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  • 99
    New England Biolabs dsrna
    Dsrna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 872 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher phi6 dsrna
    Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with <t>dsRNA</t> in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage <t>Phi6</t> (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).
    Phi6 Dsrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs dsrna ladder
    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with <t>RNase</t> A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of <t>dsRNA</t> (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.
    Dsrna Ladder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen dsrna
    AF extract suppressed BAFF and IP-10 protein secretion in BEAS-2B cells. BEAS-2B cells were treated with AF extract diluted in medium at 1:1,280, 1:640, and 1:320 wt/vol for 1 hour and stimulated with 5 μg/ml <t>dsRNA</t> or 1,000 IU/ml <t>IFN-β</t>
    Dsrna, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dsrna
    The stimulatory capacity of DCs derived from <t>HCV</t> and healthy individuals. The stimulatory capacity of DCs derived from HCV and healthy individuals was assessed in paired assays run in parallel. Following irradiation, <t>dsRNA</t> matured DCs at 10-fold increasing concentrations (0–10 4 ) were co-cultured with allogeneic PBMC (5 × 10 4 ). T-cell proliferation was determined on day 6 of culture following 18 h incubation with 3 H thymidine (c.p.m. = counts per minute). The results are expressed as mean ± SD of wells in triplicates. The same responder PBMCs from a healthy individual were used in all experiments. Two representative experiments are shown (a). In an overall analysis (b), 3 H thymidine incorporation using DCs from each HCV infected patient is plotted against 3 H thymidine incorporation for the paired healthy control (HCV/HI-1–7) at each DC concentration (10, 10 2 ,10 3 and 10 4 ). Linear regression analysis; slope = 0.9 (95% confidence interval (CI) 0.735–1.025).
    Dsrna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 851 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    SCICONS dsrna
    Colocalization of <t>dsRNA</t> and E3 protein in infected cells. (A) A549 cells on coverslips were mock infected or infected with 5 PFU per cell of vD10rev, vD9muD10mu, or vΔE3L. After 13 h, the cells were fixed, permeabilized, and stained for dsRNA with <t>J2</t> MAb (green) and for E3 with E3 MAb (red), followed by isotype-specific IgG2A and IgG3 fluorescent secondary antibodies, respectively, and DAPI. The cells were imaged by confocal microscopy. Arrows point to virus factories. The factory region within the boxed area was enlarged and is shown in the row below. Scale bar, 10 μm. (B) A549 cells were infected as described for panel A, stained with J2 MAb and fluorescent secondary antibody, and analyzed by flow cytometry. (C) The experiment is the same as that described for panel A except that cells were mock infected or infected with vD10rev-E3-GFP or vD9muD10mu-E3-GFP and stained with J2 MAb to detect dsRNA (red) followed by secondary fluorescent antibodies. E3 was visualized by GFP fluorescence. The factory region in the boxed area was enlarged and is shown in the row below. (D) A549 cells were infected with vD9muD10mu-E3-GFP, and lysates were analyzed by Western blotting with antibody to GFP and RIG-I or immunoprecipitated (IP) with J2 MAb or nonspecific IgG and then analyzed by Western blotting.
    Dsrna, supplied by SCICONS, used in various techniques. Bioz Stars score: 92/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Axolabs dsrna
    Colocalization of <t>dsRNA</t> and E3 protein in infected cells. (A) A549 cells on coverslips were mock infected or infected with 5 PFU per cell of vD10rev, vD9muD10mu, or vΔE3L. After 13 h, the cells were fixed, permeabilized, and stained for dsRNA with <t>J2</t> MAb (green) and for E3 with E3 MAb (red), followed by isotype-specific IgG2A and IgG3 fluorescent secondary antibodies, respectively, and DAPI. The cells were imaged by confocal microscopy. Arrows point to virus factories. The factory region within the boxed area was enlarged and is shown in the row below. Scale bar, 10 μm. (B) A549 cells were infected as described for panel A, stained with J2 MAb and fluorescent secondary antibody, and analyzed by flow cytometry. (C) The experiment is the same as that described for panel A except that cells were mock infected or infected with vD10rev-E3-GFP or vD9muD10mu-E3-GFP and stained with J2 MAb to detect dsRNA (red) followed by secondary fluorescent antibodies. E3 was visualized by GFP fluorescence. The factory region in the boxed area was enlarged and is shown in the row below. (D) A549 cells were infected with vD9muD10mu-E3-GFP, and lysates were analyzed by Western blotting with antibody to GFP and RIG-I or immunoprecipitated (IP) with J2 MAb or nonspecific IgG and then analyzed by Western blotting.
    Dsrna, supplied by Axolabs, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher dsrnas
    Akt phosphorylation of T308 and S473 are independent events. Panel A. A bar diagram showing independence of phosphorylation of Akt at T308 (▪) and S473 (□) in kinase assays of the 1-LN cancer cells stimulated with α 2 M* (50 pM/25 min) or insulin (200 nM/20 min). The bars are: (1) lipofectamine + buffer; (2) lipofectamine + α 2 M* (50 pM/25 min); (3) Raptor <t>dsRNA</t> (100 nM/48 h) then α 2 M*; (4) Rictor dsRNA (100 nM/48 h) then α 2 M*; (5) lipofectamine + insulin; (6) Raptor dsRNA (100 nM/48 h), then insulin (200 nM/20 min); (7) Rictor dsRNA (100 nM/48 h) + insulin; and (8) scrambled dsRNA (100 nM/48 h) then insulin. Values are mean ± SE from three experiments and are expressed as fmol [ 33 P]-γ-ATP incorporated/mg cell protein. Panel B. Levels of p-Akt T308 and p-Akt S743 in cell lysates of 1-LN cells treated with α 2 M* (50 pM/25 min) or insulin (200 nM/15 min). Representative immunoblots of p-Akt T308 and p-Akt S473 from three experiments of cells <t>transfected</t> with Raptor dsRNA or Rictor dsRNA as above are being shown.
    Dsrnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2394 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Illumina Inc dsrna
    Next-generation sequencing of totiviruses and <t>dsRNA</t> satellites from Saccharomyces cerevisiae strain BJH001. ( A ) Read quality (phred score) after <t>Illumina</t> QC shown along the length of the sequencing reads when using different concentrations of poly(A) polymerase and oligo(dT) or anchored oligo(dT) primers; 10%, 25%, 75%, and 90% quantile and median (50% quantile) read quality at each position along the reads are shown. ( B ) Sequence contigs after de novo assembled represented by contig coverage and contig length. BLAST analysis of the four contigs with the longest length and deepest coverage enabled their identification as totiviruses (ScV-L-A1 and ScV-L-BC) and a dsRNA satellite (ScV-M1), the latter was assembled as two separate contigs. Inset reverse transcriptase-PCR was used to confirm the presence of each type of dsRNA. Two primer pairs were used to amplify the ScV-L-BC. ( C ) Read depth coverage across the reference-assembled ScV-L-A, ScV-L-BC, and ScV-M1 contigs. Open reading frames present within each dsRNA are shown above the nucleotide position.
    Dsrna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Trinity Biotech dsrna
    Effects of simvastatin (Sim) and dexamethasone (Dex) on <t>IRF3</t> phosphorylation in bronchial epithelial cells from COPD donors. <t>dsRNA</t> increased phosphorylation of IRF3 (A,B) without changing IRF3 levels (B). Dexamethasone was without significant effects
    Dsrna, supplied by Trinity Biotech, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare dsrna
    RNA binding to midgut cells of western corn rootworm (WCR). (A) <t>Cy3-labeled</t> 210 dvssj1 <t>dsRNA</t> is binding to WCR midgut cells (Top panel), but 21-bp siRNA of dvssj1 is not observed (middle panel). Commercial Cy3 dye was used as a control, which showed no binding also. (B) Summary of the fluorescent intensity of midgut cells. Corrected Total Cell Fluorescence (CTCF) was calculated in ImageJ. DsRNA treatment (n = 11) is significantly different (P
    Dsrna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 422 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology dsrna
    Knockdown of Mfn1/Mfn2 and Drp1/Fis1 reduces mitochondrial fusion and fission in DM cybrid, respectively. <t>siRNA</t> transfection was performed to knockdown target gene expression. Scramble <t>dsRNA</t> (scr) was used as siRNA negative control. (a) Abundance of dynamic proteins Mfn1, Mfn2, Drp1, and Fis1 was determined using Western blotting. β -Actin served as loading control. (b) Mitochondrial morphology was visualized by transfecting cox4-DsRed (red fluorescence). An enlarged segment of each image was shown by a lower right square. (c) The MicroP algorithm categorized mitochondrial morphology into six types: small globe (blue), large globe (yellow), simple tube (green), twisted tube (orange), donut (red), and branching tube (purple). N = 75–400 mitochondria from 15–30 cells and three independent experiments.
    Dsrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Eurofins dsrnas
    Epigenetic modifications post dsRNA <t>transfection.</t> (A)Chromatin remodeling after S1, S5 or S9 dsRNA transfection. Significant reduction in MNase digestion 72 hours after S1 and S5 transfection while increase in the accessibilty after S9 transfection in HeLa cells. Cells were transfected with the respective <t>dsRNAs</t> followed by MNase-PCR 72 hours post transfection. S1:S1 dsRNA transfected cells, S5-dsRNA: S5 dsRNA transfected cells, S9-dsRNA: S9 dsRNA transfected cells, Control-dsRNA: Control dsRNA transfected cells (*) P
