubation, plaque sizes were measured by neutral red staining. Diameters of at least 30 plaques were measured. ( a ) Fumarate (50 mM) or CCCP (1 µM) was supplemented only in the growth of the bacterial inoculum. ( b ) Compared to no fumarate control (NS_NS), fumarate was added only to fibroblasts prior to infection (NS_FUM) or to both the growth of L. monocytogenes and fibroblasts prior to infection (FUM_FUM). No exogenous fumarate was added during infection. Fumarate supplementation in both L. monocytogenes and fibroblasts prior to infection resulted in complete lysis where plaque sizes were not determinable (CL, Complete Lysis). Significant differences (***, p

    Techniques Used: Incubation, Staining, Infection, Lysis

    27) Product Images from "Differential Susceptibility of Human Peripheral Blood T Cells to Suppression by Environmental Levels of Sodium Arsenite and Monomethylarsonous Acid"

    Article Title: Differential Susceptibility of Human Peripheral Blood T Cells to Suppression by Environmental Levels of Sodium Arsenite and Monomethylarsonous Acid

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109192

    Proliferation response of PHA and anti-CD3/anti-CD28 stimulated PBMC treated with As +3 . Comparison of PHA stimulated (A) and anti-CD3/anti-CD28 stimulated (B) T cell proliferation ( 3 H-thymidine) for potential As +3 inhibition in 17 donors. Data shown are the Mean (red line) and Median (black line) ± SD with *p
    Figure Legend Snippet: Proliferation response of PHA and anti-CD3/anti-CD28 stimulated PBMC treated with As +3 . Comparison of PHA stimulated (A) and anti-CD3/anti-CD28 stimulated (B) T cell proliferation ( 3 H-thymidine) for potential As +3 inhibition in 17 donors. Data shown are the Mean (red line) and Median (black line) ± SD with *p

    Techniques Used: Inhibition

    As +3 treated compared to MMA +3 treated PBMC stimulated with either PHA or anti-CD3/anti-CD28. Comparison of As +3 inhibition for PHA (A) and anti-CD3/anti-CD8 (C) and MMA +3 inhibition of T cell proliferation induced by PHA (B) and anti-CD3/anti-CD28 (D) in 8 donors. Data shown are the Mean (red line) and Median (black line) ± SD with *p
    Figure Legend Snippet: As +3 treated compared to MMA +3 treated PBMC stimulated with either PHA or anti-CD3/anti-CD28. Comparison of As +3 inhibition for PHA (A) and anti-CD3/anti-CD8 (C) and MMA +3 inhibition of T cell proliferation induced by PHA (B) and anti-CD3/anti-CD28 (D) in 8 donors. Data shown are the Mean (red line) and Median (black line) ± SD with *p

    Techniques Used: Inhibition

    28) Product Images from "Ly108 Expression Distinguishes Subsets of invariant NKT cells that Help Autoantibody Production and Secrete IL-21 from those that Secrete IL-17 in Lupus Prone NZB/W Mice"

    Article Title: Ly108 Expression Distinguishes Subsets of invariant NKT cells that Help Autoantibody Production and Secrete IL-21 from those that Secrete IL-17 in Lupus Prone NZB/W Mice

    Journal: Journal of autoimmunity

    doi: 10.1016/j.jaut.2014.01.002

    CD4 + Ly108 hi i NKT cells provide help for spontaneous antibody secretion of B cells. Sorted B cells with or without sorted i NKT cells, were cultured for 10 days, then supernatant were collected for determination of IgM, IgG and IgG anti-dsDNA antibody concentrations. For IL-21 production assays, cells were sorted and cultured (5X10 5 /ml) for 3 days with anti-CD3 and CD28 mAbs. Different populations of i NKT cells were sorted from three pooled spleens of BW mice. (A) IgM, IgG and IgG anti-dsDNA antibody production from cultures of B cells, with or without sorted Ly108 hi or Ly108 lo i NKT cells from 6 month old BW mice. (B) IL-21 production by anti-CD3/CD28 stimulated i NKT and Tcon cells of young BW mice. (C) IgM, IgG and IgG anti-dsDNA antibody production from B cells cultures, with or without sorted CD4 + Ly108 hi , CD4 + Ly108 lo or CD4 − Ly108 lo i NKT cells from 6 month old BW mice. (D) IL-21 production by sorted CD4 + Ly108 hi , CD4 + Ly108 lo or CD4- Ly108 lo i NKT from young BW mice. ND, Not Detected. *, P
    Figure Legend Snippet: CD4 + Ly108 hi i NKT cells provide help for spontaneous antibody secretion of B cells. Sorted B cells with or without sorted i NKT cells, were cultured for 10 days, then supernatant were collected for determination of IgM, IgG and IgG anti-dsDNA antibody concentrations. For IL-21 production assays, cells were sorted and cultured (5X10 5 /ml) for 3 days with anti-CD3 and CD28 mAbs. Different populations of i NKT cells were sorted from three pooled spleens of BW mice. (A) IgM, IgG and IgG anti-dsDNA antibody production from cultures of B cells, with or without sorted Ly108 hi or Ly108 lo i NKT cells from 6 month old BW mice. (B) IL-21 production by anti-CD3/CD28 stimulated i NKT and Tcon cells of young BW mice. (C) IgM, IgG and IgG anti-dsDNA antibody production from B cells cultures, with or without sorted CD4 + Ly108 hi , CD4 + Ly108 lo or CD4 − Ly108 lo i NKT cells from 6 month old BW mice. (D) IL-21 production by sorted CD4 + Ly108 hi , CD4 + Ly108 lo or CD4- Ly108 lo i NKT from young BW mice. ND, Not Detected. *, P

    Techniques Used: Cell Culture, Mouse Assay

    BW i NKT cells, produce significantly higher amounts of IL-4 and IL-17 than Tcon cells and Ly108 lo i NKT cells are predominant IL-17 producing i NKT cells. Cells were sorted from three pooled spleens of young mice and cultured (5X10 5 /ml) for 3 days with or without plate bound anti-CD3 and CD28 mAbs, culture supernatants were then collected for IFNγ, IL-4 and IL-17 measurement by ELISA or Luminex. (A) IFNγ, IL-4 and IL-17 concentrations in supernatants of stimulated i NKT and Tcon cells from BW mice. Cytokines measurements are shown on the right. (B) IFNγ, IL-4 and IL-17 production by BW CD4 + Ly108 hi , CD4 + Ly108 lo and CD4 − Ly108 lo i NKT cells. Sorting gates are shown on the left, and cytokines measurements were shown on the right. (C) IFNγ, IL-4 and IL-17 production by BW NK1.1 + and NK1.1 − i NKT cells. FACS profile is shown for the sorting gates on the left, and cytokines measurements are shown on the right. (D) IFNγ, IL-4 and IL-17 production by BW NK1.1 − Ly108 hi and NK1.1 − Ly108 lo i NKT cells. FACS profile is shown for the sort gates on the left, and cytokines measurements are shown on the right. Group differences with P > 0.05 was not considered statistically significant (ns). *, P
    Figure Legend Snippet: BW i NKT cells, produce significantly higher amounts of IL-4 and IL-17 than Tcon cells and Ly108 lo i NKT cells are predominant IL-17 producing i NKT cells. Cells were sorted from three pooled spleens of young mice and cultured (5X10 5 /ml) for 3 days with or without plate bound anti-CD3 and CD28 mAbs, culture supernatants were then collected for IFNγ, IL-4 and IL-17 measurement by ELISA or Luminex. (A) IFNγ, IL-4 and IL-17 concentrations in supernatants of stimulated i NKT and Tcon cells from BW mice. Cytokines measurements are shown on the right. (B) IFNγ, IL-4 and IL-17 production by BW CD4 + Ly108 hi , CD4 + Ly108 lo and CD4 − Ly108 lo i NKT cells. Sorting gates are shown on the left, and cytokines measurements were shown on the right. (C) IFNγ, IL-4 and IL-17 production by BW NK1.1 + and NK1.1 − i NKT cells. FACS profile is shown for the sorting gates on the left, and cytokines measurements are shown on the right. (D) IFNγ, IL-4 and IL-17 production by BW NK1.1 − Ly108 hi and NK1.1 − Ly108 lo i NKT cells. FACS profile is shown for the sort gates on the left, and cytokines measurements are shown on the right. Group differences with P > 0.05 was not considered statistically significant (ns). *, P

    Techniques Used: Mouse Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Luminex, FACS

    NK1.1 − Ly108 lo Nrp-1 + i NKT cells are IL-17 producing iNKT cells. (A) Ly108 and Nrp-1 expression on gated NK1.1 + and NK1.1 − i NKT cells from young B6, Sle1b and BW mice. (B) IFNγ, IL-4 and IL-17 production by sorted BW NK1.1 − Ly108 hi Nrp-1 + (indicated as population 1 as shown in A), NK1.1 − Ly108 lo Nrp-1 + (population 2), NK1.1 − Ly108 lo Nrp-1 − (population 3), NK1.1 − Ly108 hi Nrp-1 − (population 4) i NKT cells. Cells were sorted from three to five pooled spleens from young BW mice and cultured (5X10 5 /ml) for 3 days with anti-CD3 and CD28 mAbs. (C) Percentages of IL-17 producing i NKT cells, defined as NK1.1 − Ly108 lo Nrp-1 + TCR + CD1dTet + population, among live lymphocytes of B6, Sle1b and BW mice. nd, not detected. Group differences with P > 0.05 was not considered statistically significant (ns); ***, P
    Figure Legend Snippet: NK1.1 − Ly108 lo Nrp-1 + i NKT cells are IL-17 producing iNKT cells. (A) Ly108 and Nrp-1 expression on gated NK1.1 + and NK1.1 − i NKT cells from young B6, Sle1b and BW mice. (B) IFNγ, IL-4 and IL-17 production by sorted BW NK1.1 − Ly108 hi Nrp-1 + (indicated as population 1 as shown in A), NK1.1 − Ly108 lo Nrp-1 + (population 2), NK1.1 − Ly108 lo Nrp-1 − (population 3), NK1.1 − Ly108 hi Nrp-1 − (population 4) i NKT cells. Cells were sorted from three to five pooled spleens from young BW mice and cultured (5X10 5 /ml) for 3 days with anti-CD3 and CD28 mAbs. (C) Percentages of IL-17 producing i NKT cells, defined as NK1.1 − Ly108 lo Nrp-1 + TCR + CD1dTet + population, among live lymphocytes of B6, Sle1b and BW mice. nd, not detected. Group differences with P > 0.05 was not considered statistically significant (ns); ***, P

    Techniques Used: Expressing, Mouse Assay, Cell Culture

    29) Product Images from "Lipoplex-based targeted gene therapy for the suppression of tumours with VEGFR expression by producing anti-angiogenic molecules"

    Article Title: Lipoplex-based targeted gene therapy for the suppression of tumours with VEGFR expression by producing anti-angiogenic molecules

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/s12951-020-00610-9

    Targeting ability and Targeted transfection ability of the LPPC/RBDV complexes in vivo. a Tumour-bearing mice were i.v. injected with LPPC/DiO/RBDV or LPPC/DiO/IgG1 Fc. After 72 h, the organs and tumours were excised and imaged with an IVIS system at the appropriate wavelength (Em: 600 nm and Ex: 465 nm). b Tumour-bearing mice were i.v. injected with LPPC/pAsRed/RBDV or LPPC/pAsRed/IgG1 Fc. After 6 days, the organs and tumours were excised and imaged with an IVIS system at the appropriate wavelength (Em: 560 nm and Ex: 465 nm). c The tumours and organs were further processed by H E staining and photographed with a fluorescence microscope. LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG
    Figure Legend Snippet: Targeting ability and Targeted transfection ability of the LPPC/RBDV complexes in vivo. a Tumour-bearing mice were i.v. injected with LPPC/DiO/RBDV or LPPC/DiO/IgG1 Fc. After 72 h, the organs and tumours were excised and imaged with an IVIS system at the appropriate wavelength (Em: 600 nm and Ex: 465 nm). b Tumour-bearing mice were i.v. injected with LPPC/pAsRed/RBDV or LPPC/pAsRed/IgG1 Fc. After 6 days, the organs and tumours were excised and imaged with an IVIS system at the appropriate wavelength (Em: 560 nm and Ex: 465 nm). c The tumours and organs were further processed by H E staining and photographed with a fluorescence microscope. LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG

