Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 1. Overexpression of Mov10 decreases HIV-1 infectivity. (A) 293T cells were transfected with different amounts of Mov10 plasmid, and the expression of Mov10 was determined by Western blot. (B) 293T cells were transfected with 0.5 mg of either pCMV6-XL5 plasmid (control), Mov10 or APOBEC3G in the presence or absence of 0.5 mg vif as well as 1 mg HIV-1-GFP (Denv, Dvif, Dvpr, Dnef) and 0.5 mg p-L-VSV-G. Virus was collected 24 h later, and then added to Jurkat T cells. Virus transfer was standardized across treatment conditions by p24 levels as described (Materials and Methods). Percent infected cells was then determined using FACS analysis for GFP-expression after virus was allowed to incubate with target cells for 72 hours. Error bars represent one standard deviation. doi:10.1371/journal.pone.0009081.g001

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Over Expression, Infection, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Virus, Standard Deviation

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 2. Mov10 decreases specific infectivity of HIV-1. Supernatants of 293T cells that had been transfected with varying amounts of Mov10- expressing plasmid were assayed for (A) HIV-1.Luc p24 levels and (B) after standardization by p24 content, infectivity of target cells was determined by luciferase activity from VSVG.HIV.Luc, which encodes all the HIV accessory genes. For simplicity, the amount of Mov10 plasmid that was transfected is expressed as a ratio of HIV-1 plasmid to Mov10 plasmid and plotted logarithmically. In the experiment (see Materials and Methods for further explanation) HIV-1 plasmid levels remained constant, while Mov10 plasmid was used at levels of 1/6 to 1/1500 that of the HIV-1 plasmid. Error bars represent one standard deviation. (C) Representative plots of Jurkat T cells infected with GFP-expressing virus produced in the presence of either pcDNA3 or Mov10 (ratio of Mov10 to HIV-1 plasmid was 1/25) three days after infection. Quantification of experiments performed using GFP- expressing virus is shown in supplemental figure 1. doi:10.1371/journal.pone.0009081.g002

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Virus, Produced

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 3. Mov10 impairs HIV-1 replication in primary CD4+ T cells. Supernatants of activated CD4+ cells that had been nucleofected with a replication competent, CCR5-tropic HIV-1 viral plasmid and either Mov10 or control (pcDNA3) plasmid ([A] schematic) were assayed for p24 production (B) and then standardized by p24 concentration and used to infect CCR5+ Hut cells. (C) Infection success was determined by flow cytometry analysis of GFP expression. doi:10.1371/journal.pone.0009081.g003

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Plasmid Preparation, Control, Concentration Assay, Infection, Flow Cytometry, Expressing

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 5. An optimal concentration of Mov10 is required for HIV-1 infectivity. 293T cells were transfected with a non-targeting siRNA or Mov10-specific siRNA. At 48 h post-siRNA transfection, the cells were transfected with 1.5 mg of pHIV-RFP, 0.7 mg of p-L-VSV-G and increasing amounts of the Mov10 expression plasmid. At 96 h post-siRNA transfection: (A) the cell lysates were examined by Western blot for Mov10 levels using an anti-Mov10 antibody. Equal protein loading was confirmed by probing with anti-tubulin antibody. (B) After normalizing for p24 values, virus obtained from the transfections was used to infect HeLa cells and infectivity was measured by FACS. The % infected cells represents the percentage of RFP-positive cells in the cell population. Error bars represent one standard deviation. doi:10.1371/journal.pone.0009081.g005

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Concentration Assay, Infection, Transfection, Expressing, Plasmid Preparation, Western Blot, Virus, Standard Deviation

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 4. Broad inhibition of infectious retroviruses by Mov10. Virions derived from 293T cells transfected with various viral plasmids (as described in Materials and Methods) and either pcDNA3 or pcDNA3- Mov10 were used to infect HeLa cells. The % infected cells represents the percentage of GFP-positive cells in the cell population. doi:10.1371/journal.pone.0009081.g004

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Inhibition, Derivative Assay, Transfection, Infection

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 6. Viral glycoprotein incorporation is not affected by Mov10 overexpression. 293T cells were transfected with plasmids necessary for the production of VSV-G pseudotyped Virion-Like Particles (VLPs) containing a GFP tag (as a Gag-GFP fusion) as well as either control or Mov10. Supernatant from these cells was then collected and added to Jurkat T cells and assayed for binding to target cells. Cells bound by one or more VLPs are GFP+ by FACS. doi:10.1371/journal.pone.0009081.g006

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Over Expression, Transfection, Control, Binding Assay