    Dsrnas, supplied by Eurofins, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Shanghai GenePharma dsrnas
    Epigenetic modifications post dsRNA <t>transfection.</t> (A)Chromatin remodeling after S1, S5 or S9 dsRNA transfection. Significant reduction in MNase digestion 72 hours after S1 and S5 transfection while increase in the accessibilty after S9 transfection in HeLa cells. Cells were transfected with the respective <t>dsRNAs</t> followed by MNase-PCR 72 hours post transfection. S1:S1 dsRNA transfected cells, S5-dsRNA: S5 dsRNA transfected cells, S9-dsRNA: S9 dsRNA transfected cells, Control-dsRNA: Control dsRNA transfected cells (*) P
    Dsrnas, supplied by Shanghai GenePharma, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Horizon Discovery dsrna duplexes
    Epigenetic modifications post dsRNA <t>transfection.</t> (A)Chromatin remodeling after S1, S5 or S9 dsRNA transfection. Significant reduction in MNase digestion 72 hours after S1 and S5 transfection while increase in the accessibilty after S9 transfection in HeLa cells. Cells were transfected with the respective <t>dsRNAs</t> followed by MNase-PCR 72 hours post transfection. S1:S1 dsRNA transfected cells, S5-dsRNA: S5 dsRNA transfected cells, S9-dsRNA: S9 dsRNA transfected cells, Control-dsRNA: Control dsRNA transfected cells (*) P
    Dsrna Duplexes, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega dsrna
    zic-1 inhibits Wnt signaling to promote head outgrowth. (A) Single and <t>double-RNAi</t> as indicated to examine interactions between zic-1 and beta-catenin-1 . Total concentrations of <t>dsRNA</t> were normalized by control dsRNA so that the absolute amount of each utilized dsRNA was equivalent across treatments. zic-1(RNAi);beta-catenin-1(RNAi) animals all regenerated heads after decapitation suggesting that beta-catenin-1 inhibition is required for the zic-1 's head-promoting function. (B) Scoring percent of anterior regeneration blastema with 0, 1, or 2 photoreceptors (PR) (n > 9 animals). (C) qPCR analysis of zic-1 expression versus gapdh in animals treated with dsRNA as indicated. zic-1 dsRNA reduced zic-1 mRNA levels (p
    Dsrna, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 2857 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher fluorescent dsrna
    zic-1 inhibits Wnt signaling to promote head outgrowth. (A) Single and <t>double-RNAi</t> as indicated to examine interactions between zic-1 and beta-catenin-1 . Total concentrations of <t>dsRNA</t> were normalized by control dsRNA so that the absolute amount of each utilized dsRNA was equivalent across treatments. zic-1(RNAi);beta-catenin-1(RNAi) animals all regenerated heads after decapitation suggesting that beta-catenin-1 inhibition is required for the zic-1 's head-promoting function. (B) Scoring percent of anterior regeneration blastema with 0, 1, or 2 photoreceptors (PR) (n > 9 animals). (C) qPCR analysis of zic-1 expression versus gapdh in animals treated with dsRNA as indicated. zic-1 dsRNA reduced zic-1 mRNA levels (p
    Fluorescent Dsrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    InvivoGen ppp dsrna
    G3BP1 antagonizes RNF125-mediated degradation of RIG-I. a , b Dose-dependent effects of G3BP1 on the expression of RIG-I or IRF3. HEK293T cells (4 × 10 5 ) were transfected with the G3BP1 (0, 1, and 2 μg) and the RIG-I or IRF3 (2 μg) plasmids for 24 h. Then the cell lysates were subjected to immunoblotting with the indicated antibodies. For the HA-RIG-I, HA-G3BP1 and HA-IRF3, band intensities were determined by Image J software. c Effects of inhibitors on G3BP1-mediated stabilization of RIG-I. HEK293T cells (4 × 10 5 ) were transfected with the indicated plasmids for 18 h and then cells were treated with the indicated inhibitors for 6 h before immunoblotting analysis. For the Flag-RIG-I, band intensities were determined by Image J software. d , e Effects of G3BP1 on ubiquitination of RIG-I mediated by RNF125 or RNF125 (C72/75 A) mutant. HEK293T cells (2 × 10 6 ) were transfected with RIG-I (10 μg), G3BP1 (3 μg), HA-Ub (2 μg), and RNF125 or RNF125 (C72/75 A) (5 μg) for 24 h. Co-IP and immunoblotting analysis were performed with the indicated antibodies. f Effects of G3BP1 knockdown on RNF125-mediated ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were transfected with RIG-I (10 μg), HA-Ub (2 μg), and RNF125 (5 μg) for 24 h. Then Co-IP and immunoblotting were performed with the indicated antibodies. g Effects of G3BP1 on K48-linked ubiquitination of RIG-I mediated by RNF125. The experiments were similarly to those described in d . h Effects of G3BP1 knockdown on K48-linked ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. i Effects of G3BP1 knockout on K48-linked ubiquitination of RIG-I. The experiments were similarly to those described in h . j G3BP1 binds to <t>5´ppp-dsRNA</t> and enhances the binding of RIG-I to 5´ppp-dsRNA. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were first incubated with biotinylated-5´ppp-dsRNA and streptavidin-Sepharose. Then conjugated proteins were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Co-IP Co-immunoprecipitation, αF anti-Flag, Coni control RNAi, KO knockout, WT wild-type.
    Ppp Dsrna, supplied by InvivoGen, used in various techniques. Bioz Stars score: 95/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher control dsrnas
    <t>Smurf2</t> is required for stability and functional localization of Mad2. (a) Smurf2 silencing rapidly down-regulates Mad2 protein but not mRNA. HeLa cells in asynchronous culture were transfected with Smurf2 siRNA or nonspecific <t>dsRNAs</t> (siNS) and harvested at the indicated times after transfection for Western blotting and RT-PCR. (b) Smurf2 silencing results in increased polyubiquitination of Mad2 followed by proteasomal degradation. HeLa cells were transfected with a Mad2 expression plasmid and Smurf2 siRNA #1 or control dsRNA. Cells were then treated with 2 μM MG132 for 4 h and analyzed by immunoprecipitation (IP) with Mad2 antibody followed by Western blotting using the indicated antibodies. (c) Smurf2 physically interacts with Mad2. Untransfected HeLa cells were released from synchronization at S phase with double-thymidine treatment and were harvested at the indicated times for immunoprecipitation followed by Western blotting. Ig, Ig heavy chain; NS, nonspecific band. (d) Ectopic expression of Mad2 is unable to restore the spindle assembly checkpoint in Smurf2-depleted cells. Western blotting for mitotic regulators in HeLa cells transfected with the indicated plasmid and siRNA. Cells were synchronized with thymidine treatment, released into a nocodazole-containing medium, and harvested at the indicated times after release (see Fig. 4 b and Materials and methods). (e) Ectopically expressed Mad2 localizes at centromeres in nocodazole-treated control cells but not in Smurf2-depleted cells. Immunofluorescence microscopy with anti-Mad2 and ACAs 10 h after release from the thymidine block. The close-up images are shown with threefold higher magnifications. Bars, 10 μm.
    Control Dsrnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa dsrna synthesis
    RNAi suppression of cold exposure-induced up-regulation of PaHsp70 expression and its effect on survival. Relative levels of Pahsp70 mRNA (A), PaHsp70 protein (B) and Pahsc70 mRNA (C) were measured in the fat bodies of male Pyrrhocoris apterus at different times of recovery after the cold exposure to −5°C for 5 d. The insects were either untreated (control) or injected two days prior to heat shock with: 2 µL of the injection buffer alone (blank); or 2 µL (2 µg) of Pahsp70 <t>dsRNA.</t> (D) An example of Western blotting. (E, F) Survival in blank-injected (E, n = 49) and Pahsp70 dsRNA-injected (F, n = 48) insects after the cold exposure. (G) Cross-tolerance was assesed by observing the survival in insects ( n = 48) that were pretreated with a mild heat shock <t>(+41°C</t> for 1 h) prior to the cold exposure to −5°C for 5 d. See Figs. 1 and 2 for more information.
    Dsrna Synthesis, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Horizon Discovery control dsrna
    RNAi suppression of cold exposure-induced up-regulation of PaHsp70 expression and its effect on survival. Relative levels of Pahsp70 mRNA (A), PaHsp70 protein (B) and Pahsc70 mRNA (C) were measured in the fat bodies of male Pyrrhocoris apterus at different times of recovery after the cold exposure to −5°C for 5 d. The insects were either untreated (control) or injected two days prior to heat shock with: 2 µL of the injection buffer alone (blank); or 2 µL (2 µg) of Pahsp70 <t>dsRNA.</t> (D) An example of Western blotting. (E, F) Survival in blank-injected (E, n = 49) and Pahsp70 dsRNA-injected (F, n = 48) insects after the cold exposure. (G) Cross-tolerance was assesed by observing the survival in insects ( n = 48) that were pretreated with a mild heat shock <t>(+41°C</t> for 1 h) prior to the cold exposure to −5°C for 5 d. See Figs. 1 and 2 for more information.
    Control Dsrna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 89/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Article Snippet: The following reagents were purchased: mouse mAb J2 (Scicons); Alexa Fluor 488-goat anti-mouse IgG secondary antibody (Molecular Probes, Life Technologies); Phi6 dsRNA (Finnzymes, Thermo Fisher Scientific); dsRNA ladder (New England Biolabs); High MW poly (A:U) and poly (I:C) (InvivoGen); GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific); Silencer negative control 1 siRNA (Ambion, Life Technologies); Strep-Tactin conjugated to horseradish peroxidase (IBA Lifesciences); pNPP (Sigma-Aldrich), mMESSAGE mMACHINE T7 transcription kit (Ambion, Life Technologies), Lumi-Light western blotting substrate (Roche Diagnostics).

