Structured Review

GeneTex anti ns1
Altering mtDNA content with ddC or DNase II siRNA modulated DENV‐induced effects in DCs A–D Human DCs were pre‐incubated with ddC at the indicated concentrations for 48 h to abrogate mtDNA. Cells were collected 24 h after mock or DENV (MOI = 5) infection. The mtDNA release ( n = 4) and levels of TLR9 mRNA ( n = 3) and IFN mRNA ( n = 4 for IFN‐β1 and IFN‐λ2; n = 3 for IFN‐λ1 and IFN‐λ3) were determined (A). In parallel, DENV E protein (B, n = 4), DENV <t>NS1</t> protein (C, n = 4), and DENV mRNA (D, n = 4) were measured by different approaches, respectively. Values are means of individual measurements in each sample ± SEM. * P
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1) Product Images from "Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells"

Article Title: Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells

Journal: EMBO Reports

doi: 10.15252/embr.201846182

Altering mtDNA content with ddC or DNase II siRNA modulated DENV‐induced effects in DCs A–D Human DCs were pre‐incubated with ddC at the indicated concentrations for 48 h to abrogate mtDNA. Cells were collected 24 h after mock or DENV (MOI = 5) infection. The mtDNA release ( n = 4) and levels of TLR9 mRNA ( n = 3) and IFN mRNA ( n = 4 for IFN‐β1 and IFN‐λ2; n = 3 for IFN‐λ1 and IFN‐λ3) were determined (A). In parallel, DENV E protein (B, n = 4), DENV NS1 protein (C, n = 4), and DENV mRNA (D, n = 4) were measured by different approaches, respectively. Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: Altering mtDNA content with ddC or DNase II siRNA modulated DENV‐induced effects in DCs A–D Human DCs were pre‐incubated with ddC at the indicated concentrations for 48 h to abrogate mtDNA. Cells were collected 24 h after mock or DENV (MOI = 5) infection. The mtDNA release ( n = 4) and levels of TLR9 mRNA ( n = 3) and IFN mRNA ( n = 4 for IFN‐β1 and IFN‐λ2; n = 3 for IFN‐λ1 and IFN‐λ3) were determined (A). In parallel, DENV E protein (B, n = 4), DENV NS1 protein (C, n = 4), and DENV mRNA (D, n = 4) were measured by different approaches, respectively. Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Incubation, Infection

2) Product Images from "MicroRNA-711–Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death"

Article Title: MicroRNA-711–Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death

Journal: Journal of Neurotrauma

doi: 10.1089/neu.2017.5572

Blocking of Tie-2 and β1-integrin signaling inhibits the neuroprotective effect of Ang-1. RCNs were treated for 30 min with antibodies against Tie2 (50 μg/mL; A ); antibodies against β1-integrin (5 μg/mL; B ); combination of Tie2 (50 μg/mL) and β1-integrin (5 μg/mL; C ) and FAK inhibitor PF573228 (100 nM; D ). Next RCNs were treated with Ang-1 and etoposide (Etop) or etoposide alone. Twenty-four hours later, LDH release was measured. Data are expressed as percentage of control untreated neurons. ( E ) RCNs were treated with etoposide alone; Ang-1 and etoposide and etoposide; and Ang-1 etoposide and Akt inhibitor (2.875 μM). Cell death and cell viability were measured using the LDH and Calcein AM (calcein-acetoxymethyl ester). Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; * p
Figure Legend Snippet: Blocking of Tie-2 and β1-integrin signaling inhibits the neuroprotective effect of Ang-1. RCNs were treated for 30 min with antibodies against Tie2 (50 μg/mL; A ); antibodies against β1-integrin (5 μg/mL; B ); combination of Tie2 (50 μg/mL) and β1-integrin (5 μg/mL; C ) and FAK inhibitor PF573228 (100 nM; D ). Next RCNs were treated with Ang-1 and etoposide (Etop) or etoposide alone. Twenty-four hours later, LDH release was measured. Data are expressed as percentage of control untreated neurons. ( E ) RCNs were treated with etoposide alone; Ang-1 and etoposide and etoposide; and Ang-1 etoposide and Akt inhibitor (2.875 μM). Cell death and cell viability were measured using the LDH and Calcein AM (calcein-acetoxymethyl ester). Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; * p