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 7. Early and late reverse transcription is suppressed in virus produced from cells overexpressing Mov10. Synthesis of reverse transcripts by real-time PCR after infection by HIV-1 produced in the presence of either empty vector (pcDNA3) or Mov10 expressing plasmid was measured. No significant difference between production of (A) Early (R-U5) or (B) Late (R-Gag) DNA products of reverse transcription was observed. (C) Infectivity of virions produced in the presence of Mov10 was significantly inhibited. Input viruses were normalized for p24. Results are representative of one out of three similar experiments. doi:10.1371/journal.pone.0009081.g007

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Reverse Transcription, Virus, Produced, Real-time Polymerase Chain Reaction, Infection, Plasmid Preparation, Expressing

Journal: PloS one

Article Title: Perturbation of the P-body component Mov10 inhibits HIV-1 infectivity.

doi: 10.1371/journal.pone.0009081

Figure Lengend Snippet: Figure 8. The helicase domain of Mov10 is not required for HIV-1 restriction. Virions were produced by cotransfection of 293T cells with HIV-1 vector and either empty vector or Mov10, Mov10 N-terminus, Mov10 C-terminus or the putative helicase motif mutant expression plasmids. (A) Schematic of wild-type and mutated human Mov10 constructs. (B) Cell lysates were probed with anti-HA, anti-Mov10, and anti-tubulin. Lanes: 1) pcDNA3, 2) Mov10, 3) Mov10 Nterm, 4) Mov10 Cterm, and 5) Mov10-EQ. Arrows indicate molecular weight of native Mov10. (C) Infectivity of virions produced was examined by FACS after infection of HeLa cells. The % infectivity represents the percentage of RFP-positive cells in the cell population. Error bars represent one standard deviation. doi:10.1371/journal.pone.0009081.g008

Article Snippet: Wild type Mov10 (NCBI accession number BC009312.2, cDNA obtained from Origene) and mutant Mov10s, Mov10-Nterm (corresponds to amino acids 1–495), Mov10-Cterm (corresponds to amino acids 496–1003) and Mov10.EQ (putative helicase motif mutant) were PCR amplified and subcloned into the pcDNA3 vector that, contained an in frame 59 HA tag, using BamHI and NotI restriction sites to create HA-tagged mutants.

Techniques: Produced, Cotransfection, Plasmid Preparation, Mutagenesis, Expressing, Construct, Molecular Weight, Infection, Standard Deviation

Journal: The Journal of Neuroscience

Article Title: Downregulation of miR-23a and miR-27a following Experimental Traumatic Brain Injury Induces Neuronal Cell Death through Activation of Proapoptotic Bcl-2 Proteins

doi: 10.1523/JNEUROSCI.1260-14.2014

Figure Lengend Snippet: Activation of p53 and increased protein expression of proapoptotic Bcl2 molecules after TBI. Western blot analysis of the level of proapoptotic proteins in mouse-injured cortex after TBI. Tissue lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against p53, phosphorylated p53 (Ser 15), PUMA, Noxa, Bax, and β-actin. Levels of p53, phosphorylated p53 (Ser 15), PUMA, Noxa, and Bax were quantified as fold change to control levels after measurement of band intensity by densitometry and normalization to levels of β-actin. Data are mean ± SD. *p < 0.05 versus sham (N = 4). **p < 0.01 versus sham (N = 4). ***p < 0.001 versus sham (N = 4). Analysis by one-way ANOVA followed by multiple pairwise comparisons using Student-Newman-Keuls post hoc test.

Article Snippet: Abcam: V5 (ab27674); Histone H2A.X (ab11175); Santa Cruz Biotechnology: AIF (sc-13116); Apaf-1 (sc-65890); cytochrome c (sc-13560); FAS (sc-716); Bim (sc-11425); Cell Signaling Technology: Cleaved Caspase-3 (#9661); Cleaved PARP (#9545); Phospho-Histone H2A.X (Ser139) (#9718); Phospho-p53 (Ser15) (#9284); p53 (1C12) (#2524); Bax (#2772); XIAP (#2042); Enzo Life Sciences: GAPDH (ADI-CSA-335); Bax (active monomer) (ALX-804-224-C100); α-fodrin (BML-FG6090); ProSci: PUMA (#3041); Noxa (#2437); EMD Millipore: Bak (06-536); Sigma: β-actin (A1978).