    Techniques: Binding Assay, In Vitro, Mobility Shift, Molecular Weight

    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

    Journal: Journal of molecular biology

    Article Title: TRIM25 binds RNA to modulate cellular anti-viral defense

    doi: 10.1016/j.jmb.2018.10.003

    Figure Lengend Snippet: RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

    Article Snippet: Experiments in used RNase A (Qiagen), DNase I (Sigma), dsRNA ladder (NEB), and 100-bp dsDNA ladder (NEB).

    Techniques: Activity Assay, In Vitro, Purification, Incubation

    AF extract suppressed BAFF and IP-10 protein secretion in BEAS-2B cells. BEAS-2B cells were treated with AF extract diluted in medium at 1:1,280, 1:640, and 1:320 wt/vol for 1 hour and stimulated with 5 μg/ml dsRNA or 1,000 IU/ml IFN-β

    Journal: American Journal of Respiratory Cell and Molecular Biology

    Article Title: Suppression of Epithelial Signal Transducer and Activator of Transcription 1 Activation by Extracts of Aspergillus fumigatus

    doi: 10.1165/rcmb.2014-0333OC

    Figure Lengend Snippet: AF extract suppressed BAFF and IP-10 protein secretion in BEAS-2B cells. BEAS-2B cells were treated with AF extract diluted in medium at 1:1,280, 1:640, and 1:320 wt/vol for 1 hour and stimulated with 5 μg/ml dsRNA or 1,000 IU/ml IFN-β

    Article Snippet: The extract is a commercial antigen product used to conduct skin prick testing of allergy patients in clinical settings and contains, in addition to the extract allergens, 50% (vol/vol) glycerine, 0.5% sodium chloride, and 0.275% sodium bicarbonate. dsRNA and IFN-β1a were purchased from Invivogen (San Diego, CA).