Techniques Used: Blocking Assay

Ang-1 activates the Akt pathway in neurons through both Tie-2 and β1-integrin signaling. ( A ) RCNs were treated with Ang-1 for the indicated time. Whole-cell lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against Tie-2, phosphor-Tie2 (Y992), β1-integrin, FAK, phosphor-FAK (Tyr397), phospho-Akt (Ser473), phosphor-GSK3α/β, and β-actin. ( B ) Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared to control untreated levels. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; * p
Figure Legend Snippet: Ang-1 activates the Akt pathway in neurons through both Tie-2 and β1-integrin signaling. ( A ) RCNs were treated with Ang-1 for the indicated time. Whole-cell lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against Tie-2, phosphor-Tie2 (Y992), β1-integrin, FAK, phosphor-FAK (Tyr397), phospho-Akt (Ser473), phosphor-GSK3α/β, and β-actin. ( B ) Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared to control untreated levels. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; * p

Techniques Used:

Ang-1 is rapidly downregulated after TBI and in in vitro models of neuronal cell death. ( A ) qPCR for Ang-1 mRNA in cortex and hippocampus after TBI. Data are presented as fold change compared to uninjured controls. Data represent the mean ± SEM (one-way ANOVA, SNK post-hoc analysis); * p
Figure Legend Snippet: Ang-1 is rapidly downregulated after TBI and in in vitro models of neuronal cell death. ( A ) qPCR for Ang-1 mRNA in cortex and hippocampus after TBI. Data are presented as fold change compared to uninjured controls. Data represent the mean ± SEM (one-way ANOVA, SNK post-hoc analysis); * p

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction

Central administration of Ang-1 attenuates molecular mechanisms of neuronal apoptosis after TBI. ( A ) Whole-cell lysates from mouse cortex 24 h after TBI and i.c.v. administration of Ang-1 or vehicle (ACSF) were run on SDS-polyacrylamide gel and immunobloted with antibodies against α-fodrin, PUMA, Noxa, Bim (Bim small (S) (12 kDa) and Bim large (L) (15 kDa) isoforms were weakly detected. Bim extra-large isoform (EL; 25 kDa), active Bax, and β-actin. ( B ) Nuclear fractions were run on SDS-polyacrylamide gel and immunoblotted with antibodies against AIF-1 and H2A.X. ( C ) Protein levels were quantified by densitometry, normalized to housekeeping proteins, and presented as fold change compared to noninjured controls. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc test; *** p
Figure Legend Snippet: Central administration of Ang-1 attenuates molecular mechanisms of neuronal apoptosis after TBI. ( A ) Whole-cell lysates from mouse cortex 24 h after TBI and i.c.v. administration of Ang-1 or vehicle (ACSF) were run on SDS-polyacrylamide gel and immunobloted with antibodies against α-fodrin, PUMA, Noxa, Bim (Bim small (S) (12 kDa) and Bim large (L) (15 kDa) isoforms were weakly detected. Bim extra-large isoform (EL; 25 kDa), active Bax, and β-actin. ( B ) Nuclear fractions were run on SDS-polyacrylamide gel and immunoblotted with antibodies against AIF-1 and H2A.X. ( C ) Protein levels were quantified by densitometry, normalized to housekeeping proteins, and presented as fold change compared to noninjured controls. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc test; *** p

Techniques Used:

Treatment with Ang-1 significantly reduced neurodegeneration in 24 h after TBI. ( A and B ) Post-injury TBI-induced edema/lesion volume was quantified by T2-weighted MRI at 24 h after CCI. Treatment with Ang-1 significantly reduced the lesion volume compared to the CCI veh group ( p
Figure Legend Snippet: Treatment with Ang-1 significantly reduced neurodegeneration in 24 h after TBI. ( A and B ) Post-injury TBI-induced edema/lesion volume was quantified by T2-weighted MRI at 24 h after CCI. Treatment with Ang-1 significantly reduced the lesion volume compared to the CCI veh group ( p