Techniques: Activation Assay, Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: Downregulation of miR-23a and miR-27a following Experimental Traumatic Brain Injury Induces Neuronal Cell Death through Activation of Proapoptotic Bcl-2 Proteins

doi: 10.1523/JNEUROSCI.1260-14.2014

Figure Lengend Snippet: mRNA and protein levels of proapoptotic members of Bcl-2 family were increased in etoposide-treated primary cortical neurons. A, qPCR quantification of expression of proapoptotic genes: Noxa, Puma, and Bax in primary cortical neurons at different time points after etoposide treatment. Level of gene expression was normalized to GAPDH expression. Neuronal apoptosis were induced by etoposide as described above. Data are mean ± SD. *p < 0.05 versus control untreated RCNs (N = 4). **p < 0.01 versus control untreated RCNs (N = 4). ***p < 0.001 versus control untreated RCNs (N = 4). B, Western blot analysis of the level of proapoptotic proteins in primary cortical neurons at different time points after etoposide treatment. Cell lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against PUMA, Noxa, Bax, active Bax, and β-actin. Levels of PUMA, Noxa, Bax, and active Bax were quantified as fold change to control levels after measurement of band intensity by densitometry and normalization to levels of β-actin. Data are mean ± SD. *p < 0.05 versus control untreated RCNs (N = 4). **p < 0.01 versus control untreated RCNs (N = 4). ***p < 0.001 versus control untreated RCNs (N = 4). Analysis by one-way ANOVA followed by multiple pairwise comparisons using Student-Newman-Keuls post hoc test.

Article Snippet: Abcam: V5 (ab27674); Histone H2A.X (ab11175); Santa Cruz Biotechnology: AIF (sc-13116); Apaf-1 (sc-65890); cytochrome c (sc-13560); FAS (sc-716); Bim (sc-11425); Cell Signaling Technology: Cleaved Caspase-3 (#9661); Cleaved PARP (#9545); Phospho-Histone H2A.X (Ser139) (#9718); Phospho-p53 (Ser15) (#9284); p53 (1C12) (#2524); Bax (#2772); XIAP (#2042); Enzo Life Sciences: GAPDH (ADI-CSA-335); Bax (active monomer) (ALX-804-224-C100); α-fodrin (BML-FG6090); ProSci: PUMA (#3041); Noxa (#2437); EMD Millipore: Bak (06-536); Sigma: β-actin (A1978).

Techniques: Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: Downregulation of miR-23a and miR-27a following Experimental Traumatic Brain Injury Induces Neuronal Cell Death through Activation of Proapoptotic Bcl-2 Proteins

doi: 10.1523/JNEUROSCI.1260-14.2014

Figure Lengend Snippet: miR-23a and miR-27a mimics attenuate etoposide-induced expression of proapoptotic Bcl-2 family members. Neurons were transfected with miR-23a-3p and miR-27a-3p mimics and 4 h later treated with etoposide as described above. Whole-cell lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against PUMA, Noxa, Bax, active Bax, and β-actin (A). Levels of PUMA (B), Noxa (C), Bax (D), and active Bax (E) were quantified as fold change to control RCN level after measurement of band intensity by densitometry and normalization to levels of β-actin. Data are mean ± SD. *p < 0.05 versus mock untreated RCNs. **p < 0.01 versus mock untreated RCNs. ***p < 0.001 versus mock untreated RCNs. +p < 0.05 versus etoposide-treated mock (n = 4). ++p < 0.01 versus etoposide-treated mock (n = 4). +++p < 0.001 versus etoposide-treated mock (n = 4). Analysis by one-way ANOVA followed by multiple pairwise comparisons using Student-Newman-Keuls post hoc test.

Article Snippet: Abcam: V5 (ab27674); Histone H2A.X (ab11175); Santa Cruz Biotechnology: AIF (sc-13116); Apaf-1 (sc-65890); cytochrome c (sc-13560); FAS (sc-716); Bim (sc-11425); Cell Signaling Technology: Cleaved Caspase-3 (#9661); Cleaved PARP (#9545); Phospho-Histone H2A.X (Ser139) (#9718); Phospho-p53 (Ser15) (#9284); p53 (1C12) (#2524); Bax (#2772); XIAP (#2042); Enzo Life Sciences: GAPDH (ADI-CSA-335); Bax (active monomer) (ALX-804-224-C100); α-fodrin (BML-FG6090); ProSci: PUMA (#3041); Noxa (#2437); EMD Millipore: Bak (06-536); Sigma: β-actin (A1978).