    Techniques:

    Suppression of IP-10 mRNA expression in normal human bronchial epithelial (NHBE) primary epithelial cells by AF extract. NHBE cells were pretreated with AF extract for 1 hour and stimulated with 1,000 IU/ml of IFN-β or 5 μg/ml of dsRNA

    Journal: American Journal of Respiratory Cell and Molecular Biology

    Article Title: Suppression of Epithelial Signal Transducer and Activator of Transcription 1 Activation by Extracts of Aspergillus fumigatus

    doi: 10.1165/rcmb.2014-0333OC

    Figure Lengend Snippet: Suppression of IP-10 mRNA expression in normal human bronchial epithelial (NHBE) primary epithelial cells by AF extract. NHBE cells were pretreated with AF extract for 1 hour and stimulated with 1,000 IU/ml of IFN-β or 5 μg/ml of dsRNA

    Article Snippet: The extract is a commercial antigen product used to conduct skin prick testing of allergy patients in clinical settings and contains, in addition to the extract allergens, 50% (vol/vol) glycerine, 0.5% sodium chloride, and 0.275% sodium bicarbonate. dsRNA and IFN-β1a were purchased from Invivogen (San Diego, CA).

    Techniques: Expressing

    The stimulatory capacity of DCs derived from HCV and healthy individuals. The stimulatory capacity of DCs derived from HCV and healthy individuals was assessed in paired assays run in parallel. Following irradiation, dsRNA matured DCs at 10-fold increasing concentrations (0–10 4 ) were co-cultured with allogeneic PBMC (5 × 10 4 ). T-cell proliferation was determined on day 6 of culture following 18 h incubation with 3 H thymidine (c.p.m. = counts per minute). The results are expressed as mean ± SD of wells in triplicates. The same responder PBMCs from a healthy individual were used in all experiments. Two representative experiments are shown (a). In an overall analysis (b), 3 H thymidine incorporation using DCs from each HCV infected patient is plotted against 3 H thymidine incorporation for the paired healthy control (HCV/HI-1–7) at each DC concentration (10, 10 2 ,10 3 and 10 4 ). Linear regression analysis; slope = 0.9 (95% confidence interval (CI) 0.735–1.025).

    Journal: Journal of Viral Hepatitis

    Article Title: Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus

    doi: 10.1111/j.1365-2893.2007.00934.x

    Figure Lengend Snippet: The stimulatory capacity of DCs derived from HCV and healthy individuals. The stimulatory capacity of DCs derived from HCV and healthy individuals was assessed in paired assays run in parallel. Following irradiation, dsRNA matured DCs at 10-fold increasing concentrations (0–10 4 ) were co-cultured with allogeneic PBMC (5 × 10 4 ). T-cell proliferation was determined on day 6 of culture following 18 h incubation with 3 H thymidine (c.p.m. = counts per minute). The results are expressed as mean ± SD of wells in triplicates. The same responder PBMCs from a healthy individual were used in all experiments. Two representative experiments are shown (a). In an overall analysis (b), 3 H thymidine incorporation using DCs from each HCV infected patient is plotted against 3 H thymidine incorporation for the paired healthy control (HCV/HI-1–7) at each DC concentration (10, 10 2 ,10 3 and 10 4 ). Linear regression analysis; slope = 0.9 (95% confidence interval (CI) 0.735–1.025).

    Article Snippet: For the assessment of MD-DC in subjects with chronic HCV infection immature DCs were stimulated with dsRNA (Poly I:C, Sigma), 50 μg / mL for 36 h to generate mature DCs.

    Techniques: Derivative Assay, Irradiation, Cell Culture, Incubation, Infection, Concentration Assay

    The phenotypic characteristics of immature and mature DCs in HCV infected and healthy individuals. The expression (mean fluorescence intensity–MFI) of HLA class I, HLA class II, CD 86 and CD83 on immature (bold line) and dsRNA matured (dotted line) MD-DCs from a representative HCV infected individual (HCV-2) and a healthy control individual is shown (HI-2) (A). The expression of these markers on MD-DCs is compared between healthy and HCV infected individuals on both immature (B, upper panel) and mature (B, lower panel) MD-DCs. The table inserts give the MFI ± standard error and P values for each marker.

    Journal: Journal of Viral Hepatitis

    Article Title: Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus

    doi: 10.1111/j.1365-2893.2007.00934.x

    Figure Lengend Snippet: The phenotypic characteristics of immature and mature DCs in HCV infected and healthy individuals. The expression (mean fluorescence intensity–MFI) of HLA class I, HLA class II, CD 86 and CD83 on immature (bold line) and dsRNA matured (dotted line) MD-DCs from a representative HCV infected individual (HCV-2) and a healthy control individual is shown (HI-2) (A). The expression of these markers on MD-DCs is compared between healthy and HCV infected individuals on both immature (B, upper panel) and mature (B, lower panel) MD-DCs. The table inserts give the MFI ± standard error and P values for each marker.

    Article Snippet: For the assessment of MD-DC in subjects with chronic HCV infection immature DCs were stimulated with dsRNA (Poly I:C, Sigma), 50 μg / mL for 36 h to generate mature DCs.

    Techniques: Infection, Expressing, Fluorescence, Marker

    The efficient expansion of melan-A specific CD8+ T cells from healthy and HCV infected individuals. In three representative paired, controlled experiments, dsRNA matured MD-DCs from healthy (left hand panel; HI-5, -8, -9) and HCV infected (right hand panel; HCV-9, -10, -11) individuals were pulsed with 1 μg/mL melan-A 26-35 peptide or RPMI medium, washed thoroughly and then incubated with autologous PBMC at a ratio of 1DC: 10 PBMCs. After 13 days, cultures were stained with melan-A tetramer-PE, CD 8-FITC and 7-AAD. DCs are gated on the live population. % tetramer+/CD8+ T cells is given in each FACS plot.

    Journal: Journal of Viral Hepatitis

    Article Title: Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus

    doi: 10.1111/j.1365-2893.2007.00934.x

    Figure Lengend Snippet: The efficient expansion of melan-A specific CD8+ T cells from healthy and HCV infected individuals. In three representative paired, controlled experiments, dsRNA matured MD-DCs from healthy (left hand panel; HI-5, -8, -9) and HCV infected (right hand panel; HCV-9, -10, -11) individuals were pulsed with 1 μg/mL melan-A 26-35 peptide or RPMI medium, washed thoroughly and then incubated with autologous PBMC at a ratio of 1DC: 10 PBMCs. After 13 days, cultures were stained with melan-A tetramer-PE, CD 8-FITC and 7-AAD. DCs are gated on the live population. % tetramer+/CD8+ T cells is given in each FACS plot.

    Article Snippet: For the assessment of MD-DC in subjects with chronic HCV infection immature DCs were stimulated with dsRNA (Poly I:C, Sigma), 50 μg / mL for 36 h to generate mature DCs.

    Techniques: Infection, Incubation, Staining, FACS

    Cytokine production by maturing DCs derived from HCV infected and healthy individuals. Culture supernatants were sampled 36 h after the addition of dsRNA to immature DCs derived from HCV and healthy individuals. IL-10 and IL-12(p70) concentrations were measured by ELISA.

    Journal: Journal of Viral Hepatitis

    Article Title: Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus

    doi: 10.1111/j.1365-2893.2007.00934.x

    Figure Lengend Snippet: Cytokine production by maturing DCs derived from HCV infected and healthy individuals. Culture supernatants were sampled 36 h after the addition of dsRNA to immature DCs derived from HCV and healthy individuals. IL-10 and IL-12(p70) concentrations were measured by ELISA.