    Techniques Used: Transfection, In Vivo, Mouse Assay, Injection, Staining, Fluorescence, Microscopy

    Inhibition of tumour growth by LPPC/pRBDV/RBDV and the RBDV protein. Female C57BL/6 mice (6–8 weeks of age) were subcutaneously inoculated with 1 × 10 6 cells in 100 mL of PBS. When the average tumour volume reached 30 mm 3 , the mice were i.v. injected with LPPC/pRBDV/RBDV or RBDV protein. Inverted filled triangle means the day of complex injection. Tumour volume was measured every 2 days after injection of the complexes, and the mice were sacrificed when the tumour grew to a size of 2500 mm 3 . LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG. The data represent the mean ± SD (n = 7). Significant differences are evaluated by Student’s t -test and are labelled as *P
    Figure Legend Snippet: Inhibition of tumour growth by LPPC/pRBDV/RBDV and the RBDV protein. Female C57BL/6 mice (6–8 weeks of age) were subcutaneously inoculated with 1 × 10 6 cells in 100 mL of PBS. When the average tumour volume reached 30 mm 3 , the mice were i.v. injected with LPPC/pRBDV/RBDV or RBDV protein. Inverted filled triangle means the day of complex injection. Tumour volume was measured every 2 days after injection of the complexes, and the mice were sacrificed when the tumour grew to a size of 2500 mm 3 . LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG. The data represent the mean ± SD (n = 7). Significant differences are evaluated by Student’s t -test and are labelled as *P

    Techniques Used: Inhibition, Mouse Assay, Injection

    In vivo expression of RBDV or IgG1 Fc. The LPPC/RBDV-IgG1 Fc protein complexes, LPPC/IgG1 Fc plasmid/RBDV-IgG1 Fc protein complexes, LPPC/RBDV-IgG1 Fc plasmid/IgG1 Fc protein complexes, LPPC/RBDV-IgG1 Fc plasmid/RBDV-IgG1 Fc protein complexes and PBS were i.v. injected into mice. Mouse sera were collected at different times post-administration and analysed by ELISA. LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG. The data represent the mean ± SD (n = 3). Significant differences were evaluated by ANOVA with the Bonferroni test
    Figure Legend Snippet: In vivo expression of RBDV or IgG1 Fc. The LPPC/RBDV-IgG1 Fc protein complexes, LPPC/IgG1 Fc plasmid/RBDV-IgG1 Fc protein complexes, LPPC/RBDV-IgG1 Fc plasmid/IgG1 Fc protein complexes, LPPC/RBDV-IgG1 Fc plasmid/RBDV-IgG1 Fc protein complexes and PBS were i.v. injected into mice. Mouse sera were collected at different times post-administration and analysed by ELISA. LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG. The data represent the mean ± SD (n = 3). Significant differences were evaluated by ANOVA with the Bonferroni test

    Techniques Used: In Vivo, Expressing, Plasmid Preparation, Injection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of tumour growth by the LPPC/RBDV-IgG1 Fc plasmid/RBDV-IgG1 Fc protein. a Female C57BL/6 mice (6–8 weeks of age) were subcutaneously inoculated with 1 × 10 6 cells in 100 mL of PBS. When the average tumour volume reached 30 mm 3 , the mice were i.v. injected with LPPC/RBDV-IgG1 Fc plasmid/RBDV-IgG1 Fc protein or other groups and injected again after four days. Inverted filled triangle means the day of complex injection. Tumour volume was measured every 2 days after injecting the complexes, and the mice were sacrificed when the tumour grew to a size of 2500 mm 3 . The data represent the mean tumour volume ± SD (n = 7). b The survival rate of C57BL/6 mice bearing B16-F10 tumours treated with LPPC/RBDV-IgG1 Fc plasmid/RBDV-IgG1 Fc protein or other groups. LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG
    Figure Legend Snippet: Inhibition of tumour growth by the LPPC/RBDV-IgG1 Fc plasmid/RBDV-IgG1 Fc protein. a Female C57BL/6 mice (6–8 weeks of age) were subcutaneously inoculated with 1 × 10 6 cells in 100 mL of PBS. When the average tumour volume reached 30 mm 3 , the mice were i.v. injected with LPPC/RBDV-IgG1 Fc plasmid/RBDV-IgG1 Fc protein or other groups and injected again after four days. Inverted filled triangle means the day of complex injection. Tumour volume was measured every 2 days after injecting the complexes, and the mice were sacrificed when the tumour grew to a size of 2500 mm 3 . The data represent the mean tumour volume ± SD (n = 7). b The survival rate of C57BL/6 mice bearing B16-F10 tumours treated with LPPC/RBDV-IgG1 Fc plasmid/RBDV-IgG1 Fc protein or other groups. LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG

    Techniques Used: Inhibition, Plasmid Preparation, Mouse Assay, Injection

    The binding activities of the LPPC/DiO/RBDV-IgG1 Fc complexes. a BALB/3T3 cells and b B16-F10 cells were stained with DiO-labelled LPPC complexes without PEG 1.500 complexed. c BALB/3T3 cells and d B16-F10 cells were stained with PEG-complexed LPPC/DiO complexes. e DiO-labelled LPPC complexes (50 μg) were incubated with different amounts of RBDV-IgG1 Fc or IgG1 Fc proteins for 30 min, and then the complexes were added to B16-F10 cells. The binding intensity was analysed by flow cytometry. The data represent the mean ± SD (n = 6). Significant differences are evaluated by Student’s t -test and are labelled as *P
    Figure Legend Snippet: The binding activities of the LPPC/DiO/RBDV-IgG1 Fc complexes. a BALB/3T3 cells and b B16-F10 cells were stained with DiO-labelled LPPC complexes without PEG 1.500 complexed. c BALB/3T3 cells and d B16-F10 cells were stained with PEG-complexed LPPC/DiO complexes. e DiO-labelled LPPC complexes (50 μg) were incubated with different amounts of RBDV-IgG1 Fc or IgG1 Fc proteins for 30 min, and then the complexes were added to B16-F10 cells. The binding intensity was analysed by flow cytometry. The data represent the mean ± SD (n = 6). Significant differences are evaluated by Student’s t -test and are labelled as *P

    Techniques Used: Binding Assay, Staining, Incubation, Flow Cytometry

    In vitro transfection efficiency of the LPPC/DNA/RBDV complexes. Different concentrations of a RBDV complexes or b IgG1 Fc with 5 μg of DNA and 50 μg of LPPC were transfected into B16-F10 cells, and the cells were observed under a microscope 48 h after transfection. LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG
    Figure Legend Snippet: In vitro transfection efficiency of the LPPC/DNA/RBDV complexes. Different concentrations of a RBDV complexes or b IgG1 Fc with 5 μg of DNA and 50 μg of LPPC were transfected into B16-F10 cells, and the cells were observed under a microscope 48 h after transfection. LPPC, which encapsulated with RBDV or IgG1 Fc, were all complexed by PEG

    Techniques Used: In Vitro, Transfection, Microscopy

    30) Product Images from "Automated Quantification of Mitochondrial Fragmentation in an In-Vitro Parkinson’s Disease Model"

    Article Title: Automated Quantification of Mitochondrial Fragmentation in an In-Vitro Parkinson’s Disease Model

    Journal: bioRxiv

    doi: 10.1101/2020.05.15.093369

    Identify SN4741 Cytoplasm as Tertiary Object. Images ( A ), ( C ) and ( F ) illustrate the masked image result of sequential steps/modules from identification of primary object a (Nuclei), identification of secondary object (cell body) and tertiary object f (cell cytoplasm), respectively. SN4741 cytoplasm is the result of removing the Nuclei areas from the areas of entire cell bodies. Micro-pictograph ( G ) highlights the outlines of the resulting SN4741 cell cytoplasm. Dashed white boxed in images ( A ), ( C ), ( E ) and ( G ) indicated the areas which have been enlarged for illustrative purposes and are represented by images ( B ), ( D ), ( F ) and ( H ), respectively. Images ( A - H ) are micro-pictograms acquired and saved from the CellProfiler software.
    Figure Legend Snippet: Identify SN4741 Cytoplasm as Tertiary Object. Images ( A ), ( C ) and ( F ) illustrate the masked image result of sequential steps/modules from identification of primary object a (Nuclei), identification of secondary object (cell body) and tertiary object f (cell cytoplasm), respectively. SN4741 cytoplasm is the result of removing the Nuclei areas from the areas of entire cell bodies. Micro-pictograph ( G ) highlights the outlines of the resulting SN4741 cell cytoplasm. Dashed white boxed in images ( A ), ( C ), ( E ) and ( G ) indicated the areas which have been enlarged for illustrative purposes and are represented by images ( B ), ( D ), ( F ) and ( H ), respectively. Images ( A - H ) are micro-pictograms acquired and saved from the CellProfiler software.

    Techniques Used: Software

    Identify SN4741 Cell Body as Secondary Object. Figure 6 ( A ) shows the input Illumination Corrected x40 magnification Cy3 channel image. ( C ) Illustrates the result of propagation method of identifying cell (body) edge with respect to Cy3 image staining intensity thresholding. Identified secondary objects/Cell bodies are illustrated in the form of coloured masked objects. Image ( E ) shows the input Cy3 channel image with identified cell body outlines in magenta and nuclei outlines in green. Dashed white line box in ( E ) highlights the area enlarged to produce image ( F ). White box in image ( F ) indicates enlarged area to produce image ( G ). Images ( F ) and ( G ) were generated to illustrate image segmentation. Dashed white line boxes in ( A ) and ( C ) highlight areas that are enlarged and illustrated in ( B ) and ( D ), respectively. Images ( A - G ) are micro pictograms acquired and saved from the CellProfiler software.
    Figure Legend Snippet: Identify SN4741 Cell Body as Secondary Object. Figure 6 ( A ) shows the input Illumination Corrected x40 magnification Cy3 channel image. ( C ) Illustrates the result of propagation method of identifying cell (body) edge with respect to Cy3 image staining intensity thresholding. Identified secondary objects/Cell bodies are illustrated in the form of coloured masked objects. Image ( E ) shows the input Cy3 channel image with identified cell body outlines in magenta and nuclei outlines in green. Dashed white line box in ( E ) highlights the area enlarged to produce image ( F ). White box in image ( F ) indicates enlarged area to produce image ( G ). Images ( F ) and ( G ) were generated to illustrate image segmentation. Dashed white line boxes in ( A ) and ( C ) highlight areas that are enlarged and illustrated in ( B ) and ( D ), respectively. Images ( A - G ) are micro pictograms acquired and saved from the CellProfiler software.

    Techniques Used: Staining, Generated, Software

    Experimental Outline and Associated Timelines. This diagram illustrates the outlines key steps of basic protocol 1 and basic protocol 2. This includes wet-laboratory preparation of SN4741 cells; cell staining process; automated image acquisition; and execution of CellProfiler image analysis software for image segmentation and data output.
    Figure Legend Snippet: Experimental Outline and Associated Timelines. This diagram illustrates the outlines key steps of basic protocol 1 and basic protocol 2. This includes wet-laboratory preparation of SN4741 cells; cell staining process; automated image acquisition; and execution of CellProfiler image analysis software for image segmentation and data output.