Techniques: Expressing, Transfection

Journal: The Journal of Neuroscience

Article Title: Downregulation of miR-23a and miR-27a following Experimental Traumatic Brain Injury Induces Neuronal Cell Death through Activation of Proapoptotic Bcl-2 Proteins

doi: 10.1523/JNEUROSCI.1260-14.2014

Figure Lengend Snippet: miR-23a and miR-27a target PUMA, Noxa, and Bax. SH-SY5Y cells were transfected with either negative control miR (-ve cnt) or miR-23a-3p or miR-27a-3p mimics. Cells were also cotransfected with reporter plasmids with inserted 3′ UTRs of mouse PUMA (A), Noxa (B), and Bax (C). Twenty-four hours after transfection, cells were analyzed for Luciferase activity. Normalized Luciferase activities were shown as the percentage relative to the cells transfected with reporter plasmid and -ve Con miR mimic, which was set as 1. Experiments were performed in triplicate. Data are mean ± SD. **p < 0.01 (n = 3). ***p < 0.001 (n = 3). Analysis by one-way ANOVA followed by multiple pairwise comparisons using Student-Newman-Keuls post hoc test.

Article Snippet: Abcam: V5 (ab27674); Histone H2A.X (ab11175); Santa Cruz Biotechnology: AIF (sc-13116); Apaf-1 (sc-65890); cytochrome c (sc-13560); FAS (sc-716); Bim (sc-11425); Cell Signaling Technology: Cleaved Caspase-3 (#9661); Cleaved PARP (#9545); Phospho-Histone H2A.X (Ser139) (#9718); Phospho-p53 (Ser15) (#9284); p53 (1C12) (#2524); Bax (#2772); XIAP (#2042); Enzo Life Sciences: GAPDH (ADI-CSA-335); Bax (active monomer) (ALX-804-224-C100); α-fodrin (BML-FG6090); ProSci: PUMA (#3041); Noxa (#2437); EMD Millipore: Bak (06-536); Sigma: β-actin (A1978).

Techniques: Transfection, Negative Control, Luciferase, Activity Assay, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: Downregulation of miR-23a and miR-27a following Experimental Traumatic Brain Injury Induces Neuronal Cell Death through Activation of Proapoptotic Bcl-2 Proteins

doi: 10.1523/JNEUROSCI.1260-14.2014

Figure Lengend Snippet: Intracerebroventricular injection of miR-23a and miR-27a mimics attenuate expression of PUMA, Noxa, and Bax in injured cortex after TBI. qPCR quantification of miR-23a-3p, miR-27a-3p (A) and PUMA, Noxa, and Bax (B) expressions in mouse cortex 6 h after TBI and intracerebroventricular injection of miR-23a-3p or miR-27a-3p or negative control miR (-ve Con) mimics. Levels of miRs were normalized to U6 snRNA; levels of PUMA, Noxa, and Bax were normalized to GAPDH. Data are mean ± SD. *p < 0.05 versus sham animals. **p < 0.01 versus sham animals. ***p < 0.001 versus sham animals. +p < 0.05 versus injured -ve Con injected group (N = 4–6). ++p < 0.01 versus injured -ve Con injected group (N = 4–6). C, Whole-tissue lysates from mouse cortex 24 h after TBI and intracerebroventricular injection of miR-23a-3p or miR-27a-3p or -ve cnt mimics were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against PUMA, Noxa, Bax, active Bax, and β-actin. Levels of PUMA, Noxa, Bax, and active Bax in total lysates were quantified as fold change to the levels of sham after measurement of band intensity by densitometry. Data are mean ± SD. *p < 0.05 versus sham animals. **p < 0.01 versus sham animals. ***p < 0.001 versus sham animals. +p < 0.05 versus injured -ve cnt-injected group (N = 4–6). ++p < 0.01 versus injured -ve cnt-injected group (N = 4–6).

Article Snippet: Abcam: V5 (ab27674); Histone H2A.X (ab11175); Santa Cruz Biotechnology: AIF (sc-13116); Apaf-1 (sc-65890); cytochrome c (sc-13560); FAS (sc-716); Bim (sc-11425); Cell Signaling Technology: Cleaved Caspase-3 (#9661); Cleaved PARP (#9545); Phospho-Histone H2A.X (Ser139) (#9718); Phospho-p53 (Ser15) (#9284); p53 (1C12) (#2524); Bax (#2772); XIAP (#2042); Enzo Life Sciences: GAPDH (ADI-CSA-335); Bax (active monomer) (ALX-804-224-C100); α-fodrin (BML-FG6090); ProSci: PUMA (#3041); Noxa (#2437); EMD Millipore: Bak (06-536); Sigma: β-actin (A1978).

Techniques: Injection, Expressing, Negative Control