Techniques Used: Magnetic Resonance Imaging

Ang-1 attenuates molecular mechanisms of neuronal cell death. Neurons were treated with etoposide (Etop) alone or with etoposide and recombinant Ang-1 and harvested 24 h after treatment. Whole-cell lysates ( A ), cytosolic ( B ), and nuclear fractions ( C ) were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against PUMA, Noxa, Bim (Bim (Bim small (S) (12 kDa) and Bim large (L) (15 kDa) (Bim extra-large isoform (EL) (25 kDa) was weakly detected), active Bax, cleaved fragment of caspase-3, cleaved (PARP), and α-fodrin, β-actin, AIF-1, cytochrome c, GAPDH, and H2A.X. ( D ) Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared to control untreated levels. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; * p
Figure Legend Snippet: Ang-1 attenuates molecular mechanisms of neuronal cell death. Neurons were treated with etoposide (Etop) alone or with etoposide and recombinant Ang-1 and harvested 24 h after treatment. Whole-cell lysates ( A ), cytosolic ( B ), and nuclear fractions ( C ) were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against PUMA, Noxa, Bim (Bim (Bim small (S) (12 kDa) and Bim large (L) (15 kDa) (Bim extra-large isoform (EL) (25 kDa) was weakly detected), active Bax, cleaved fragment of caspase-3, cleaved (PARP), and α-fodrin, β-actin, AIF-1, cytochrome c, GAPDH, and H2A.X. ( D ) Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared to control untreated levels. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; * p

Techniques Used: Recombinant

Ang-1 reverses the attenuation of the Akt pathway in neuronal cell death. Neurons were treated with etoposide (Etop) alone or with etoposide and recombinant Ang-1, and harvested 24 h after treatment. Whole-cell lysates ( A ) and nuclear fractions ( B ) were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against Akt, phospho-Akt (Ser473), phosphor-GSK3α/β β-actin, FoxO3a, and H2A.X. ( C ) Protein levels were quantified by densitometry, normalized to β-actin (whole cell lysates) or to H2A.X (nuclear fractions), and presented as fold change compared to control untreated levels. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; * p
Figure Legend Snippet: Ang-1 reverses the attenuation of the Akt pathway in neuronal cell death. Neurons were treated with etoposide (Etop) alone or with etoposide and recombinant Ang-1, and harvested 24 h after treatment. Whole-cell lysates ( A ) and nuclear fractions ( B ) were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against Akt, phospho-Akt (Ser473), phosphor-GSK3α/β β-actin, FoxO3a, and H2A.X. ( C ) Protein levels were quantified by densitometry, normalized to β-actin (whole cell lysates) or to H2A.X (nuclear fractions), and presented as fold change compared to control untreated levels. Data represent the mean ± SEM. One-way ANOVA, SNK post-hoc analysis; * p

Techniques Used: Recombinant

3) Product Images from "Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells"

Article Title: Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells

Journal: EMBO Reports

doi: 10.15252/embr.201846182

DENV infection induced release of oxidized and non‐oxidized mtDNA into the cytosol Human DCs (1 × 10 6 cells/ml) were infected by mock or DENV (MOI = 5) for 24 h. The mtDNA levels in supernatants as total ( n = 7), free form ( n = 5), or vesicle‐encapsulated mtDNA ( n = 5) from mock‐ or DENV‐infected DCs were measured. Mitochondrial particles in supernatants were monitored by fluorescence probe MitoTracker Green ( n = 6). There appeared no statistical significance with the parameters measured between mock and DENV infection by unpaired, two‐tailed Student's t ‐test analysis. Human DCs infected with mock or DENV (MOI = 5) for 12 or 24 h were collected, and both total DNA and cytosolic DNA were extracted and quantified using qPCR with specific primers to measure the mtDNA and nDNA levels ( n = 3). mtDNA bound to TLR9 was analyzed via immunoprecipitation of TLR9 followed by qPCR of mtDNA ( n = 4). The relative abundance of cytosolic mtDNA and cytosolic nDNA was determined by normalization with the exogenously added 20 ng of a purified plasmid encoding FLAG gene (PCR3.1‐flag) as described in Materials and Methods. Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: DENV infection induced release of oxidized and non‐oxidized mtDNA into the cytosol Human DCs (1 × 10 6 cells/ml) were infected by mock or DENV (MOI = 5) for 24 h. The mtDNA levels in supernatants as total ( n = 7), free form ( n = 5), or vesicle‐encapsulated mtDNA ( n = 5) from mock‐ or DENV‐infected DCs were measured. Mitochondrial particles in supernatants were monitored by fluorescence probe MitoTracker Green ( n = 6). There appeared no statistical significance with the parameters measured between mock and DENV infection by unpaired, two‐tailed Student's t ‐test analysis. Human DCs infected with mock or DENV (MOI = 5) for 12 or 24 h were collected, and both total DNA and cytosolic DNA were extracted and quantified using qPCR with specific primers to measure the mtDNA and nDNA levels ( n = 3). mtDNA bound to TLR9 was analyzed via immunoprecipitation of TLR9 followed by qPCR of mtDNA ( n = 4). The relative abundance of cytosolic mtDNA and cytosolic nDNA was determined by normalization with the exogenously added 20 ng of a purified plasmid encoding FLAG gene (PCR3.1‐flag) as described in Materials and Methods. Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Infection, Fluorescence, Two Tailed Test, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, Plasmid Preparation