    Article Snippet: For the assessment of MD-DC in subjects with chronic HCV infection immature DCs were stimulated with dsRNA (Poly I:C, Sigma), 50 μg / mL for 36 h to generate mature DCs.

    Techniques: Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay

    Colocalization of dsRNA and E3 protein in infected cells. (A) A549 cells on coverslips were mock infected or infected with 5 PFU per cell of vD10rev, vD9muD10mu, or vΔE3L. After 13 h, the cells were fixed, permeabilized, and stained for dsRNA with J2 MAb (green) and for E3 with E3 MAb (red), followed by isotype-specific IgG2A and IgG3 fluorescent secondary antibodies, respectively, and DAPI. The cells were imaged by confocal microscopy. Arrows point to virus factories. The factory region within the boxed area was enlarged and is shown in the row below. Scale bar, 10 μm. (B) A549 cells were infected as described for panel A, stained with J2 MAb and fluorescent secondary antibody, and analyzed by flow cytometry. (C) The experiment is the same as that described for panel A except that cells were mock infected or infected with vD10rev-E3-GFP or vD9muD10mu-E3-GFP and stained with J2 MAb to detect dsRNA (red) followed by secondary fluorescent antibodies. E3 was visualized by GFP fluorescence. The factory region in the boxed area was enlarged and is shown in the row below. (D) A549 cells were infected with vD9muD10mu-E3-GFP, and lysates were analyzed by Western blotting with antibody to GFP and RIG-I or immunoprecipitated (IP) with J2 MAb or nonspecific IgG and then analyzed by Western blotting.

    Journal: Journal of Virology

    Article Title: Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants

    doi: 10.1128/JVI.00869-16

    Figure Lengend Snippet: Colocalization of dsRNA and E3 protein in infected cells. (A) A549 cells on coverslips were mock infected or infected with 5 PFU per cell of vD10rev, vD9muD10mu, or vΔE3L. After 13 h, the cells were fixed, permeabilized, and stained for dsRNA with J2 MAb (green) and for E3 with E3 MAb (red), followed by isotype-specific IgG2A and IgG3 fluorescent secondary antibodies, respectively, and DAPI. The cells were imaged by confocal microscopy. Arrows point to virus factories. The factory region within the boxed area was enlarged and is shown in the row below. Scale bar, 10 μm. (B) A549 cells were infected as described for panel A, stained with J2 MAb and fluorescent secondary antibody, and analyzed by flow cytometry. (C) The experiment is the same as that described for panel A except that cells were mock infected or infected with vD10rev-E3-GFP or vD9muD10mu-E3-GFP and stained with J2 MAb to detect dsRNA (red) followed by secondary fluorescent antibodies. E3 was visualized by GFP fluorescence. The factory region in the boxed area was enlarged and is shown in the row below. (D) A549 cells were infected with vD9muD10mu-E3-GFP, and lysates were analyzed by Western blotting with antibody to GFP and RIG-I or immunoprecipitated (IP) with J2 MAb or nonspecific IgG and then analyzed by Western blotting.

    Article Snippet: The following antibodies were purchased: phospho-(Ser396)-IRF3 (catalog number 4947), IRF3 (catalog number 11904), phospho-(Ser51)-eIF2α (catalog number 3398), and eIF2α (catalog number 5324) (all from Cell Signaling Technology); phospho (T446)-PKR (catalog number ab32036; Abcam); PKR (catalog number 700286; Life Technologies); mouse J2 MAb to dsRNA (SCICONS English and Scientific Consulting Kft); mouse anti-FLAG M2 (catalog number F1804) and actin (catalog number A2066) (both from Sigma); Xrn1 (catalog number A300-443A; Bethyl Laboratories).

    Techniques: Infection, Staining, Confocal Microscopy, Flow Cytometry, Cytometry, Fluorescence, Western Blot, Immunoprecipitation

    Synthesis of dsRNA in Xrn1 KO cells. (A) Control and Xrn1 KO A549 cells were mock infected or infected with 5 PFU per cell of purified vD10rev, vD9muD10mu, or vΔE3L, harvested after 13 h, and stained with J2 MAb and a secondary fluorescent antibody. Fluorescent cells were analyzed by flow cytometry. (B) A549 Xrn1 KO cells were infected as described for panel A, stained with J2 MAb, secondary fluorescent antibody, and DAPI, and examined by confocal microscopy. Scale bar, 10 µm.

    Journal: Journal of Virology

    Article Title: Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants

    doi: 10.1128/JVI.00869-16

    Figure Lengend Snippet: Synthesis of dsRNA in Xrn1 KO cells. (A) Control and Xrn1 KO A549 cells were mock infected or infected with 5 PFU per cell of purified vD10rev, vD9muD10mu, or vΔE3L, harvested after 13 h, and stained with J2 MAb and a secondary fluorescent antibody. Fluorescent cells were analyzed by flow cytometry. (B) A549 Xrn1 KO cells were infected as described for panel A, stained with J2 MAb, secondary fluorescent antibody, and DAPI, and examined by confocal microscopy. Scale bar, 10 µm.

    Article Snippet: The following antibodies were purchased: phospho-(Ser396)-IRF3 (catalog number 4947), IRF3 (catalog number 11904), phospho-(Ser51)-eIF2α (catalog number 3398), and eIF2α (catalog number 5324) (all from Cell Signaling Technology); phospho (T446)-PKR (catalog number ab32036; Abcam); PKR (catalog number 700286; Life Technologies); mouse J2 MAb to dsRNA (SCICONS English and Scientific Consulting Kft); mouse anti-FLAG M2 (catalog number F1804) and actin (catalog number A2066) (both from Sigma); Xrn1 (catalog number A300-443A; Bethyl Laboratories).

    Techniques: Infection, Purification, Staining, Flow Cytometry, Cytometry, Confocal Microscopy

    Akt phosphorylation of T308 and S473 are independent events. Panel A. A bar diagram showing independence of phosphorylation of Akt at T308 (▪) and S473 (□) in kinase assays of the 1-LN cancer cells stimulated with α 2 M* (50 pM/25 min) or insulin (200 nM/20 min). The bars are: (1) lipofectamine + buffer; (2) lipofectamine + α 2 M* (50 pM/25 min); (3) Raptor dsRNA (100 nM/48 h) then α 2 M*; (4) Rictor dsRNA (100 nM/48 h) then α 2 M*; (5) lipofectamine + insulin; (6) Raptor dsRNA (100 nM/48 h), then insulin (200 nM/20 min); (7) Rictor dsRNA (100 nM/48 h) + insulin; and (8) scrambled dsRNA (100 nM/48 h) then insulin. Values are mean ± SE from three experiments and are expressed as fmol [ 33 P]-γ-ATP incorporated/mg cell protein. Panel B. Levels of p-Akt T308 and p-Akt S743 in cell lysates of 1-LN cells treated with α 2 M* (50 pM/25 min) or insulin (200 nM/15 min). Representative immunoblots of p-Akt T308 and p-Akt S473 from three experiments of cells transfected with Raptor dsRNA or Rictor dsRNA as above are being shown.