    Techniques Used: Staining, Software

    Rotenone-Based Parkinson’s disease model results in Cell Loss, Decreases Viability and Increases SN4741 Cell Cytotoxicity and Mitochondrial Fragmentation. Figure 1 ( A ), ( B ), ( C ) and ( D ) represents SN4741 cell count, cytotoxicity, mitochondrial fragments per cell, and viability data respectively. A decrease in cell number ( A ); increase in cell cytotoxicity ( B ) and mitochondria fragments per cell ( C ); and a decreased cell viability ( D ) is observed following 24hr challenge with 10nM rotenone. SN4741 cell number (A) is expressed as mean cell count from cells treated with in quadruplicate (n=4); SN4741 cytotoxicity, mitochondrial fragmentation and viability data ( B - D ) expressed as mean cell count from cells treated in triplicate for 3 experimental repeats (n=3). Cell Cytotoxicity ( B ) and viability data ( D ) are expressed as percentage of control (0nM). Statistical analysis was performed by One-way ANOVA with Dunnett’s post-hoc analysis ( A - D ) in graph pad prism 6 software, with P
    Figure Legend Snippet: Rotenone-Based Parkinson’s disease model results in Cell Loss, Decreases Viability and Increases SN4741 Cell Cytotoxicity and Mitochondrial Fragmentation. Figure 1 ( A ), ( B ), ( C ) and ( D ) represents SN4741 cell count, cytotoxicity, mitochondrial fragments per cell, and viability data respectively. A decrease in cell number ( A ); increase in cell cytotoxicity ( B ) and mitochondria fragments per cell ( C ); and a decreased cell viability ( D ) is observed following 24hr challenge with 10nM rotenone. SN4741 cell number (A) is expressed as mean cell count from cells treated with in quadruplicate (n=4); SN4741 cytotoxicity, mitochondrial fragmentation and viability data ( B - D ) expressed as mean cell count from cells treated in triplicate for 3 experimental repeats (n=3). Cell Cytotoxicity ( B ) and viability data ( D ) are expressed as percentage of control (0nM). Statistical analysis was performed by One-way ANOVA with Dunnett’s post-hoc analysis ( A - D ) in graph pad prism 6 software, with P

    Techniques Used: Cell Counting, Software

    31) Product Images from "Arsenic exposure associated T cell proliferation, smoking, and vitamin D in Bangladeshi men and women"

    Article Title: Arsenic exposure associated T cell proliferation, smoking, and vitamin D in Bangladeshi men and women

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0234965

    Age and BMI adjusted association of urinary arsenic with T cell proliferation (TCP) in male smokers and non-smokers with sufficient or deficient serum VitD levels. Male Smoker (n = 173) and Male Non-smoker (n = 61) by High/sufficient VitD serum concentration ( > 20 ng/ml), indicated by black solid line, and low/deficient serum VitD (
    Figure Legend Snippet: Age and BMI adjusted association of urinary arsenic with T cell proliferation (TCP) in male smokers and non-smokers with sufficient or deficient serum VitD levels. Male Smoker (n = 173) and Male Non-smoker (n = 61) by High/sufficient VitD serum concentration ( > 20 ng/ml), indicated by black solid line, and low/deficient serum VitD (

    Techniques Used: Concentration Assay

    32) Product Images from "Dual-sensing genetically encoded fluorescent indicators resolve the spatiotemporal coordination of cytosolic abscisic acid and second messenger dynamics in Arabidopsis"

    Article Title: Dual-sensing genetically encoded fluorescent indicators resolve the spatiotemporal coordination of cytosolic abscisic acid and second messenger dynamics in Arabidopsis

    Journal: bioRxiv

    doi: 10.1101/844118

    Development of ABAleonSD1-3L21. (A) FRET-pair and sensory domain, and (B) linker screening of ABA indicator variants after expression in HEK293T cells. Shown are emission ratio changes in response to 60 min treatments with 0 and 100 µM ABA. Reference indicators are shown in cyan and new candidates in red. Information on ABA indicator topologies is given in Supplemental Figure 1 . (C to E) Representative ABA-dependent normalized in vitro emission spectra of ABAleons with indicated maximum emission ratio change (ΔR (max) /R 0 ). (F) ABA-dependent in vitro emission ratios and apparent ABA affinities (k’ d ) of ABAleons. (G and H) Comparison of ABA-dependent ABAleon emission ratios: (G) in vitro and (H) in HEK293T cells. (I) In vitro and HEK293T cell comparison of ABA-induced maximum emission ratio change. All data are shown as mean ± SD, n = 3.
    Figure Legend Snippet: Development of ABAleonSD1-3L21. (A) FRET-pair and sensory domain, and (B) linker screening of ABA indicator variants after expression in HEK293T cells. Shown are emission ratio changes in response to 60 min treatments with 0 and 100 µM ABA. Reference indicators are shown in cyan and new candidates in red. Information on ABA indicator topologies is given in Supplemental Figure 1 . (C to E) Representative ABA-dependent normalized in vitro emission spectra of ABAleons with indicated maximum emission ratio change (ΔR (max) /R 0 ). (F) ABA-dependent in vitro emission ratios and apparent ABA affinities (k’ d ) of ABAleons. (G and H) Comparison of ABA-dependent ABAleon emission ratios: (G) in vitro and (H) in HEK293T cells. (I) In vitro and HEK293T cell comparison of ABA-induced maximum emission ratio change. All data are shown as mean ± SD, n = 3.

    Techniques Used: Expressing, In Vitro

    33) Product Images from "LsrB-based and temperature-dependent identification of bacterial AI-2 receptor"

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    Journal: AMB Express

    doi: 10.1186/s13568-017-0486-y

    Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control
    Figure Legend Snippet: Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Techniques Used: Binding Assay, Purification, Incubation, Activity Assay, Recombinant, Produced, Mutagenesis, Negative Control, Positive Control

    The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as a positive control and E. coli DH5α as a negative control. The figure represents the means of the results from three independent experiments. The error bars indicate standard deviations
    Figure Legend Snippet: The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as a positive control and E. coli DH5α as a negative control. The figure represents the means of the results from three independent experiments. The error bars indicate standard deviations

    Techniques Used: Activity Assay, Mutagenesis, Positive Control, Negative Control

    34) Product Images from "RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells"

    Article Title: RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells

    Journal: Parasites & Vectors

    doi: 10.1186/s13071-016-1944-0

    Effect of knockdown of I. scapularis transcripts on LGTV infection of ISE6 cells. a ISE6 cell viability following transfection with 10ng dsRNA for 60 h and 12 h post-infection (hpi) with LGTV, compared to the pGEM negative control. Results represent 2–5 technical replicates and 3 biological replicates. b Effect of I. scapularis transcript knockdown on LGTV genome replication in ISE6 cells following transfection with 10ng dsRNA for 60 h and 12 hpi with LGTV as compared to the negative control. Comparison of fold change in transcripts for the LGTV negative strand were normalized to the I. scapularis β-tubulin gene and expressed relative to percentage of pGEM control. Results represent 2 technical replicates (each with 3 machine replicates) and 2 biological replicates. c Effect of I. scapularis transcript knockdown on release of infectious LGTV from ISE6 cells transfected with dsRNA for 60 h prior to LGTV infections as assessed by plaque assay and normalized to the pGEM dsRNA negative control. Results represent 5 technical replicates and 2 biological replicates. d Effect of I. scapularis transcript knockdown on release of infectious LGTV from ISE6 cells infected with LGTV and transfected with dsRNA for 60 h. Results represent 2–5 technical replicates and 3 biological replicates. Error bars represent SEM for unpaired t-tests comparing cell viability, negative strand levels or pfu/ml of the negative pGEM control versus each gene of interest. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Abbreviations : FAH, fumarylacetoacetase; ERP29, endoplasmic reticulum protein 29; ALDH, aldehyde dehydrogenase; VNN, carbon-nitrogen hydrolase/vanin-like; MDH2, malate dehydrogenase; PARP, poly [ADP-ribose] polymerase; CMPK, cytidine/urdine monophosphate kinase; ACAT1, acetyl-CoA acetyltransferase; Hypo195 and Hypo576, hypothetical proteins 195 and 576; pGEM, pGEM plasmid negative control ( light gray bars); LGTV 3UTR, 3’ UTR of LGTV positive control ( dark gray bars ); RLU, relative light units
    Figure Legend Snippet: Effect of knockdown of I. scapularis transcripts on LGTV infection of ISE6 cells. a ISE6 cell viability following transfection with 10ng dsRNA for 60 h and 12 h post-infection (hpi) with LGTV, compared to the pGEM negative control. Results represent 2–5 technical replicates and 3 biological replicates. b Effect of I. scapularis transcript knockdown on LGTV genome replication in ISE6 cells following transfection with 10ng dsRNA for 60 h and 12 hpi with LGTV as compared to the negative control. Comparison of fold change in transcripts for the LGTV negative strand were normalized to the I. scapularis β-tubulin gene and expressed relative to percentage of pGEM control. Results represent 2 technical replicates (each with 3 machine replicates) and 2 biological replicates. c Effect of I. scapularis transcript knockdown on release of infectious LGTV from ISE6 cells transfected with dsRNA for 60 h prior to LGTV infections as assessed by plaque assay and normalized to the pGEM dsRNA negative control. Results represent 5 technical replicates and 2 biological replicates. d Effect of I. scapularis transcript knockdown on release of infectious LGTV from ISE6 cells infected with LGTV and transfected with dsRNA for 60 h. Results represent 2–5 technical replicates and 3 biological replicates. Error bars represent SEM for unpaired t-tests comparing cell viability, negative strand levels or pfu/ml of the negative pGEM control versus each gene of interest. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Abbreviations : FAH, fumarylacetoacetase; ERP29, endoplasmic reticulum protein 29; ALDH, aldehyde dehydrogenase; VNN, carbon-nitrogen hydrolase/vanin-like; MDH2, malate dehydrogenase; PARP, poly [ADP-ribose] polymerase; CMPK, cytidine/urdine monophosphate kinase; ACAT1, acetyl-CoA acetyltransferase; Hypo195 and Hypo576, hypothetical proteins 195 and 576; pGEM, pGEM plasmid negative control ( light gray bars); LGTV 3UTR, 3’ UTR of LGTV positive control ( dark gray bars ); RLU, relative light units

    Techniques Used: Infection, Transfection, Negative Control, Plaque Assay, Plasmid Preparation, Positive Control

    dsRNA-mediated knockdown of transcripts for I. scapularis genes of interest in ISE6 cells. Following transfection of ISE6 cells with 10 ng dsRNA for 60 h, total RNA was prepared from ~ 1 × 10 5 cells and cDNA was amplified via a two-step RT-PCR reaction. mRNA levels were normalized to I. scapularis β-actin and expressed relative to the percentage of pGEM control cDNA. Results show relative expression of ten I. scapularis genes of interest following knockdown ( white bars) relative to the pGEM dsRNA negative control ( gray bars). Error bars represent standard error of the mean (SEM). Statistical analysis was performed using an unpaired t-test between the negative pGEM control and dsRNA treatments for each gene of interest. Results represent 2–3 technical replicates (each with 2–3 machine replicates) and 2 biological replicates. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Abbreviations : FAH, fumarylacetoacetase (ISCW020196); ERP29, endoplasmic reticulum protein 29 (ISCW018425); ALDH, aldehyde dehydrogenase (ISCW015982); VNN, carbon-nitrogen hydrolase/vanin-like (ISCW004822); MDH2, malate dehydrogenase (ISCW003528); PARP, poly [ADP-ribose] polymerase (ISCW019519); CMPK, cytidine/uridine monophosphate kinase (ISCW012446); ACAT1, acetyl-CoA acetyltransferase (ISCW016117); Hypo195, hypothetical protein (ISCW011195); Hypo576, hypothetical protein (ISCW020576); pGEM, pGEM plasmid (negative control)
    Figure Legend Snippet: dsRNA-mediated knockdown of transcripts for I. scapularis genes of interest in ISE6 cells. Following transfection of ISE6 cells with 10 ng dsRNA for 60 h, total RNA was prepared from ~ 1 × 10 5 cells and cDNA was amplified via a two-step RT-PCR reaction. mRNA levels were normalized to I. scapularis β-actin and expressed relative to the percentage of pGEM control cDNA. Results show relative expression of ten I. scapularis genes of interest following knockdown ( white bars) relative to the pGEM dsRNA negative control ( gray bars). Error bars represent standard error of the mean (SEM). Statistical analysis was performed using an unpaired t-test between the negative pGEM control and dsRNA treatments for each gene of interest. Results represent 2–3 technical replicates (each with 2–3 machine replicates) and 2 biological replicates. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Abbreviations : FAH, fumarylacetoacetase (ISCW020196); ERP29, endoplasmic reticulum protein 29 (ISCW018425); ALDH, aldehyde dehydrogenase (ISCW015982); VNN, carbon-nitrogen hydrolase/vanin-like (ISCW004822); MDH2, malate dehydrogenase (ISCW003528); PARP, poly [ADP-ribose] polymerase (ISCW019519); CMPK, cytidine/uridine monophosphate kinase (ISCW012446); ACAT1, acetyl-CoA acetyltransferase (ISCW016117); Hypo195, hypothetical protein (ISCW011195); Hypo576, hypothetical protein (ISCW020576); pGEM, pGEM plasmid (negative control)