4) Product Images from "Activation of adiponectin receptors has negative impact on muscle mass in C2C12 myotubes and fast-type mouse skeletal muscle"

Article Title: Activation of adiponectin receptors has negative impact on muscle mass in C2C12 myotubes and fast-type mouse skeletal muscle

Journal: PLoS ONE

doi: 10.1371/journal.pone.0205645

Effects of AdipoRon administration on C2C12 myotubes with or without knockdown of adiponectin receptor (AdipoR). Final concentration of AdipoRon was set at 0, 5, 10 and 20 μM in the differentiation medium. A: Effects of AdipoRon on C2C12 myotubes morphology with or without AdipoR knockdown. B: Effects of AdipoRon on protein content in C2C12 myotubes with or without AdipoR knockdown. siScramble: scrambled non-targeting control siRNA, siAdipoR1: siRNA for AdipoR1, siAdipoR: siRNA for AdipoR2, siAdipoR1+R2: siRNA for AdipoR1 and AdipoR2. n = 5 in each condition of each treated cells. Values are expressed means with SEM. *: p
Figure Legend Snippet: Effects of AdipoRon administration on C2C12 myotubes with or without knockdown of adiponectin receptor (AdipoR). Final concentration of AdipoRon was set at 0, 5, 10 and 20 μM in the differentiation medium. A: Effects of AdipoRon on C2C12 myotubes morphology with or without AdipoR knockdown. B: Effects of AdipoRon on protein content in C2C12 myotubes with or without AdipoR knockdown. siScramble: scrambled non-targeting control siRNA, siAdipoR1: siRNA for AdipoR1, siAdipoR: siRNA for AdipoR2, siAdipoR1+R2: siRNA for AdipoR1 and AdipoR2. n = 5 in each condition of each treated cells. Values are expressed means with SEM. *: p

Techniques Used: Concentration Assay

5) Product Images from "Activation of adiponectin receptors has negative impact on muscle mass in C2C12 myotubes and fast-type mouse skeletal muscle"

Article Title: Activation of adiponectin receptors has negative impact on muscle mass in C2C12 myotubes and fast-type mouse skeletal muscle

Journal: PLoS ONE

doi: 10.1371/journal.pone.0205645

Effects of AdipoRon administration on C2C12 myotubes with or without knockdown of adiponectin receptor (AdipoR). Final concentration of AdipoRon was set at 0, 5, 10 and 20 μM in the differentiation medium. A: Effects of AdipoRon on C2C12 myotubes morphology with or without AdipoR knockdown. B: Effects of AdipoRon on protein content in C2C12 myotubes with or without AdipoR knockdown. siScramble: scrambled non-targeting control siRNA, siAdipoR1: siRNA for AdipoR1, siAdipoR: siRNA for AdipoR2, siAdipoR1+R2: siRNA for AdipoR1 and AdipoR2. n = 5 in each condition of each treated cells. Values are expressed means with SEM. *: p
Figure Legend Snippet: Effects of AdipoRon administration on C2C12 myotubes with or without knockdown of adiponectin receptor (AdipoR). Final concentration of AdipoRon was set at 0, 5, 10 and 20 μM in the differentiation medium. A: Effects of AdipoRon on C2C12 myotubes morphology with or without AdipoR knockdown. B: Effects of AdipoRon on protein content in C2C12 myotubes with or without AdipoR knockdown. siScramble: scrambled non-targeting control siRNA, siAdipoR1: siRNA for AdipoR1, siAdipoR: siRNA for AdipoR2, siAdipoR1+R2: siRNA for AdipoR1 and AdipoR2. n = 5 in each condition of each treated cells. Values are expressed means with SEM. *: p

Techniques Used: Concentration Assay

6) Product Images from "Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells"

Article Title: Infection with the dengue RNA virus activates TLR9 signaling in human dendritic cells