    Journal: PLoS ONE

    Article Title: Receptor-Recognized ?2-Macroglobulin Binds to Cell Surface-Associated GRP78 and Activates mTORC1 and mTORC2 Signaling in Prostate Cancer Cells

    doi: 10.1371/journal.pone.0051735

    Figure Lengend Snippet: Akt phosphorylation of T308 and S473 are independent events. Panel A. A bar diagram showing independence of phosphorylation of Akt at T308 (▪) and S473 (□) in kinase assays of the 1-LN cancer cells stimulated with α 2 M* (50 pM/25 min) or insulin (200 nM/20 min). The bars are: (1) lipofectamine + buffer; (2) lipofectamine + α 2 M* (50 pM/25 min); (3) Raptor dsRNA (100 nM/48 h) then α 2 M*; (4) Rictor dsRNA (100 nM/48 h) then α 2 M*; (5) lipofectamine + insulin; (6) Raptor dsRNA (100 nM/48 h), then insulin (200 nM/20 min); (7) Rictor dsRNA (100 nM/48 h) + insulin; and (8) scrambled dsRNA (100 nM/48 h) then insulin. Values are mean ± SE from three experiments and are expressed as fmol [ 33 P]-γ-ATP incorporated/mg cell protein. Panel B. Levels of p-Akt T308 and p-Akt S743 in cell lysates of 1-LN cells treated with α 2 M* (50 pM/25 min) or insulin (200 nM/15 min). Representative immunoblots of p-Akt T308 and p-Akt S473 from three experiments of cells transfected with Raptor dsRNA or Rictor dsRNA as above are being shown.

    Article Snippet: Cells for the negative control were transfected with scrambled dsRNA (100 nM/48 h, Ambion) and then stimulated with either buffer, α2 M* or insulin as above.

    Techniques: Western Blot, Transfection

    α 2 M*-induced activation of mTORC1 as measured by phosphorylation of S6 Kinase (▪) and 4EBP1 (□) in prostate cancer cells. The phosphorylation of S6-Kinase and 4EBP1 was determined by [ 33 P]-γ-ATP incorporation and autoradiography (AR) and by immunoblotting (IB). Panel A. A bar diagram showing α 2 M*-induced activation of mTORC1 and its modulation by LY294002 and rapamycin in mTOR immunoprecipitates as determined by autoradiography. The bars and lanes in the autoradiographs are: (1) buffer; (2) α 2 M* (50 pM/25 min); (3) LY294002 (20 µM/20 min) then α 2 M* (50 pM/25 min); and (4) rapamycin (100 nM/15 min) then α 2 M* (50 pM/25 min). A representative autoradiograph (AR) of three individual experiments is shown below the bar diagram. The phosphorylation of S6-Kinase (▪) and 4EBP1 (□) is expressed in arbitrary fluorescence units and is the mean ± from three independent experiments. Also shown below AR is a representative immunoblot (IB) of three experiments of p-S6-Kinase and p-4EBP1 obtained from mTOR immunoprecipitates of cells treated as in autoradiographic analysis in Panel A. Panel B. A bar diagram showing the effect of silencing GRP78 expression by RNAi on α 2 M*-induced activation of mTORC1 in mTOR immunopreciptates in 1-LN cells as determined by autoradiography. The bars and the lanes in the autoradiograph (AR) are: (1) lipofectamine + buffer; (2) lipfectamine + α 2 M (50 pM/25 mins); (3) GRP78 dsRNA (100 nM/48h) then α 2 M* (to pM/25 min); and (4) scrambled dsRNA (100 nM/48h) than α 2 M* (to pM/25 min). A representative autoradiograph of S6-Kinase (▪) and 4EBP1 (□) of three experiments is shown below the bar diagram. The phosphorylation of S6-Kinase (▪) and 4EBP1 (□) is expressed in arbitrary fluorescence units as mean ± SE from three independent experiments. Also shown below the autoradiograph (AR) is a representative immunoblot (IB) of p-S6-Kinase and p-4EBP1 obtained from mTOR immunoprecipitates of 1-LN cells treated as in autoradiographic analysis in Panel B. An immunoblot representative of three experiments showing the expression of GRP78 in cells transfected with GRP78 dsRNA is also shown. The lanes in the immunoblot are: (1) lipofectamine + buffer; (2) lipofectamine + α 2 M* (50 pm/25 min); (3) GRP78 dsRNA (100 nM/48h) + α 2 M*; and (4) scrambled dsRNAi (100 nM/48h) + α 2 M*. Panel C. A bar diagram showing the effect of silencing GRP78 expression by RNAi on α 2 M*-induced phosphorylation of S6-Kinase (▪) and 4EBP1 (□) in Raptor immunoprecipitates of 1-LN cells as determined by autoradiography. The bars and lanes in the autoradiograph (AR) are identical to the bar diagram and autoradiograph in Panel B. A representative autoradiograph (AR) of S6-Kinase and 4EBP1 of three experiments is shown below the bar diagram. The phosphorylation of S6-Kinase (▪) and 4EBP1 (□) is expressed in arbitrary fluorescence units as the mean ± SE from three experiments. Also shown below the autoradiograph (AR) is a representative immunoblot (IB) of three experiments of p-S6-Kinase and p-4EBP1 obtained from Raptor immunoprecipitates of 1-LN cells treated as in the autoradiographic analysis in Panel C. Panel D. Autoradiograph showing α 2 M*-induced phosphorylation of S6-Kinase and 4EBP1 in DU-145 prostate cancer cells but not in PC-3 prostate cancer cells. The lanes in the autoradiograph are: (1) buffer; (2) α 2 M*. For details see

    Journal: PLoS ONE

    Article Title: Receptor-Recognized ?2-Macroglobulin Binds to Cell Surface-Associated GRP78 and Activates mTORC1 and mTORC2 Signaling in Prostate Cancer Cells