    Techniques Used: Transfection, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Plasmid Preparation

    35) Product Images from "Dicer Regulates the Balance of Short-Lived Effector and Long-Lived Memory CD8 T Cell Lineages"

    Article Title: Dicer Regulates the Balance of Short-Lived Effector and Long-Lived Memory CD8 T Cell Lineages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0162674

    Decreased numbers of dicer CKO P14 effector cells demonstrate an intrinsic role of Dicer during CD8 T cell expansion. P14 chimeric mice containing 5x10 4 D b GP33-specific WT P14 as well as dicer CKO P14 CD8 T cells were infected with LCMV and sacrificed at day 7 post-infection. (A) FACS plots depict WT and dicer CKO frequencies of donor antigen-specific CD8 T cells. Bar graphs show total numbers of WT and dicer CKO donor antigen-specific CD8 T cells in spleen, inguinal lymph nodes, lung, and liver. (B) Histogram shows GzmB expression in naïve (grey), WT (black line), and dicer CKO (dashed line) CD8 T cells on day 7 post-infection in spleen. Bar graphs shows GzmB mean fluorescence intensity (MFI) in spleen, lymph node, lung, liver, and blood. (C) Antigen-specific CD8 T cells were analyzed via 5 hour direct ex vivo peptide re-stimulation. IFN-γ, TNF-α, and IL-2 production was analyzed using intracellular cytokine staining. FACS plots show donor WT or dicer CKO IFN-γ+ CD8 T cells. Bar graphs depict percent of TNF-α+ or IL-2+ of IFN-γ+ CD8 T cells of WT or dicer CKO donor antigen-specific T cells. Bar graphs display mean and SEM. Paired Student’s t-test was used with statistical significance in difference of means represented as * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). Experiments are representative of 2 experiments with 3 mice per group.
    Figure Legend Snippet: Decreased numbers of dicer CKO P14 effector cells demonstrate an intrinsic role of Dicer during CD8 T cell expansion. P14 chimeric mice containing 5x10 4 D b GP33-specific WT P14 as well as dicer CKO P14 CD8 T cells were infected with LCMV and sacrificed at day 7 post-infection. (A) FACS plots depict WT and dicer CKO frequencies of donor antigen-specific CD8 T cells. Bar graphs show total numbers of WT and dicer CKO donor antigen-specific CD8 T cells in spleen, inguinal lymph nodes, lung, and liver. (B) Histogram shows GzmB expression in naïve (grey), WT (black line), and dicer CKO (dashed line) CD8 T cells on day 7 post-infection in spleen. Bar graphs shows GzmB mean fluorescence intensity (MFI) in spleen, lymph node, lung, liver, and blood. (C) Antigen-specific CD8 T cells were analyzed via 5 hour direct ex vivo peptide re-stimulation. IFN-γ, TNF-α, and IL-2 production was analyzed using intracellular cytokine staining. FACS plots show donor WT or dicer CKO IFN-γ+ CD8 T cells. Bar graphs depict percent of TNF-α+ or IL-2+ of IFN-γ+ CD8 T cells of WT or dicer CKO donor antigen-specific T cells. Bar graphs display mean and SEM. Paired Student’s t-test was used with statistical significance in difference of means represented as * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). Experiments are representative of 2 experiments with 3 mice per group.

    Techniques Used: Mouse Assay, Infection, FACS, Expressing, Fluorescence, Ex Vivo, Staining

    Ablation of Dicer in CD8 T cells results in reduced memory CD8 T cell numbers. P14 chimeric mice containing 5x10 4 D b GP33-specific WT P14 as well as dicer CKO P14 CD8 T cells were infected with LCMV and sacrificed at memory (D44). (A) FACS plots depict WT and dicer CKO frequencies of donor antigen-specific CD8 T cells. Bar graphs show total numbers as well as percent contraction between effector and memory time points of WT and dicer CKO donor antigen-specific CD8 T cells in spleen, inguinal lymph nodes, lung, and liver. (B) Antigen-specific CD8 T cells were analyzed via direct ex vivo re-stimulation as described before and production of IFN-γ, TNF-α, and IL-2 is presented. FACS plots show donor WT or dicer CKO IFN-γ+ CD8 T cells. Bar graphs depict percent of TNF-α+ or IL-2+ of IFN-γ+ CD8 T cells of WT or dicer CKO donor antigen-specific T cells. Bar graphs display mean and SEM. Paired Student’s t-test was used with statistical significance in difference of means represented as * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). Experiments are representative of 2 experiments with 3 mice per group.
    Figure Legend Snippet: Ablation of Dicer in CD8 T cells results in reduced memory CD8 T cell numbers. P14 chimeric mice containing 5x10 4 D b GP33-specific WT P14 as well as dicer CKO P14 CD8 T cells were infected with LCMV and sacrificed at memory (D44). (A) FACS plots depict WT and dicer CKO frequencies of donor antigen-specific CD8 T cells. Bar graphs show total numbers as well as percent contraction between effector and memory time points of WT and dicer CKO donor antigen-specific CD8 T cells in spleen, inguinal lymph nodes, lung, and liver. (B) Antigen-specific CD8 T cells were analyzed via direct ex vivo re-stimulation as described before and production of IFN-γ, TNF-α, and IL-2 is presented. FACS plots show donor WT or dicer CKO IFN-γ+ CD8 T cells. Bar graphs depict percent of TNF-α+ or IL-2+ of IFN-γ+ CD8 T cells of WT or dicer CKO donor antigen-specific T cells. Bar graphs display mean and SEM. Paired Student’s t-test was used with statistical significance in difference of means represented as * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). Experiments are representative of 2 experiments with 3 mice per group.

    Techniques Used: Mouse Assay, Infection, FACS, Ex Vivo

    Compromised antigen-driven proliferation upon ablation of Dicer following activation and effector differentiation. WT and dicer CKO CD8 T cells were labeled with CFSE and adoptively transferred into C57BL/6 recipients. Mice were infected with LCMV and sacrificed 2.75 days later. (A) FACS plots show WT and dicer CKO donor CD8 T cells. Bar graphs show the total number of WT and dicer CKO donor CD8 T cells. (B) Histograms show cell proliferation indicated by CFSE dilution. Bar graphs show frequency of cells per round of cell division at day 2.75 post-infection. (C) Representative histogram plots show WT and dicer CKO donor CD8 T cells from P14 chimeras on day 2.75 and day 7 post-infection. Black numbers in histograms represent MFI. Grey histograms represent a naïve control. (D) WT and dicer CKO P14 splenocytes were stimulated with GP33 and αCD28 in vitro for 2.5 days. BrdU was administered 2h prior to the end of the incubation period. Numbers in histograms show percent of BrdU incorporating cells. Unpaired Student’s t-test was used with statistical significance in difference of means represented as * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). Experiments are representative of 2 experiments with 3 mice per group.
    Figure Legend Snippet: Compromised antigen-driven proliferation upon ablation of Dicer following activation and effector differentiation. WT and dicer CKO CD8 T cells were labeled with CFSE and adoptively transferred into C57BL/6 recipients. Mice were infected with LCMV and sacrificed 2.75 days later. (A) FACS plots show WT and dicer CKO donor CD8 T cells. Bar graphs show the total number of WT and dicer CKO donor CD8 T cells. (B) Histograms show cell proliferation indicated by CFSE dilution. Bar graphs show frequency of cells per round of cell division at day 2.75 post-infection. (C) Representative histogram plots show WT and dicer CKO donor CD8 T cells from P14 chimeras on day 2.75 and day 7 post-infection. Black numbers in histograms represent MFI. Grey histograms represent a naïve control. (D) WT and dicer CKO P14 splenocytes were stimulated with GP33 and αCD28 in vitro for 2.5 days. BrdU was administered 2h prior to the end of the incubation period. Numbers in histograms show percent of BrdU incorporating cells. Unpaired Student’s t-test was used with statistical significance in difference of means represented as * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). Experiments are representative of 2 experiments with 3 mice per group.

    Techniques Used: Activation Assay, Labeling, Mouse Assay, Infection, FACS, In Vitro, Incubation

    Characterization of effector and memory lineage differentiation of dicer CKO antigen-specific CD8 T cells. P14 chimeric mice containing 5x10 4 D b GP33-specific WT P14 as well as dicer CKO P14 CD8 T cells were infected with LCMV and sacrificed at the peak of effector expansion and memory. (A) Line graphs show the longitudinal frequency of WT and dicer CKO CD8 T cell subsets in blood based on their expression of CD127 and KLRG-1. (B) FACS plots show CD127 and KLRG-1 expression of WT and dicer CKO donor CD8 T cells on day 7 and 44 post-LCMV infection in spleen, lymph node, lung, liver, and blood. (C) Bar graphs show numbers of MPEC (CD127+), T CM (CD127+KLRG-1-), T EM (CD127+KLRG-1+), SLEC (CD127-KLRG-1+) donor WT and dicer CKO antigen-specific CD8 T cells at day 7 and day 44 post-infection in spleen, inguinal lymph nodes, lung, and liver. (D) Bar graphs show numbers of CD62L+ and CD62L- donor WT and dicer CKO antigen-specific CD8 T cells at day 70 post-infection in spleen and inguinal lymph nodes. Line and bar graph display mean and SEM. Mixed ANOVA was used to compare longitudinal PBMC data (A). Otherwise paired Student’s t-test was used. Statistical significance in difference of means is represented as * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). Experiments are representative of 2 experiments with 3 mice per group.
    Figure Legend Snippet: Characterization of effector and memory lineage differentiation of dicer CKO antigen-specific CD8 T cells. P14 chimeric mice containing 5x10 4 D b GP33-specific WT P14 as well as dicer CKO P14 CD8 T cells were infected with LCMV and sacrificed at the peak of effector expansion and memory. (A) Line graphs show the longitudinal frequency of WT and dicer CKO CD8 T cell subsets in blood based on their expression of CD127 and KLRG-1. (B) FACS plots show CD127 and KLRG-1 expression of WT and dicer CKO donor CD8 T cells on day 7 and 44 post-LCMV infection in spleen, lymph node, lung, liver, and blood. (C) Bar graphs show numbers of MPEC (CD127+), T CM (CD127+KLRG-1-), T EM (CD127+KLRG-1+), SLEC (CD127-KLRG-1+) donor WT and dicer CKO antigen-specific CD8 T cells at day 7 and day 44 post-infection in spleen, inguinal lymph nodes, lung, and liver. (D) Bar graphs show numbers of CD62L+ and CD62L- donor WT and dicer CKO antigen-specific CD8 T cells at day 70 post-infection in spleen and inguinal lymph nodes. Line and bar graph display mean and SEM. Mixed ANOVA was used to compare longitudinal PBMC data (A). Otherwise paired Student’s t-test was used. Statistical significance in difference of means is represented as * (P ≤ 0.05), ** (P ≤ 0.01), *** (P ≤ 0.001). Experiments are representative of 2 experiments with 3 mice per group.