Journal: EMBO Reports

doi: 10.15252/embr.201846182

Introduction of mtDNA simulated DENV infection‐mediated effects in DCs A–E Both mtDNA and nDNA were prepared as described in the Materials and Methods. Either mtDNA or nDNA at 1 μg was delivered into DCs via electroporation. Null‐electroporated cells were used as controls. Several parameters, including IFN mRNA (A, n = 5 for IFN‐β1 and IFN‐λ1, n = 3 for IFN‐λ2 and n = 4 for IFN‐λ3), TLR9 mRNA (B, n = 4), and TLR9 protein expression (C, n = 9 for MFI measurement and n = 4 for percentages analysis), were measured. The levels of phosphorylated and unphosphorylated p65, p38, and IRF7 (D, n = 3) and expression of CCR7 mRNA (E, n = 5) were determined. DCs infected by mock or DENV (MOI = 5) for 24 h served for additional comparisons in (A). Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: Introduction of mtDNA simulated DENV infection‐mediated effects in DCs A–E Both mtDNA and nDNA were prepared as described in the Materials and Methods. Either mtDNA or nDNA at 1 μg was delivered into DCs via electroporation. Null‐electroporated cells were used as controls. Several parameters, including IFN mRNA (A, n = 5 for IFN‐β1 and IFN‐λ1, n = 3 for IFN‐λ2 and n = 4 for IFN‐λ3), TLR9 mRNA (B, n = 4), and TLR9 protein expression (C, n = 9 for MFI measurement and n = 4 for percentages analysis), were measured. The levels of phosphorylated and unphosphorylated p65, p38, and IRF7 (D, n = 3) and expression of CCR7 mRNA (E, n = 5) were determined. DCs infected by mock or DENV (MOI = 5) for 24 h served for additional comparisons in (A). Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Infection, Electroporation, Expressing

Altering mtDNA content with ddC or DNase II siRNA modulated DENV‐induced effects in DCs A–D Human DCs were pre‐incubated with ddC at the indicated concentrations for 48 h to abrogate mtDNA. Cells were collected 24 h after mock or DENV (MOI = 5) infection. The mtDNA release ( n = 4) and levels of TLR9 mRNA ( n = 3) and IFN mRNA ( n = 4 for IFN‐β1 and IFN‐λ2; n = 3 for IFN‐λ1 and IFN‐λ3) were determined (A). In parallel, DENV E protein (B, n = 4), DENV NS1 protein (C, n = 4), and DENV mRNA (D, n = 4) were measured by different approaches, respectively. Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: Altering mtDNA content with ddC or DNase II siRNA modulated DENV‐induced effects in DCs A–D Human DCs were pre‐incubated with ddC at the indicated concentrations for 48 h to abrogate mtDNA. Cells were collected 24 h after mock or DENV (MOI = 5) infection. The mtDNA release ( n = 4) and levels of TLR9 mRNA ( n = 3) and IFN mRNA ( n = 4 for IFN‐β1 and IFN‐λ2; n = 3 for IFN‐λ1 and IFN‐λ3) were determined (A). In parallel, DENV E protein (B, n = 4), DENV NS1 protein (C, n = 4), and DENV mRNA (D, n = 4) were measured by different approaches, respectively. Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Incubation, Infection

DENV infection induced release of oxidized and non‐oxidized mtDNA into the cytosol Human DCs (1 × 10 6 cells/ml) were infected by mock or DENV (MOI = 5) for 24 h. The mtDNA levels in supernatants as total ( n = 7), free form ( n = 5), or vesicle‐encapsulated mtDNA ( n = 5) from mock‐ or DENV‐infected DCs were measured. Mitochondrial particles in supernatants were monitored by fluorescence probe MitoTracker Green ( n = 6). There appeared no statistical significance with the parameters measured between mock and DENV infection by unpaired, two‐tailed Student's t ‐test analysis. Human DCs infected with mock or DENV (MOI = 5) for 12 or 24 h were collected, and both total DNA and cytosolic DNA were extracted and quantified using qPCR with specific primers to measure the mtDNA and nDNA levels ( n = 3). mtDNA bound to TLR9 was analyzed via immunoprecipitation of TLR9 followed by qPCR of mtDNA ( n = 4). The relative abundance of cytosolic mtDNA and cytosolic nDNA was determined by normalization with the exogenously added 20 ng of a purified plasmid encoding FLAG gene (PCR3.1‐flag) as described in Materials and Methods. Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: DENV infection induced release of oxidized and non‐oxidized mtDNA into the cytosol Human DCs (1 × 10 6 cells/ml) were infected by mock or DENV (MOI = 5) for 24 h. The mtDNA levels in supernatants as total ( n = 7), free form ( n = 5), or vesicle‐encapsulated mtDNA ( n = 5) from mock‐ or DENV‐infected DCs were measured. Mitochondrial particles in supernatants were monitored by fluorescence probe MitoTracker Green ( n = 6). There appeared no statistical significance with the parameters measured between mock and DENV infection by unpaired, two‐tailed Student's t ‐test analysis. Human DCs infected with mock or DENV (MOI = 5) for 12 or 24 h were collected, and both total DNA and cytosolic DNA were extracted and quantified using qPCR with specific primers to measure the mtDNA and nDNA levels ( n = 3). mtDNA bound to TLR9 was analyzed via immunoprecipitation of TLR9 followed by qPCR of mtDNA ( n = 4). The relative abundance of cytosolic mtDNA and cytosolic nDNA was determined by normalization with the exogenously added 20 ng of a purified plasmid encoding FLAG gene (PCR3.1‐flag) as described in Materials and Methods. Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Infection, Fluorescence, Two Tailed Test, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, Plasmid Preparation