    doi: 10.1371/journal.pone.0051735

    Figure Lengend Snippet: α 2 M*-induced activation of mTORC1 as measured by phosphorylation of S6 Kinase (▪) and 4EBP1 (□) in prostate cancer cells. The phosphorylation of S6-Kinase and 4EBP1 was determined by [ 33 P]-γ-ATP incorporation and autoradiography (AR) and by immunoblotting (IB). Panel A. A bar diagram showing α 2 M*-induced activation of mTORC1 and its modulation by LY294002 and rapamycin in mTOR immunoprecipitates as determined by autoradiography. The bars and lanes in the autoradiographs are: (1) buffer; (2) α 2 M* (50 pM/25 min); (3) LY294002 (20 µM/20 min) then α 2 M* (50 pM/25 min); and (4) rapamycin (100 nM/15 min) then α 2 M* (50 pM/25 min). A representative autoradiograph (AR) of three individual experiments is shown below the bar diagram. The phosphorylation of S6-Kinase (▪) and 4EBP1 (□) is expressed in arbitrary fluorescence units and is the mean ± from three independent experiments. Also shown below AR is a representative immunoblot (IB) of three experiments of p-S6-Kinase and p-4EBP1 obtained from mTOR immunoprecipitates of cells treated as in autoradiographic analysis in Panel A. Panel B. A bar diagram showing the effect of silencing GRP78 expression by RNAi on α 2 M*-induced activation of mTORC1 in mTOR immunopreciptates in 1-LN cells as determined by autoradiography. The bars and the lanes in the autoradiograph (AR) are: (1) lipofectamine + buffer; (2) lipfectamine + α 2 M (50 pM/25 mins); (3) GRP78 dsRNA (100 nM/48h) then α 2 M* (to pM/25 min); and (4) scrambled dsRNA (100 nM/48h) than α 2 M* (to pM/25 min). A representative autoradiograph of S6-Kinase (▪) and 4EBP1 (□) of three experiments is shown below the bar diagram. The phosphorylation of S6-Kinase (▪) and 4EBP1 (□) is expressed in arbitrary fluorescence units as mean ± SE from three independent experiments. Also shown below the autoradiograph (AR) is a representative immunoblot (IB) of p-S6-Kinase and p-4EBP1 obtained from mTOR immunoprecipitates of 1-LN cells treated as in autoradiographic analysis in Panel B. An immunoblot representative of three experiments showing the expression of GRP78 in cells transfected with GRP78 dsRNA is also shown. The lanes in the immunoblot are: (1) lipofectamine + buffer; (2) lipofectamine + α 2 M* (50 pm/25 min); (3) GRP78 dsRNA (100 nM/48h) + α 2 M*; and (4) scrambled dsRNAi (100 nM/48h) + α 2 M*. Panel C. A bar diagram showing the effect of silencing GRP78 expression by RNAi on α 2 M*-induced phosphorylation of S6-Kinase (▪) and 4EBP1 (□) in Raptor immunoprecipitates of 1-LN cells as determined by autoradiography. The bars and lanes in the autoradiograph (AR) are identical to the bar diagram and autoradiograph in Panel B. A representative autoradiograph (AR) of S6-Kinase and 4EBP1 of three experiments is shown below the bar diagram. The phosphorylation of S6-Kinase (▪) and 4EBP1 (□) is expressed in arbitrary fluorescence units as the mean ± SE from three experiments. Also shown below the autoradiograph (AR) is a representative immunoblot (IB) of three experiments of p-S6-Kinase and p-4EBP1 obtained from Raptor immunoprecipitates of 1-LN cells treated as in the autoradiographic analysis in Panel C. Panel D. Autoradiograph showing α 2 M*-induced phosphorylation of S6-Kinase and 4EBP1 in DU-145 prostate cancer cells but not in PC-3 prostate cancer cells. The lanes in the autoradiograph are: (1) buffer; (2) α 2 M*. For details see "Experimental Procedures" section. The autoradiograph shown is representative of four experiments. Panel E. A bar diagram showing that antibodies against the carboxyl-terminal domain of GRP78 inhibit α 2 M*-induced phosphorylation of S6-Kinase (▪) and 4EBP1 (□) in mTOR immunoprecipitates of 1-LN cells. The bars are: (1) buffer; (2) α 2 M* (50 pM/25 min); (3) antibodies against the carboxyl-terminal domain of GRP78 (3 µg/ml/1 h) then α 2 M*. The phosphorylation of S6-Kinase and 4EBP1 was determined by autoradiographic analysis and is expressed in arbitrary units and is the mean ± SE from three experiments. The values significantly different at 5% for α 2 M* and scrambled dsRNA-treated cells are denoted by an asterisk (*) in all the Panels A to E.

    Article Snippet: Cells for the negative control were transfected with scrambled dsRNA (100 nM/48 h, Ambion) and then stimulated with either buffer, α2 M* or insulin as above.

    Techniques: Activation Assay, Autoradiography, Fluorescence, Expressing, Transfection

    Effect of silencing Raptor expression by RNAi on phosphorylation of S6 kinase and 4EBP1 in 1-LN cells stimulated with α 2 M* and insulin. Panel A. Bar diagram showing levels of Raptor in cells transfected with Raptor dsRNA and stimulated with α 2 M* or insulin. The bars are: (1) lipofectamine + buffer; (2) lipofectamine + α 2 M* (50 pM/25 min); (3) lipofectamine + insulin (200 nM/20 min); (4) scrambled dsRNA (100 nM/48 h) + insulin; (5) Raptor dsRNA (100 nM/48 h); (6) Raptor dsRNA (100 nM/48 h) then α 2 M*; (7) Raptor dsRNA (100 nM/48 h) then insulin (200 nM/20 min); (8) rapamycin (100 nM/20 min) then insulin. Values are mean ± SE from three to four independent experiments and are expressed as arbitrary fluorescence units. Values significantly different at 5% level for α 2 M*, insulin and scrambled dsRNA treated cells are indicated by an asterisk (*). A representative immunoblot of Raptor from three to four experiments along with its protein loading control actin is shown below the bar diagram. Panel B. A bar diagram showing mTORC1 activation in cells treated with α 2 M* and insulin and effect of transfection with Raptor dsRNA on its activation as measured by assaying phosphorylation of S6-Kinase by autoradiography. The bars and lanes in autoradiograph are: (1) lipofectamine + buffer; (2) lipofectamine + α 2 M* (50 pM/25 min); (3) Raptor dsRNA (100 nM/48 h); (4) Raptor dsRNA + α 2 M*; (5) lipofectamine + insulin (200 nM/20 min); (6) scrambled dsRNA (100 nM/48 h) + insulin; (7) Raptor dsRNA (100 nM/20 min) then insulin; (8) rapamycin (100 nM/20 min) then α 2 M*; (9) LY294002 (20 mM/20 min) then insulin; (10) rapamycin (100 nM/20 min) then insulin. A representative autoradiograph of three experiments of S6-Kinase phosphorylation is shown below the bar diagram. Values are the mean ± SE from three experiments and are expressed in arbitrary units. Values significantly different at 5% levels for α 2 M*, insulin and scrambled dsRNA treated cells are indicated by an asterisk (*). Panel C. A bar diagram showing levels of S6-Kinase phosphorylated at T389 (▪); T229 (see grey square) and T 235/236 (□) in cells stimulated with α 2 M* or insulin and transfected with Raptor dsRNA. The bars are as in Panel A. Representative immunoblots of p-S6-Kinase T389 , p-S6-Kinase T229 and p-S6-Kinase T235/236 of three experiments along with its protein loading control is shown below the bar diagram. Values are the mean ± SE from three experiments and are expressed in arbitrary fluorescence units. Values significantly different at 5% level for α 2 M*, insulin or scrambled dsRNA are indicated by an asterisk (*). Panel D. A bar diagram showing phosphorylation levels of 4EBP1, in cells treated with α 2 M* and insulin or transfected with Raptor dsRNA. The bars are as in Panel A. A representative immunoblot of p-4EBP1 of three experiments along with its protein loading control is shown below the bar diagram. Values are the mean ± SE from three experiments and are expressed in arbitrary fluorescence units. Values significantly different at 5% level for α 2 M*, insulin and scrambled dsRNA-treated cells are indicated by an asterisk (*).