    Techniques Used: Mouse Assay, Infection, Expressing, FACS

    36) Product Images from "A versatile drug delivery system targeting senescent cells"

    Article Title: A versatile drug delivery system targeting senescent cells

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201809355

    Release of gal‐encapsulated fluorophores in xenografts GalNP beads are based on a mesoporous silica scaffold (MCM‐41) that can be loaded with different cargoes encapsulated by a coat of 6‐mer β(1,4)‐galacto‐oligosaccharides. Cellular uptake of the GalNP beads occurs via endocytosis and, after fusion with lysosomal vesicles, the beads are released by exocytosis. The high lysosomal β‐galactosidase activity of senescent cells allows a preferential release of the cargo by a β‐galactosidase‐mediated hydrolysis of the cap. SK‐MEL‐103 melanoma cells were treated with palbociclib (1 μM) for 1 week, and senescence induction was assessed by SAβgal staining. Next, cultures were exposed to GalNP(rho) (50 μg/ml, for 16 h). Pictures show representative images illustrating rhodamine release by confocal microscopy. Cells were co‐stained with Calcein, and nuclei were stained with Hoechst. Graphs to the right show the rhodamine intensity relative to cell surface in senescent cells and non‐senescent (control) cells. Each assay was repeated at least three times with similar results. Scale bar: 50 μm. Subcutaneous tumor xenografts of SK‐MEL‐103 melanoma cells in athymic female nude mice. Upon tumor formation, mice were treated daily with palbociclib (oral gavage, 100 mg/kg) during 7 days. The left panel picture shows representative whole tissue portions of tumors after SAβGal staining. The right panel shows sections of control and palbociclib‐treated tumors processed for SAβGal staining, and Ki67 and phosphorylated Rb (p‐Rb) immunohistochemistry. This experiment has been repeated at least two times with similar results. Scale bar: 50 μm. Mice bearing SK‐MEL‐103 xenografts, control or treated with palbociclib for 7 days, as in (C), were tail vein injected with 200 μl of a solution containing GalNP(rho) (4 mg/ml). At 6 h post‐injection, mice were sacrificed, tumors were collected, and fluorescence was analyzed by an IVIS spectrum imaging system. The graph indicates the average difference in tumor radiance between GalNP‐injected control and palbociclib‐treated groups. The inset shows the absolute values of radiance (p/s/cm 2 /sr × 10 6 ) for each group. The corresponding differences are highlighted in black or red. Values are expressed as mean ± SD, and statistical significance was assessed by the two‐tailed Student's t ‐test.
    Figure Legend Snippet: Release of gal‐encapsulated fluorophores in xenografts GalNP beads are based on a mesoporous silica scaffold (MCM‐41) that can be loaded with different cargoes encapsulated by a coat of 6‐mer β(1,4)‐galacto‐oligosaccharides. Cellular uptake of the GalNP beads occurs via endocytosis and, after fusion with lysosomal vesicles, the beads are released by exocytosis. The high lysosomal β‐galactosidase activity of senescent cells allows a preferential release of the cargo by a β‐galactosidase‐mediated hydrolysis of the cap. SK‐MEL‐103 melanoma cells were treated with palbociclib (1 μM) for 1 week, and senescence induction was assessed by SAβgal staining. Next, cultures were exposed to GalNP(rho) (50 μg/ml, for 16 h). Pictures show representative images illustrating rhodamine release by confocal microscopy. Cells were co‐stained with Calcein, and nuclei were stained with Hoechst. Graphs to the right show the rhodamine intensity relative to cell surface in senescent cells and non‐senescent (control) cells. Each assay was repeated at least three times with similar results. Scale bar: 50 μm. Subcutaneous tumor xenografts of SK‐MEL‐103 melanoma cells in athymic female nude mice. Upon tumor formation, mice were treated daily with palbociclib (oral gavage, 100 mg/kg) during 7 days. The left panel picture shows representative whole tissue portions of tumors after SAβGal staining. The right panel shows sections of control and palbociclib‐treated tumors processed for SAβGal staining, and Ki67 and phosphorylated Rb (p‐Rb) immunohistochemistry. This experiment has been repeated at least two times with similar results. Scale bar: 50 μm. Mice bearing SK‐MEL‐103 xenografts, control or treated with palbociclib for 7 days, as in (C), were tail vein injected with 200 μl of a solution containing GalNP(rho) (4 mg/ml). At 6 h post‐injection, mice were sacrificed, tumors were collected, and fluorescence was analyzed by an IVIS spectrum imaging system. The graph indicates the average difference in tumor radiance between GalNP‐injected control and palbociclib‐treated groups. The inset shows the absolute values of radiance (p/s/cm 2 /sr × 10 6 ) for each group. The corresponding differences are highlighted in black or red. Values are expressed as mean ± SD, and statistical significance was assessed by the two‐tailed Student's t ‐test.

    Techniques Used: Activity Assay, Staining, Confocal Microscopy, Mouse Assay, Immunohistochemistry, Injection, Fluorescence, Imaging, Two Tailed Test

    37) Product Images from "The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes"

    Article Title: The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06607-1

    Candidalysin induces IL-1β release in murine mononuclear cells. a IL-1β release measured by ELISA in culture supernatants LPS-primed mBMDMs infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 6) or co-incubated with synthetic Candidalysin for 5 h. b Levels of bioactive IL-1 measured in culture supernatants of LPS-primed mBMDMs that were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 6) or co-incubated with synthetic Candidalysin. Bioactive IL-1 was quantified by stimulation of EL4.NOB-1 cells culture supernatants and correlation of the secreted murine IL-2 to a concentration range of recombinant human IL-1β. c The presence of processed IL-1β (p17) detected by western blotting in the supernatant of LPS-primed or unprimed (no LPS) mBMDMs that were infected with C . albicans Wt re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 10) or co-incubated with synthetic Candidalysin for 5 h. A representative image of three independent experiments or donors is shown. d IL-1β and e TNF levels measured by ELISA in culture supernatants of LPS-primed or unprimed mBMDCs respectively, that were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 5) or co-incubated with synthetic Candidalysin for 5 h (mBMDMs) or 4 h (mBMDCs). Secreted IL-1β ( a , d ) and TNF ( e ) were determined by ELISA. Values are represented as scatterplots and the median of at least three different replicates ( n ≥ 3). nd not detectable
    Figure Legend Snippet: Candidalysin induces IL-1β release in murine mononuclear cells. a IL-1β release measured by ELISA in culture supernatants LPS-primed mBMDMs infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 6) or co-incubated with synthetic Candidalysin for 5 h. b Levels of bioactive IL-1 measured in culture supernatants of LPS-primed mBMDMs that were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 6) or co-incubated with synthetic Candidalysin. Bioactive IL-1 was quantified by stimulation of EL4.NOB-1 cells culture supernatants and correlation of the secreted murine IL-2 to a concentration range of recombinant human IL-1β. c The presence of processed IL-1β (p17) detected by western blotting in the supernatant of LPS-primed or unprimed (no LPS) mBMDMs that were infected with C . albicans Wt re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 10) or co-incubated with synthetic Candidalysin for 5 h. A representative image of three independent experiments or donors is shown. d IL-1β and e TNF levels measured by ELISA in culture supernatants of LPS-primed or unprimed mBMDCs respectively, that were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 5) or co-incubated with synthetic Candidalysin for 5 h (mBMDMs) or 4 h (mBMDCs). Secreted IL-1β ( a , d ) and TNF ( e ) were determined by ELISA. Values are represented as scatterplots and the median of at least three different replicates ( n ≥ 3). nd not detectable

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection, Mutagenesis, Incubation, Concentration Assay, Recombinant, Western Blot

    Candidalysin-dependent release of bioactive, mature IL-1β by primed hMDMs. a Levels of bioactive IL-1 measured in culture supernatants of LPS-primed hMDMs that were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 10) or co-incubated with synthetic Candidalysin. Bioactive IL-1 was quantified by stimulation of EL4.NOB-1 cells culture supernatants and correlation of the secreted murine IL-2 to a concentration range of recombinant human IL-1β. b The presence of processed IL-1β (p17) detected by western blotting in the supernatant of LPS-primed or unprimed (no LPS) hMDMs that were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 10) or co-incubated with synthetic Candidalysin for 5 h. A representative image of three independent experiments or donors is shown. c IL-1β levels were determined by ELISA in culture supernatants of human MDMs that were primed for 16 h with heat-killed C . albicans yeasts or hyphae, Zymosan ( Saccharomyces cerevisiae cell wall), WGP (whole glucan particles; S . cerevisiae β-glucan) or Curdlan (β-1,3 glucan) followed by treatment with synthetic Candidalysin or Nigericin for 5 h. Values are represented as scatterplot and the median of at least three different donors in at least two independent experiments. For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparison test was used. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, nd not detectable. Significance compared to Wt ( a ) or to unprimed cells ( c )
    Figure Legend Snippet: Candidalysin-dependent release of bioactive, mature IL-1β by primed hMDMs. a Levels of bioactive IL-1 measured in culture supernatants of LPS-primed hMDMs that were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 10) or co-incubated with synthetic Candidalysin. Bioactive IL-1 was quantified by stimulation of EL4.NOB-1 cells culture supernatants and correlation of the secreted murine IL-2 to a concentration range of recombinant human IL-1β. b The presence of processed IL-1β (p17) detected by western blotting in the supernatant of LPS-primed or unprimed (no LPS) hMDMs that were infected with C . albicans Wt, re-integrant ( ece1 Δ/Δ + ECE1 ) or mutant strains ( ece1 Δ/Δ, ece1 Δ/Δ + ECE1 Δ 184–279 ) (MOI 10) or co-incubated with synthetic Candidalysin for 5 h. A representative image of three independent experiments or donors is shown. c IL-1β levels were determined by ELISA in culture supernatants of human MDMs that were primed for 16 h with heat-killed C . albicans yeasts or hyphae, Zymosan ( Saccharomyces cerevisiae cell wall), WGP (whole glucan particles; S . cerevisiae β-glucan) or Curdlan (β-1,3 glucan) followed by treatment with synthetic Candidalysin or Nigericin for 5 h. Values are represented as scatterplot and the median of at least three different donors in at least two independent experiments. For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparison test was used. *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, nd not detectable. Significance compared to Wt ( a ) or to unprimed cells ( c )

    Techniques Used: Infection, Mutagenesis, Incubation, Concentration Assay, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay

    38) Product Images from "Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo"

    Article Title: Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01017

    Periodontal outer membrane vesicle (OMV) cytotoxicity differs between unprimed and primed THP-1 cells. Cytotoxicity assays were performed on THP-1 monocytes, M(naïve), and M(IFNγ) macrophages left unprimed (A,C,E) or primed for 3 h with a low dose of Porphyromonas gingivalis OMVs (B,D,F) . Cells were treated with periodontal OMVs in increasing OMV to cell ratios (10:1, 50:1, and 100:1) in cell suspension for 4 h. Cell viability was determined by trypan blue exclusion and deteriorating cell counts determined by hemocytometer and a Z1 Coulter Particle Counter. Results are displayed as the number of remaining viable cells after 4 h. Data are represented as mean ± SEM of three replicates. *represents a significant ( p
    Figure Legend Snippet: Periodontal outer membrane vesicle (OMV) cytotoxicity differs between unprimed and primed THP-1 cells. Cytotoxicity assays were performed on THP-1 monocytes, M(naïve), and M(IFNγ) macrophages left unprimed (A,C,E) or primed for 3 h with a low dose of Porphyromonas gingivalis OMVs (B,D,F) . Cells were treated with periodontal OMVs in increasing OMV to cell ratios (10:1, 50:1, and 100:1) in cell suspension for 4 h. Cell viability was determined by trypan blue exclusion and deteriorating cell counts determined by hemocytometer and a Z1 Coulter Particle Counter. Results are displayed as the number of remaining viable cells after 4 h. Data are represented as mean ± SEM of three replicates. *represents a significant ( p

    Techniques Used:

    Inflammasome activation in immortalized bone marrow-derived macrophages (IBMDM) knockout cell lines. Wild-type IBMDM and NLRP3−/−, AIM2−/−, ASC−/−, and Caspase 1−/− knockout IBMDM strains were primed with Porphyromonas gingivalis outer membrane vesicles (OMVs, 3 h) and treated with positive controls nigericin, silica, and Poly(dA:dT) or P. gingivalis, Treponema denticola , and Tannerella forsythia OMVs at OMV:cell ratios of 10:1, 50:1, and 100:1 for 6 h. Cellular supernatants were collected and IL-1β secretion detected by an IL-1β ELISA Kit. Data are represented as mean ± SEM of three replicates. *represents a significant ( p
    Figure Legend Snippet: Inflammasome activation in immortalized bone marrow-derived macrophages (IBMDM) knockout cell lines. Wild-type IBMDM and NLRP3−/−, AIM2−/−, ASC−/−, and Caspase 1−/− knockout IBMDM strains were primed with Porphyromonas gingivalis outer membrane vesicles (OMVs, 3 h) and treated with positive controls nigericin, silica, and Poly(dA:dT) or P. gingivalis, Treponema denticola , and Tannerella forsythia OMVs at OMV:cell ratios of 10:1, 50:1, and 100:1 for 6 h. Cellular supernatants were collected and IL-1β secretion detected by an IL-1β ELISA Kit. Data are represented as mean ± SEM of three replicates. *represents a significant ( p