Regulation of IFN‐β1 and DENV mRNA in bone marrow‐derived DCs (BMDCs) prepared from Tlr9 ‐knockout and wild‐type mice BMDCs (1 × 10 6 cells/ml) prepared from wild‐type and Tlr9 ‐knockout mice were infected with mock or DENV (MOI = 5) for 12 or 24 h. Treatment with CpG (0.5 μM) served as the control. The expression of TLR9 mRNA, IFN‐β1 mRNA, and DENV mRNA was measured with qPCR. One pair of mice (one wild‐type mouse and one Tlr9‐ knockout mouse) were included for each independent experiment. The results showed two out of five independent experiments. Values are means of individual measurements (in triplicate) in each sample ± SD. * P
Figure Legend Snippet: Regulation of IFN‐β1 and DENV mRNA in bone marrow‐derived DCs (BMDCs) prepared from Tlr9 ‐knockout and wild‐type mice BMDCs (1 × 10 6 cells/ml) prepared from wild‐type and Tlr9 ‐knockout mice were infected with mock or DENV (MOI = 5) for 12 or 24 h. Treatment with CpG (0.5 μM) served as the control. The expression of TLR9 mRNA, IFN‐β1 mRNA, and DENV mRNA was measured with qPCR. One pair of mice (one wild‐type mouse and one Tlr9‐ knockout mouse) were included for each independent experiment. The results showed two out of five independent experiments. Values are means of individual measurements (in triplicate) in each sample ± SD. * P

Techniques Used: Derivative Assay, Knock-Out, Mouse Assay, Infection, Expressing, Real-time Polymerase Chain Reaction

Effects of minocycline and NIM811 on DENV‐induced mtDNA release, TLR9 mRNA expression, and IFN mRNA expression A–D Human DCs (2 ×10 4 ) were infected with mock or DENV (MOI = 5) for 24 h in the absence or presence of minocycline (1 μM), NIM811 (10 μM) or DMSO. The relative abundance of mtDNA was measured using the exogenously added FLAG gene as a control for normalization (A, n = 2). The expression of TLR9 mRNA (B, n = 2), IFN‐β1 and IFN‐λ1 mRNA (C, n = 2), and LDH release (D, n = 2) were measured accordingly. Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: Effects of minocycline and NIM811 on DENV‐induced mtDNA release, TLR9 mRNA expression, and IFN mRNA expression A–D Human DCs (2 ×10 4 ) were infected with mock or DENV (MOI = 5) for 24 h in the absence or presence of minocycline (1 μM), NIM811 (10 μM) or DMSO. The relative abundance of mtDNA was measured using the exogenously added FLAG gene as a control for normalization (A, n = 2). The expression of TLR9 mRNA (B, n = 2), IFN‐β1 and IFN‐λ1 mRNA (C, n = 2), and LDH release (D, n = 2) were measured accordingly. Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Expressing, Infection

Effects of cGAS knockdown with or without concomitant mtDNA depletion in DENV‐infected DCs A–C Human DCs were transfected with control siRNA (Ctl) or three sets of cGAS siRNA duplexes for 24 h and then infected with mock or DENV (MOI = 5) for an additional 24 h. As indicated, ddC treatment to deplete mtDNA was introduced in some conditions. The cGAS, viral NS3 and β‐actin protein levels (A, n = 4), cGAS mRNA (B, n = 4) and TLR9 mRNA, DENV mRNA, and IFN‐λ1 mRNA (C, n = 4) were determined. Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: Effects of cGAS knockdown with or without concomitant mtDNA depletion in DENV‐infected DCs A–C Human DCs were transfected with control siRNA (Ctl) or three sets of cGAS siRNA duplexes for 24 h and then infected with mock or DENV (MOI = 5) for an additional 24 h. As indicated, ddC treatment to deplete mtDNA was introduced in some conditions. The cGAS, viral NS3 and β‐actin protein levels (A, n = 4), cGAS mRNA (B, n = 4) and TLR9 mRNA, DENV mRNA, and IFN‐λ1 mRNA (C, n = 4) were determined. Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Infection, Transfection, CTL Assay