    Techniques Used: Activation Assay, Derivative Assay, Knock-Out, Enzyme-linked Immunosorbent Assay

    Periodontal outer membrane vesicles (OMVs) prime and activate inflammasome formation in vitro . THP-1 monocytes (A) , M(naïve) (B) , and M(IFNγ) (C) macrophages were primed with Porphyromonas gingivalis OMVs for 3 h then stimulated with either positive controls nigericin, silica, and Poly(dA:dT) or P. gingivalis, Treponema denticola , and Tannerella forsythia OMVs. Data are represented as mean ± SEM of three replicates. *represents a significant ( p
    Figure Legend Snippet: Periodontal outer membrane vesicles (OMVs) prime and activate inflammasome formation in vitro . THP-1 monocytes (A) , M(naïve) (B) , and M(IFNγ) (C) macrophages were primed with Porphyromonas gingivalis OMVs for 3 h then stimulated with either positive controls nigericin, silica, and Poly(dA:dT) or P. gingivalis, Treponema denticola , and Tannerella forsythia OMVs. Data are represented as mean ± SEM of three replicates. *represents a significant ( p

    Techniques Used: In Vitro

    Porphyromonas gingivalis outer membrane vesicles (OMVs) prime and activate inflammasome formation in vivo . (A) Intraperitoneal cells from naive mice (unprimed and unactivated) were cultured overnight and stimulated ex vivo with nigericin, silica, P. gingivalis, Treponema denticola , or Tannerella forsythia OMVs, IL-1β secretion was determined by ELISA. (B) For in vivo inflammasome activation C57BL/6 J mice received intraperitoneal injections of phosphate-buffered saline (PBS, naïve), Escherichia coli lipopolysaccharide, or P. gingivalis OMVs 72 h prior to harvest to recruit immune cells to the peritoneal cavity. A second intraperitoneal injection of PBS, silica, nigericin, or P. gingivalis OMVs was administered 15 min prior to killing to active inflammasomes in peritoneal macrophages, IL-1β secretion was determined by ELISA. Data are represented as mean ± SEM of three replicates. *represents a significant ( p
    Figure Legend Snippet: Porphyromonas gingivalis outer membrane vesicles (OMVs) prime and activate inflammasome formation in vivo . (A) Intraperitoneal cells from naive mice (unprimed and unactivated) were cultured overnight and stimulated ex vivo with nigericin, silica, P. gingivalis, Treponema denticola , or Tannerella forsythia OMVs, IL-1β secretion was determined by ELISA. (B) For in vivo inflammasome activation C57BL/6 J mice received intraperitoneal injections of phosphate-buffered saline (PBS, naïve), Escherichia coli lipopolysaccharide, or P. gingivalis OMVs 72 h prior to harvest to recruit immune cells to the peritoneal cavity. A second intraperitoneal injection of PBS, silica, nigericin, or P. gingivalis OMVs was administered 15 min prior to killing to active inflammasomes in peritoneal macrophages, IL-1β secretion was determined by ELISA. Data are represented as mean ± SEM of three replicates. *represents a significant ( p

    Techniques Used: In Vivo, Mouse Assay, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay, Activation Assay, Injection

    Periodontal pathogen outer membrane vesicles (OMVs) are phagocytosed by THP-1 cell subsets. Cell phagocytosis assays were performed with THP-1 cell suspensions (monocytes) and monolayers [M(naïve) and M(IFNγ)] incubated with pHrodo-labeled Porphyromonas gingivalis, Treponema denticola , and Tannerella forsythia OMVs at three OMV to cell ratios (10:1, 50:1, and 100:1). Cells were incubated for 60 min, washed, and OMV phagocytosis determined by flow cytometry. Results are expressed as the percentage of THP-1 cells with at least one bound OMV (A,C,E) or the mean fluorescence intensity (MFI) of each sample (B,D,F) , which indicates the quantity of OMVs bound per cell. Data are represented as mean ± SEM of three replicates. *represents a result significantly higher ( p
    Figure Legend Snippet: Periodontal pathogen outer membrane vesicles (OMVs) are phagocytosed by THP-1 cell subsets. Cell phagocytosis assays were performed with THP-1 cell suspensions (monocytes) and monolayers [M(naïve) and M(IFNγ)] incubated with pHrodo-labeled Porphyromonas gingivalis, Treponema denticola , and Tannerella forsythia OMVs at three OMV to cell ratios (10:1, 50:1, and 100:1). Cells were incubated for 60 min, washed, and OMV phagocytosis determined by flow cytometry. Results are expressed as the percentage of THP-1 cells with at least one bound OMV (A,C,E) or the mean fluorescence intensity (MFI) of each sample (B,D,F) , which indicates the quantity of OMVs bound per cell. Data are represented as mean ± SEM of three replicates. *represents a result significantly higher ( p

    Techniques Used: Incubation, Labeling, Flow Cytometry, Cytometry, Fluorescence

    Periodontal pathogen outer membrane vesicles (OMVs) differentially activate NF-κB in THP-1-differentiated cells. THP-Blue cells [monocytes, M(naïve) and M(IFNγ) cell subsets] were incubated with either Porphyromonas gingivalis, Treponema denticola , or Tannerella forsythia OMVs (in fivefold dilutions) or positive control ligands Pam3CSK4 and lipopolysaccharide (LPS)-EB. Alkaline phosphatase secretion was determined with Quanti-Blue after 20 h incubation at 620 nm on a spectrophotometer. Data are represented as mean ± SEM of three biological replicates and results are presented as the number of OMVs required to achieve ED 50 . All Pam3CSK4 and LPS-EB controls gave positive results and were not included in the final data.
    Figure Legend Snippet: Periodontal pathogen outer membrane vesicles (OMVs) differentially activate NF-κB in THP-1-differentiated cells. THP-Blue cells [monocytes, M(naïve) and M(IFNγ) cell subsets] were incubated with either Porphyromonas gingivalis, Treponema denticola , or Tannerella forsythia OMVs (in fivefold dilutions) or positive control ligands Pam3CSK4 and lipopolysaccharide (LPS)-EB. Alkaline phosphatase secretion was determined with Quanti-Blue after 20 h incubation at 620 nm on a spectrophotometer. Data are represented as mean ± SEM of three biological replicates and results are presented as the number of OMVs required to achieve ED 50 . All Pam3CSK4 and LPS-EB controls gave positive results and were not included in the final data.

    Techniques Used: Incubation, Positive Control, Spectrophotometry

    Periodontal pathogen outer membrane vesicles (OMVs) induce cytokine release from THP-1 cell subsets. THP-1 cytokine responses to Porphyromonas gingivalis, Treponema denticola , and Tannerella forsythia were determined following a 60 min incubation of OMVs (10:1, 50:1, and 100:1 OMV to cell ratios) on a THP-1 suspension (monocytes) or cell monolayer [M(naïve) and M(IFNγ)], followed by a 20-h incubation at 37°C. Results are expressed as picograms per milliliter of cytokine/chemokine TNFα (A–C) , IL-1β (D–F) , IL-8 (G–I) , and IL-10 (J–L) in the resulting supernatant. Data are represented as mean ± SEM of three replicates. *represents a significant ( p
    Figure Legend Snippet: Periodontal pathogen outer membrane vesicles (OMVs) induce cytokine release from THP-1 cell subsets. THP-1 cytokine responses to Porphyromonas gingivalis, Treponema denticola , and Tannerella forsythia were determined following a 60 min incubation of OMVs (10:1, 50:1, and 100:1 OMV to cell ratios) on a THP-1 suspension (monocytes) or cell monolayer [M(naïve) and M(IFNγ)], followed by a 20-h incubation at 37°C. Results are expressed as picograms per milliliter of cytokine/chemokine TNFα (A–C) , IL-1β (D–F) , IL-8 (G–I) , and IL-10 (J–L) in the resulting supernatant. Data are represented as mean ± SEM of three replicates. *represents a significant ( p

    Techniques Used: Incubation

    Porphyromonas gingivalis outer membrane vesicles (OMVs) and lipopolysaccharide (LPS) are priming agents for macrophage inflammasome activation. THP-1 monocytes (A) , M(naïve) (B) , and M(IFNγ) (C) macrophages were primed for 3 h with Escherichia coli, P. gingivalis, Treponema denticola , and Tannerella forsythia LPS and OMVs or left unprimed. Cells were then stimulated with positive controls nigericin, silica, and Poly(dA:dT) or E. coli, P. gingivalis, T. denticola , and T. forsythia LPS as required to match the priming material. Cellular supernatants were collected and IL-1β secretion detected by ELISA. Data are represented as mean ± SEM of three replicates. *represents a significant ( p
    Figure Legend Snippet: Porphyromonas gingivalis outer membrane vesicles (OMVs) and lipopolysaccharide (LPS) are priming agents for macrophage inflammasome activation. THP-1 monocytes (A) , M(naïve) (B) , and M(IFNγ) (C) macrophages were primed for 3 h with Escherichia coli, P. gingivalis, Treponema denticola , and Tannerella forsythia LPS and OMVs or left unprimed. Cells were then stimulated with positive controls nigericin, silica, and Poly(dA:dT) or E. coli, P. gingivalis, T. denticola , and T. forsythia LPS as required to match the priming material. Cellular supernatants were collected and IL-1β secretion detected by ELISA. Data are represented as mean ± SEM of three replicates. *represents a significant ( p

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay

    Periodontal pathogen outer membrane vesicles (OMVs) bind THP-1 cell subsets. Cell binding assays were performed with THP-1 cell suspensions of monocytes and monolayers of M(naïve) and M(IFNγ) macrophages incubated with PKH26-labeled Porphyromonas gingivalis, Treponema denticola , and Tannerella forsythia OMVs at three OMV to cell ratios (10:1, 50:1, and 100:1). Cells were incubated for 60 min, washed, and OMV binding determined by flow cytometry. Results are expressed as the percentage of THP-1 cells with at least one bound OMV (A,C,E) or the mean fluorescence intensity (MFI) of each sample (B,D,F) , which indicates the quantity of OMVs bound per cell. Data are represented as mean ± SEM of three replicates. *represents a result significantly higher ( p
    Figure Legend Snippet: Periodontal pathogen outer membrane vesicles (OMVs) bind THP-1 cell subsets. Cell binding assays were performed with THP-1 cell suspensions of monocytes and monolayers of M(naïve) and M(IFNγ) macrophages incubated with PKH26-labeled Porphyromonas gingivalis, Treponema denticola , and Tannerella forsythia OMVs at three OMV to cell ratios (10:1, 50:1, and 100:1). Cells were incubated for 60 min, washed, and OMV binding determined by flow cytometry. Results are expressed as the percentage of THP-1 cells with at least one bound OMV (A,C,E) or the mean fluorescence intensity (MFI) of each sample (B,D,F) , which indicates the quantity of OMVs bound per cell. Data are represented as mean ± SEM of three replicates. *represents a result significantly higher ( p

    Techniques Used: Binding Assay, Incubation, Labeling, Flow Cytometry, Cytometry, Fluorescence

    Analysis of ASC speck formation in peritoneal macrophages ( in vivo activation). C57BL/6 J mice received intraperitoneal injections of phosphate-buffered saline (PBS, naïve), Escherichia coli lipopolysaccharide (LPS), or Porphyromonas gingivalis outer membrane vesicles (OMVs) 72 h prior to harvest to recruit immune cells to the peritoneal cavity. Intraperitoneal washes were counted using a Z1 Coulter Particle Counter to observe increase in cell recruitment following LPS and OMV priming (A) . Data are represented as mean ± SEM of three replicates. *represents a significant ( p
    Figure Legend Snippet: Analysis of ASC speck formation in peritoneal macrophages ( in vivo activation). C57BL/6 J mice received intraperitoneal injections of phosphate-buffered saline (PBS, naïve), Escherichia coli lipopolysaccharide (LPS), or Porphyromonas gingivalis outer membrane vesicles (OMVs) 72 h prior to harvest to recruit immune cells to the peritoneal cavity. Intraperitoneal washes were counted using a Z1 Coulter Particle Counter to observe increase in cell recruitment following LPS and OMV priming (A) . Data are represented as mean ± SEM of three replicates. *represents a significant ( p

    Techniques Used: In Vivo, Activation Assay, Mouse Assay

    39) Product Images from "Three distinct developmental pathways for adaptive and two IFN-γ-producing γδ T subsets in adult thymus"

    Article Title: Three distinct developmental pathways for adaptive and two IFN-γ-producing γδ T subsets in adult thymus

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01963-w

    Progression through the D, E and F populations is induced by TCR signalling. a – c Changes in surface marker expression of TCRδ + cells from sorted population A to G cells after 2 days of culture on OP9-DL1 monolayers in the presence or absence of immobilised anti-CD3. The data are visualised as a histograms and b bar plots of the percentage of cells positive for each individual marker as well as c gated into the A to G populations. Bars depict the mean ± SEM from three independent experiments with cells sorted from four to eight mice. Statistical analyses were performed using the paired two-sided Student’s t test, with significance defined as * p
    Figure Legend Snippet: Progression through the D, E and F populations is induced by TCR signalling. a – c Changes in surface marker expression of TCRδ + cells from sorted population A to G cells after 2 days of culture on OP9-DL1 monolayers in the presence or absence of immobilised anti-CD3. The data are visualised as a histograms and b bar plots of the percentage of cells positive for each individual marker as well as c gated into the A to G populations. Bars depict the mean ± SEM from three independent experiments with cells sorted from four to eight mice. Statistical analyses were performed using the paired two-sided Student’s t test, with significance defined as * p

    Techniques Used: Marker, Expressing, Mouse Assay

    40) Product Images from "Three distinct developmental pathways for adaptive and two IFN-γ-producing γδ T subsets in adult thymus"

    Article Title: Three distinct developmental pathways for adaptive and two IFN-γ-producing γδ T subsets in adult thymus

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01963-w

    Progression through the D, E and F populations is induced by TCR signalling. a – c Changes in surface marker expression of TCRδ + cells from sorted population A to G cells after 2 days of culture on OP9-DL1 monolayers in the presence or absence of immobilised anti-CD3. The data are visualised as a histograms and b bar plots of the percentage of cells positive for each individual marker as well as c gated into the A to G populations. Bars depict the mean ± SEM from three independent experiments with cells sorted from four to eight mice. Statistical analyses were performed using the paired two-sided Student’s t test, with significance defined as * p
    Figure Legend Snippet: Progression through the D, E and F populations is induced by TCR signalling. a – c Changes in surface marker expression of TCRδ + cells from sorted population A to G cells after 2 days of culture on OP9-DL1 monolayers in the presence or absence of immobilised anti-CD3. The data are visualised as a histograms and b bar plots of the percentage of cells positive for each individual marker as well as c gated into the A to G populations. Bars depict the mean ± SEM from three independent experiments with cells sorted from four to eight mice. Statistical analyses were performed using the paired two-sided Student’s t test, with significance defined as * p

    Techniques Used: Marker, Expressing, Mouse Assay

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  • 99
    Thermo Fisher 96 well flat bottomed microtiter plates
    Carvacrol influences the ATP-recovery effect on Vac-induced cell death Semi-confluent #12537-GB cells (seeded in <t>96-well</t> flat-bottomed <t>microtiter</t> plates) were treated with DMSO diluted in medium at 10 −4 served as control, Vac (7 μM), Vac+ATP or ATP without carvacrol or with 50 μM and 100 μM carvacrol. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 24 h (x-axis) (IncuCyteZOOM®) (A, B) . Quantitative analysis at 8 h for glioma cell lines (* in A and B) (C) . Vac+ATP vs. Vac+ATP+carvacrol 100 μM (multiple comparison two way ANOVA, p=0.02). All values are given as means ± SD (6× replicates). All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 3 independent experiments.
    96 Well Flat Bottomed Microtiter Plates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 96 well flat bottom uv transparent plate
    Changes in PANC-1 cell aggregate volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for cell aggregate formation followed by 3 days of treatment). ( B ) Cell aggregate volume was measured daily following treatment using V = (4/3) πr 3  where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Cell aggregate volume shown in the graph represents a cell aggregate volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results.  Notes:  The control is represented by the spheroids that were untreated. Indicated  p -values are a comparison between treated vs. untreated cells ( n  = 26 cell aggregates). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not a complete representation of all the spheroids that were measured and plotted in the bar graph.  Abbreviations:  SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin; CA, cell aggregate.
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    Thermo Fisher high binding microplates
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    Carvacrol influences the ATP-recovery effect on Vac-induced cell death Semi-confluent #12537-GB cells (seeded in 96-well flat-bottomed microtiter plates) were treated with DMSO diluted in medium at 10 −4 served as control, Vac (7 μM), Vac+ATP or ATP without carvacrol or with 50 μM and 100 μM carvacrol. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 24 h (x-axis) (IncuCyteZOOM®) (A, B) . Quantitative analysis at 8 h for glioma cell lines (* in A and B) (C) . Vac+ATP vs. Vac+ATP+carvacrol 100 μM (multiple comparison two way ANOVA, p=0.02). All values are given as means ± SD (6× replicates). All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 3 independent experiments.

    Journal: Oncotarget

    Article Title: Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP

    doi: 10.18632/oncotarget.16703

    Figure Lengend Snippet: Carvacrol influences the ATP-recovery effect on Vac-induced cell death Semi-confluent #12537-GB cells (seeded in 96-well flat-bottomed microtiter plates) were treated with DMSO diluted in medium at 10 −4 served as control, Vac (7 μM), Vac+ATP or ATP without carvacrol or with 50 μM and 100 μM carvacrol. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 24 h (x-axis) (IncuCyteZOOM®) (A, B) . Quantitative analysis at 8 h for glioma cell lines (* in A and B) (C) . Vac+ATP vs. Vac+ATP+carvacrol 100 μM (multiple comparison two way ANOVA, p=0.02). All values are given as means ± SD (6× replicates). All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 3 independent experiments.

    Article Snippet: Semi-confluent cell layers seeded into 96-well flat-bottomed microtiter plates ( ThermoFisher.com ) were treated with a final concentration of 7 μM Vac in the presence of 10 μg/ml PI ( Sigma.com ) with and without ATP, carvacrol ( Sigma.com ), suramin ( Tocris/Biotechné.com , UK) or A-438079 ( Sellekchem.com ).

    Techniques: Imaging

    Vac leads to caspase 3/7 activation in glioma cells, counter regulated by ATP Caspase 3/7 activity given as relative color units (RCU) per μM 2 /image. Semi-confluent glioma cells (seeded in 96-well flat-bottomed microtiter plates) were treated with DMSO diluted in medium at 10 −4 served as control; 7 μM Vac; Vac+ATP 1 mM; and 1 mM ATP alone. Caspase 3/7 activity was monitored over 16 h (x-axis) (IncuCyteZOOM®) by adding IncuCyte® Caspase-3/7 reagent (y-axis, RCU×μM 2 /image) (A) . Quantitative analysis at 2 h for glioma cells (* in A). Vac vs. control and Vac vs. Vac+ATP (both p

    Journal: Oncotarget

    Article Title: Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP

    doi: 10.18632/oncotarget.16703

    Figure Lengend Snippet: Vac leads to caspase 3/7 activation in glioma cells, counter regulated by ATP Caspase 3/7 activity given as relative color units (RCU) per μM 2 /image. Semi-confluent glioma cells (seeded in 96-well flat-bottomed microtiter plates) were treated with DMSO diluted in medium at 10 −4 served as control; 7 μM Vac; Vac+ATP 1 mM; and 1 mM ATP alone. Caspase 3/7 activity was monitored over 16 h (x-axis) (IncuCyteZOOM®) by adding IncuCyte® Caspase-3/7 reagent (y-axis, RCU×μM 2 /image) (A) . Quantitative analysis at 2 h for glioma cells (* in A). Vac vs. control and Vac vs. Vac+ATP (both p

    Article Snippet: Semi-confluent cell layers seeded into 96-well flat-bottomed microtiter plates ( ThermoFisher.com ) were treated with a final concentration of 7 μM Vac in the presence of 10 μg/ml PI ( Sigma.com ) with and without ATP, carvacrol ( Sigma.com ), suramin ( Tocris/Biotechné.com , UK) or A-438079 ( Sellekchem.com ).

    Techniques: Activation Assay, Activity Assay

    Vac- vs STS-mediated cell death Semi-confluent glioma cells (seeded in 96-well flat-bottomed microtiter plates) were treated with 2×10 −3 DMSO diluted in medium served as control, 7 μM Vac or 1 μM STS. Cell death was monitored over 20 h (x-axis) by PI staining (y-axis, RCU×μM 2 /image). All values are given as means ± SD (8× replicates) (A) . All imaging was performed using IncuCyteZOOM® at 10× objective. Caspase 3/7 activity was determined by luminescence assay (RLU, y-axis by Caspase Glo 3/7 assay, Promega.com ) after 4 h (*, A); control vs. Vac (t-test, p=0.0002); control vs. STS (t-test, p

    Journal: Oncotarget

    Article Title: Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP

    doi: 10.18632/oncotarget.16703

    Figure Lengend Snippet: Vac- vs STS-mediated cell death Semi-confluent glioma cells (seeded in 96-well flat-bottomed microtiter plates) were treated with 2×10 −3 DMSO diluted in medium served as control, 7 μM Vac or 1 μM STS. Cell death was monitored over 20 h (x-axis) by PI staining (y-axis, RCU×μM 2 /image). All values are given as means ± SD (8× replicates) (A) . All imaging was performed using IncuCyteZOOM® at 10× objective. Caspase 3/7 activity was determined by luminescence assay (RLU, y-axis by Caspase Glo 3/7 assay, Promega.com ) after 4 h (*, A); control vs. Vac (t-test, p=0.0002); control vs. STS (t-test, p

    Article Snippet: Semi-confluent cell layers seeded into 96-well flat-bottomed microtiter plates ( ThermoFisher.com ) were treated with a final concentration of 7 μM Vac in the presence of 10 μg/ml PI ( Sigma.com ) with and without ATP, carvacrol ( Sigma.com ), suramin ( Tocris/Biotechné.com , UK) or A-438079 ( Sellekchem.com ).

    Techniques: Staining, Imaging, Activity Assay, Luminescence Assay, Caspase-Glo Assay, T-Test

    ATP concentrations interfering with Vac-induced cell death, inhibition by purinergic receptor inhibitors Semi-confluent #12537-GB cells (seeded in 96-well flat-bottomed microtiter plates) were treated with 7 μM Vac with or without 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 1 mM ATP, or DMSO diluted in medium at 10 −4 served as control. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 17 h (x-axis). All values are means of PI fluorescence ± SD (triplicate values) (A) . Vac-induced cell death (7 μM) (y-axis, RCU×μM 2 /image) recorded for 24 h (x-axis) was attenuated by ATP (1 mM) but was further increased by ATP in the presence of the universal purinergic inhibitor Suramin (30 μM). All values are means of PI fluorescence ± SD (triplicate values) (B) . Vac-induced cell death (7 μM) (y-axis: RCU×μM 2 /image) recorded for 24 h (x-axis) was attenuated by ATP (1 mM) but was further increased by ATP in the presence of the selective P2×7 inhibitor A-438079 (100 μM). All values are means of PI fluorescence ± SD (6× replicates) (C) . All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 2 independent experiments.