DENV infection activated TLR9 in human DCs A–D Human DCs (1 × 10 6 cells/ml) were infected by mock or DENV (NGC strain) at MOI 5 for the indicated periods of time or treated with CpG (0.5 μM) for 2 h, and the cells or cellular extracts were collected for determination of TLR9 mRNA expression (A, n = 5) or TLR9 protein expression via Western blotting (B, n = 8) or flow cytometry (C, n = 4). Heat‐ or UV‐inactivated DENV was used as controls (D, n = 4). Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: DENV infection activated TLR9 in human DCs A–D Human DCs (1 × 10 6 cells/ml) were infected by mock or DENV (NGC strain) at MOI 5 for the indicated periods of time or treated with CpG (0.5 μM) for 2 h, and the cells or cellular extracts were collected for determination of TLR9 mRNA expression (A, n = 5) or TLR9 protein expression via Western blotting (B, n = 8) or flow cytometry (C, n = 4). Heat‐ or UV‐inactivated DENV was used as controls (D, n = 4). Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Infection, Expressing, Western Blot, Flow Cytometry, Cytometry

ROS and inflammasomes were involved in DENV‐induced mtDNA release A–G Human DCs were infected with mock or DENV (MOI = 5) in the absence or presence of a mitochondrial ROS scavenger, MitoTEMPO (100 μM). The intensity of MitoSOX fluorescence observed via flow cytometry was used as an indicator of mitochondrial ROS levels (A, n = 5). The levels of 8‐OHdG were measured by immunostaining (Scale bar = 20 μm) and flow cytometry analysis (B, n = 6). The levels of both cytosolic 8‐OHdG (C, n = 6) and mtDNA release (D, n = 4) were measured by ELISA and qPCR, respectively. Meanwhile, the expression of TLR9 mRNA (E, n = 4), DENV mRNA (F, n = 6), and IFN‐β1 and IFN‐λ1 mRNA (G, n = 4) was determined. Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: ROS and inflammasomes were involved in DENV‐induced mtDNA release A–G Human DCs were infected with mock or DENV (MOI = 5) in the absence or presence of a mitochondrial ROS scavenger, MitoTEMPO (100 μM). The intensity of MitoSOX fluorescence observed via flow cytometry was used as an indicator of mitochondrial ROS levels (A, n = 5). The levels of 8‐OHdG were measured by immunostaining (Scale bar = 20 μm) and flow cytometry analysis (B, n = 6). The levels of both cytosolic 8‐OHdG (C, n = 6) and mtDNA release (D, n = 4) were measured by ELISA and qPCR, respectively. Meanwhile, the expression of TLR9 mRNA (E, n = 4), DENV mRNA (F, n = 6), and IFN‐β1 and IFN‐λ1 mRNA (G, n = 4) was determined. Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Infection, Fluorescence, Flow Cytometry, Cytometry, Immunostaining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing

Side‐by‐side comparisons of the effects of TLR9 and cGAS knockdown in DENV‐induced IFN mRNA expression Human DCs were transfected with control siRNA (Ctl), TLR9 siRNA, cGAS siRNA, or both TLR9 and cGAS siRNA duplexes for 24 h and then infected with mock or DENV (MOI = 5) for an additional 24 h. The mRNA levels of TLR9, cGAS, IFN‐β1, and IFN‐λ1 were determined ( n = 4). Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: Side‐by‐side comparisons of the effects of TLR9 and cGAS knockdown in DENV‐induced IFN mRNA expression Human DCs were transfected with control siRNA (Ctl), TLR9 siRNA, cGAS siRNA, or both TLR9 and cGAS siRNA duplexes for 24 h and then infected with mock or DENV (MOI = 5) for an additional 24 h. The mRNA levels of TLR9, cGAS, IFN‐β1, and IFN‐λ1 were determined ( n = 4). Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Expressing, Transfection, CTL Assay, Infection