    Journal: Oncotarget

    Article Title: Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP

    doi: 10.18632/oncotarget.16703

    Figure Lengend Snippet: ATP concentrations interfering with Vac-induced cell death, inhibition by purinergic receptor inhibitors Semi-confluent #12537-GB cells (seeded in 96-well flat-bottomed microtiter plates) were treated with 7 μM Vac with or without 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 1 mM ATP, or DMSO diluted in medium at 10 −4 served as control. PI-positive (dead) cells are given as RCU (y-axis, RCU×μM 2 /image). Glioma cells were followed for 17 h (x-axis). All values are means of PI fluorescence ± SD (triplicate values) (A) . Vac-induced cell death (7 μM) (y-axis, RCU×μM 2 /image) recorded for 24 h (x-axis) was attenuated by ATP (1 mM) but was further increased by ATP in the presence of the universal purinergic inhibitor Suramin (30 μM). All values are means of PI fluorescence ± SD (triplicate values) (B) . Vac-induced cell death (7 μM) (y-axis: RCU×μM 2 /image) recorded for 24 h (x-axis) was attenuated by ATP (1 mM) but was further increased by ATP in the presence of the selective P2×7 inhibitor A-438079 (100 μM). All values are means of PI fluorescence ± SD (6× replicates) (C) . All imaging was performed using IncuCyteZOOM® at 10× objective. Results are representative of 2 independent experiments.

    Article Snippet: Semi-confluent cell layers seeded into 96-well flat-bottomed microtiter plates ( ThermoFisher.com ) were treated with a final concentration of 7 μM Vac in the presence of 10 μg/ml PI ( Sigma.com ) with and without ATP, carvacrol ( Sigma.com ), suramin ( Tocris/Biotechné.com , UK) or A-438079 ( Sellekchem.com ).

    Techniques: Inhibition, Fluorescence, Imaging

    Effect of azithromycin up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Azithromycin at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h under anaerobic conditions. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.

    Journal: Journal of Oral Microbiology

    Article Title: Effect of azithromycin on a red complex polymicrobial biofilm

    doi: 10.1080/20002297.2017.1339579

    Figure Lengend Snippet: Effect of azithromycin up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Azithromycin at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h under anaerobic conditions. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.

    Article Snippet: Two hundred microliters of P. gingivalis , T. denticola , or T. forsythia as a monospecies inoculum and the combination of each two bacterial species at equal volumes (100 µL each), as well as all three species (67 µL each) as a polymicrobial inoculum, were aliquoted into 96-well flat-bottom plates (Nunc; Thermo Scientific) to provide the same total number of bacterial cells per inoculum.

    Techniques: Incubation

    Formation of mono- and polymicrobial biofilms in a 96-well plate model after 48 h of incubation at 37°C under anaerobic condition. Native bacterial growth with addition of uncultured growth medium and no antibiotic served as controls. Adherent biofilms were stained with 0.1% crystal violet and the optical density at AU 620 was measured. Data represent the mean AU 620 value of a minimum of three biological replicates.

    Journal: Journal of Oral Microbiology

    Article Title: Effect of azithromycin on a red complex polymicrobial biofilm

    doi: 10.1080/20002297.2017.1339579

    Figure Lengend Snippet: Formation of mono- and polymicrobial biofilms in a 96-well plate model after 48 h of incubation at 37°C under anaerobic condition. Native bacterial growth with addition of uncultured growth medium and no antibiotic served as controls. Adherent biofilms were stained with 0.1% crystal violet and the optical density at AU 620 was measured. Data represent the mean AU 620 value of a minimum of three biological replicates.

    Article Snippet: Two hundred microliters of P. gingivalis , T. denticola , or T. forsythia as a monospecies inoculum and the combination of each two bacterial species at equal volumes (100 µL each), as well as all three species (67 µL each) as a polymicrobial inoculum, were aliquoted into 96-well flat-bottom plates (Nunc; Thermo Scientific) to provide the same total number of bacterial cells per inoculum.

    Techniques: Incubation, Staining

    Effects of azithromycin and amoxicillin + metronidazole (1:1 ratio) up to 5.0 mg/L on formation polymicrobial biofilms after 48 h of anaerobic incubation at 37°C in a 96-well plate model. Azithromycin and amoxicillin + metronidazole (1:1 ratio) at concentrations 0–100 mg/L were incubated with bacterial cultures. Data points represent the mean AU 620 value of a minimum of three biological replicates and the standard deviation. * p

    Journal: Journal of Oral Microbiology

    Article Title: Effect of azithromycin on a red complex polymicrobial biofilm

    doi: 10.1080/20002297.2017.1339579

    Figure Lengend Snippet: Effects of azithromycin and amoxicillin + metronidazole (1:1 ratio) up to 5.0 mg/L on formation polymicrobial biofilms after 48 h of anaerobic incubation at 37°C in a 96-well plate model. Azithromycin and amoxicillin + metronidazole (1:1 ratio) at concentrations 0–100 mg/L were incubated with bacterial cultures. Data points represent the mean AU 620 value of a minimum of three biological replicates and the standard deviation. * p

    Article Snippet: Two hundred microliters of P. gingivalis , T. denticola , or T. forsythia as a monospecies inoculum and the combination of each two bacterial species at equal volumes (100 µL each), as well as all three species (67 µL each) as a polymicrobial inoculum, were aliquoted into 96-well flat-bottom plates (Nunc; Thermo Scientific) to provide the same total number of bacterial cells per inoculum.

    Techniques: Incubation, Standard Deviation

    Effect of amoxicillin + metronidazole up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Amoxicillin + metronidazole in a 1:1 ratio at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h at 37°C anaerobically. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.

    Journal: Journal of Oral Microbiology

    Article Title: Effect of azithromycin on a red complex polymicrobial biofilm

    doi: 10.1080/20002297.2017.1339579

    Figure Lengend Snippet: Effect of amoxicillin + metronidazole up to 5.0 mg/L on the red complex mono- and polymicrobial biofilms in a 96-well plate model. Amoxicillin + metronidazole in a 1:1 ratio at concentrations 0–100 mg/L was incubated with bacterial cultures for 48 h at 37°C anaerobically. Data points represent the mean AU 620 value of a minimum of three biological replicates. Note the categorical scale.

    Article Snippet: Two hundred microliters of P. gingivalis , T. denticola , or T. forsythia as a monospecies inoculum and the combination of each two bacterial species at equal volumes (100 µL each), as well as all three species (67 µL each) as a polymicrobial inoculum, were aliquoted into 96-well flat-bottom plates (Nunc; Thermo Scientific) to provide the same total number of bacterial cells per inoculum.

    Techniques: Incubation

    Changes in PANC-1 cell aggregate volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for cell aggregate formation followed by 3 days of treatment). ( B ) Cell aggregate volume was measured daily following treatment using V = (4/3) πr 3  where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Cell aggregate volume shown in the graph represents a cell aggregate volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results.  Notes:  The control is represented by the spheroids that were untreated. Indicated  p -values are a comparison between treated vs. untreated cells ( n  = 26 cell aggregates). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not a complete representation of all the spheroids that were measured and plotted in the bar graph.  Abbreviations:  SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin; CA, cell aggregate.

    Journal: Nanomaterials

    Article Title: Functionalized Folic Acid-Conjugated Amphiphilic Alternating Copolymer Actively Targets 3D Multicellular Tumour Spheroids and Delivers the Hydrophobic Drug to the Inner Core

    doi: 10.3390/nano8080588

    Figure Lengend Snippet: Changes in PANC-1 cell aggregate volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for cell aggregate formation followed by 3 days of treatment). ( B ) Cell aggregate volume was measured daily following treatment using V = (4/3) πr 3 where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Cell aggregate volume shown in the graph represents a cell aggregate volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results. Notes: The control is represented by the spheroids that were untreated. Indicated p -values are a comparison between treated vs. untreated cells ( n = 26 cell aggregates). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not a complete representation of all the spheroids that were measured and plotted in the bar graph. Abbreviations: SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin; CA, cell aggregate.

    Article Snippet: An amount of 100 μL of the dissolved precipitate was transferred in triplicate to a 96-well flat bottom UV-transparent plate and was measured using Thermo Scientific Varioskan Flash Microplate Reader.

    Techniques:

    Changes in MDA-MB231 spheroid volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM unloaded and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for spheroid formation followed by 3 days of treatment). ( B ) Spheroid volume was measured daily following treatment using V = (4/3) πr 3  where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Spheroid volume shown in the graph represent spheroid volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results.  Notes:  The control is represented by the spheroids that were untreated. Indicated  p -values vs. untreated cells ( n  = 38). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not the complete representation of all the spheroids that were measured.  Abbreviations : SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin.

    Journal: Nanomaterials

    Article Title: Functionalized Folic Acid-Conjugated Amphiphilic Alternating Copolymer Actively Targets 3D Multicellular Tumour Spheroids and Delivers the Hydrophobic Drug to the Inner Core

    doi: 10.3390/nano8080588

    Figure Lengend Snippet: Changes in MDA-MB231 spheroid volume following exposure to 3 μM empty and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. ( A ) Phase contrast images at 4× objective of changes in spheroid volume over the course of three days following exposure to 3 μM unloaded and curcumin loaded SMA and FA-DABA-SMA and 3 μM curcumin only. A total of 10,000 cells were plated per well in a 96 well plate for a total of 7 days (4 days for spheroid formation followed by 3 days of treatment). ( B ) Spheroid volume was measured daily following treatment using V = (4/3) πr 3 where π = 3.1415 and r = average radius (μm). Radius was measured using a scale bar. Spheroid volume shown in the graph represent spheroid volume ± standard error (error bars). Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. The data presented in the graph are the combined results of two independent experiments that showed similar results. Notes: The control is represented by the spheroids that were untreated. Indicated p -values vs. untreated cells ( n = 38). The image scale bar represents 100 μm. The insets are magnified 300%. Phase contrast images are not the complete representation of all the spheroids that were measured. Abbreviations : SMA, poly(styrene- alt -maleic anhydride); FA, folic acid; DABA, 2,4-diaminobutyric acid; Cur, curcumin.

    Article Snippet: An amount of 100 μL of the dissolved precipitate was transferred in triplicate to a 96-well flat bottom UV-transparent plate and was measured using Thermo Scientific Varioskan Flash Microplate Reader.

    Techniques: Multiple Displacement Amplification

    POS binds directly to PeV-A3. Microplates were coated with 0.5 μg/well rabbit anti-PeV-A3 pAb (16-1071) and incubated overnight at 4°C. The next day, 100 CCID 50 PeV-A3 (US-WI-09) was incubated with 10 μM drug (A and B), drug titration (C and D), or without drug (A to D) for 2 h at room temperature. The contents were transferred to pAb-coated microplates and incubated for 1 h. Unbound drug and PeV-A3 were removed by washing, and bound components were incubated with mouse anti-PeV-A3 MAb (clone AB6-BA9-AH7) for 1 h. The effects of drug, PeV-A3, and MAb were detected using a goat anti-mouse IgG (H+L) peroxidase-labeled antibody and TMB substrate. (A and C) The effect of POS binding to PeV-A3. (B and D) The effect of VRC binding to PeV-A3. Data represent mean absorbance at 620 nm from three independent experiments.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Antifungal Triazole Posaconazole Targets an Early Stage of the Parechovirus A3 Life Cycle

    doi: 10.1128/AAC.02372-19

    Figure Lengend Snippet: POS binds directly to PeV-A3. Microplates were coated with 0.5 μg/well rabbit anti-PeV-A3 pAb (16-1071) and incubated overnight at 4°C. The next day, 100 CCID 50 PeV-A3 (US-WI-09) was incubated with 10 μM drug (A and B), drug titration (C and D), or without drug (A to D) for 2 h at room temperature. The contents were transferred to pAb-coated microplates and incubated for 1 h. Unbound drug and PeV-A3 were removed by washing, and bound components were incubated with mouse anti-PeV-A3 MAb (clone AB6-BA9-AH7) for 1 h. The effects of drug, PeV-A3, and MAb were detected using a goat anti-mouse IgG (H+L) peroxidase-labeled antibody and TMB substrate. (A and C) The effect of POS binding to PeV-A3. (B and D) The effect of VRC binding to PeV-A3. Data represent mean absorbance at 620 nm from three independent experiments.

    Article Snippet: High-binding microplates (Immulon 2HB, 96-well polystyrene, flat bottom; Thermo-Fisher) were coated with 0.5 μg/well rabbit anti-PeV-A3 pAb (CDC no. 16-1071) and incubated overnight at 4°C.

    Techniques: Incubation, Titration, Labeling, Binding Assay