TLR9 activation contributed to DENV‐induced IFN production A–C Human DCs were transfected with control siRNA (Ctl) or three sets of TLR9 siRNA duplexes for 24 h and then infected with mock or DENV (MOI = 5) for additional 24 h. The TLR9 mRNA (A, n = 3) and protein expression (B, n = 4) were determined by qPCR and flow cytometry, respectively. The levels of phosphorylated and unphosphorylated p65 ( n = 5) and p38 ( n = 3) and TLR9 in total cell lysates were analyzed and the relative band intensity measured (C). Values are means of individual measurements in each sample ± SEM. * P
Figure Legend Snippet: TLR9 activation contributed to DENV‐induced IFN production A–C Human DCs were transfected with control siRNA (Ctl) or three sets of TLR9 siRNA duplexes for 24 h and then infected with mock or DENV (MOI = 5) for additional 24 h. The TLR9 mRNA (A, n = 3) and protein expression (B, n = 4) were determined by qPCR and flow cytometry, respectively. The levels of phosphorylated and unphosphorylated p65 ( n = 5) and p38 ( n = 3) and TLR9 in total cell lysates were analyzed and the relative band intensity measured (C). Values are means of individual measurements in each sample ± SEM. * P

Techniques Used: Activation Assay, Transfection, CTL Assay, Infection, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry

Related Articles

Real-time Polymerase Chain Reaction:

Article Title: MicroRNA-711–Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death
Article Snippet: .. Treatment with angiopoietin-1 attenuated the decrease of tight junction protein expression 24 h after traumatic brain injury We examined the effect of Ang-1 on expression of tight junction proteins (TJs) after TBI by qPCR. .. Treatment with angiopoietin-1 attenuated the decrease of tight junction protein expression 24 h after traumatic brain injury We examined the effect of Ang-1 on expression of tight junction proteins (TJs) after TBI by qPCR.

Expressing:

Article Title: MicroRNA-711–Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death
Article Snippet: .. Treatment with angiopoietin-1 attenuated the decrease of tight junction protein expression 24 h after traumatic brain injury We examined the effect of Ang-1 on expression of tight junction proteins (TJs) after TBI by qPCR. .. Treatment with angiopoietin-1 attenuated the decrease of tight junction protein expression 24 h after traumatic brain injury We examined the effect of Ang-1 on expression of tight junction proteins (TJs) after TBI by qPCR.

Article Title: MicroRNA-711–Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death
Article Snippet: .. Ang-1 expression displays a different profile, rapidly decreasing after cerebral ischemia followed by prolonged upregulation starting 72 h post-ischemia., Interestingly, Ang-1 is not downregulated in the blood of patients after TBI., There is no evidence that the brain and, specifically, neurons are a main source of Ang-1 in the blood. .. Ang-1 expression displays a different profile, rapidly decreasing after cerebral ischemia followed by prolonged upregulation starting 72 h post-ischemia., Interestingly, Ang-1 is not downregulated in the blood of patients after TBI., There is no evidence that the brain and, specifically, neurons are a main source of Ang-1 in the blood.

other:

Article Title: MicroRNA-711–Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death
Article Snippet: Neither Ang-1 nor etoposide treatments changed levels of Tie-2 and integrin β1 ( ).

Article Title: Activation of adiponectin receptors has negative impact on muscle mass in C2C12 myotubes and fast-type mouse skeletal muscle
Article Snippet: AdipoRon-associated decrease in myotube dimeter and number of nuclei per myotube was partially rescued by knockdown of AdipoR1 and/or AdipoR2 ( ; effect of treatment, p < 0.05).

SDS Page:

Article Title: Deconvolution of pro- and antiviral genomic responses in Zika virus-infected and bystander macrophages
Article Snippet: .. Protein lysates were separated by 10% SDS/PAGE, electrophoretically transferred to a nitrocellulose membrane, and immunoblotted at 4 °C overnight with antibodies (1:1,000) against STAT1, STAT1-P, β-actin (Cell Signaling Technology), and NS2B (GTX133308; GeneTex). .. Membranes were then incubated with an HRP-conjugated second antibody (1:10,000) for 1 h at room temperature followed by detection by enhanced chemiluminescence (Bio-Rad).

Blocking Assay:

Article Title: MicroRNA-711–Induced Downregulation of Angiopoietin-1 Mediates Neuronal Cell Death
Article Snippet: .. Akt inhibitor attenuated, but did not completely block, the Ang-1 neuroprotective effects. .. MicroRNA-711 is upregulated the mouse brain after traumatic brain injury and in in vitro model of neuronal cell death Ang-1 is a predicted target for miR-711.

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