    Journal: PLoS ONE

    Article Title: Receptor-Recognized ?2-Macroglobulin Binds to Cell Surface-Associated GRP78 and Activates mTORC1 and mTORC2 Signaling in Prostate Cancer Cells

    doi: 10.1371/journal.pone.0051735

    Figure Lengend Snippet: Effect of silencing Raptor expression by RNAi on phosphorylation of S6 kinase and 4EBP1 in 1-LN cells stimulated with α 2 M* and insulin. Panel A. Bar diagram showing levels of Raptor in cells transfected with Raptor dsRNA and stimulated with α 2 M* or insulin. The bars are: (1) lipofectamine + buffer; (2) lipofectamine + α 2 M* (50 pM/25 min); (3) lipofectamine + insulin (200 nM/20 min); (4) scrambled dsRNA (100 nM/48 h) + insulin; (5) Raptor dsRNA (100 nM/48 h); (6) Raptor dsRNA (100 nM/48 h) then α 2 M*; (7) Raptor dsRNA (100 nM/48 h) then insulin (200 nM/20 min); (8) rapamycin (100 nM/20 min) then insulin. Values are mean ± SE from three to four independent experiments and are expressed as arbitrary fluorescence units. Values significantly different at 5% level for α 2 M*, insulin and scrambled dsRNA treated cells are indicated by an asterisk (*). A representative immunoblot of Raptor from three to four experiments along with its protein loading control actin is shown below the bar diagram. Panel B. A bar diagram showing mTORC1 activation in cells treated with α 2 M* and insulin and effect of transfection with Raptor dsRNA on its activation as measured by assaying phosphorylation of S6-Kinase by autoradiography. The bars and lanes in autoradiograph are: (1) lipofectamine + buffer; (2) lipofectamine + α 2 M* (50 pM/25 min); (3) Raptor dsRNA (100 nM/48 h); (4) Raptor dsRNA + α 2 M*; (5) lipofectamine + insulin (200 nM/20 min); (6) scrambled dsRNA (100 nM/48 h) + insulin; (7) Raptor dsRNA (100 nM/20 min) then insulin; (8) rapamycin (100 nM/20 min) then α 2 M*; (9) LY294002 (20 mM/20 min) then insulin; (10) rapamycin (100 nM/20 min) then insulin. A representative autoradiograph of three experiments of S6-Kinase phosphorylation is shown below the bar diagram. Values are the mean ± SE from three experiments and are expressed in arbitrary units. Values significantly different at 5% levels for α 2 M*, insulin and scrambled dsRNA treated cells are indicated by an asterisk (*). Panel C. A bar diagram showing levels of S6-Kinase phosphorylated at T389 (▪); T229 (see grey square) and T 235/236 (□) in cells stimulated with α 2 M* or insulin and transfected with Raptor dsRNA. The bars are as in Panel A. Representative immunoblots of p-S6-Kinase T389 , p-S6-Kinase T229 and p-S6-Kinase T235/236 of three experiments along with its protein loading control is shown below the bar diagram. Values are the mean ± SE from three experiments and are expressed in arbitrary fluorescence units. Values significantly different at 5% level for α 2 M*, insulin or scrambled dsRNA are indicated by an asterisk (*). Panel D. A bar diagram showing phosphorylation levels of 4EBP1, in cells treated with α 2 M* and insulin or transfected with Raptor dsRNA. The bars are as in Panel A. A representative immunoblot of p-4EBP1 of three experiments along with its protein loading control is shown below the bar diagram. Values are the mean ± SE from three experiments and are expressed in arbitrary fluorescence units. Values significantly different at 5% level for α 2 M*, insulin and scrambled dsRNA-treated cells are indicated by an asterisk (*).

    Article Snippet: Cells for the negative control were transfected with scrambled dsRNA (100 nM/48 h, Ambion) and then stimulated with either buffer, α2 M* or insulin as above.

    Techniques: Expressing, Transfection, Fluorescence, Activation Assay, Autoradiography, Western Blot

    Next-generation sequencing of totiviruses and dsRNA satellites from Saccharomyces cerevisiae strain BJH001. ( A ) Read quality (phred score) after Illumina QC shown along the length of the sequencing reads when using different concentrations of poly(A) polymerase and oligo(dT) or anchored oligo(dT) primers; 10%, 25%, 75%, and 90% quantile and median (50% quantile) read quality at each position along the reads are shown. ( B ) Sequence contigs after de novo assembled represented by contig coverage and contig length. BLAST analysis of the four contigs with the longest length and deepest coverage enabled their identification as totiviruses (ScV-L-A1 and ScV-L-BC) and a dsRNA satellite (ScV-M1), the latter was assembled as two separate contigs. Inset reverse transcriptase-PCR was used to confirm the presence of each type of dsRNA. Two primer pairs were used to amplify the ScV-L-BC. ( C ) Read depth coverage across the reference-assembled ScV-L-A, ScV-L-BC, and ScV-M1 contigs. Open reading frames present within each dsRNA are shown above the nucleotide position.

    Journal: Viruses

    Article Title: A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae

    doi: 10.3390/v11010070

    Figure Lengend Snippet: Next-generation sequencing of totiviruses and dsRNA satellites from Saccharomyces cerevisiae strain BJH001. ( A ) Read quality (phred score) after Illumina QC shown along the length of the sequencing reads when using different concentrations of poly(A) polymerase and oligo(dT) or anchored oligo(dT) primers; 10%, 25%, 75%, and 90% quantile and median (50% quantile) read quality at each position along the reads are shown. ( B ) Sequence contigs after de novo assembled represented by contig coverage and contig length. BLAST analysis of the four contigs with the longest length and deepest coverage enabled their identification as totiviruses (ScV-L-A1 and ScV-L-BC) and a dsRNA satellite (ScV-M1), the latter was assembled as two separate contigs. Inset reverse transcriptase-PCR was used to confirm the presence of each type of dsRNA. Two primer pairs were used to amplify the ScV-L-BC. ( C ) Read depth coverage across the reference-assembled ScV-L-A, ScV-L-BC, and ScV-M1 contigs. Open reading frames present within each dsRNA are shown above the nucleotide position.

    Article Snippet: Discussion The methods that we describe constitute a broadly applicable approach to the sequencing of dsRNA purified from fungi using Illumina NGS.

    Techniques: Next-Generation Sequencing, Sequencing, Polymerase Chain Reaction

    Effects of simvastatin (Sim) and dexamethasone (Dex) on IRF3 phosphorylation in bronchial epithelial cells from COPD donors. dsRNA increased phosphorylation of IRF3 (A,B) without changing IRF3 levels (B). Dexamethasone was without significant effects

    Journal: British Journal of Pharmacology

    Article Title: Selective inhibition by simvastatin of IRF3 phosphorylation and TSLP production in dsRNA-challenged bronchial epithelial cells from COPD donors

    doi: 10.1111/j.1476-5381.2012.02131.x

    Figure Lengend Snippet: Effects of simvastatin (Sim) and dexamethasone (Dex) on IRF3 phosphorylation in bronchial epithelial cells from COPD donors. dsRNA increased phosphorylation of IRF3 (A,B) without changing IRF3 levels (B). Dexamethasone was without significant effects

    Article Snippet: IRF3 is phosphorylated in response to viral infection or dsRNA (Fitzgerald et al ., ; Chow et al ., ) (this study).

    Techniques:

    RNA binding to midgut cells of western corn rootworm (WCR). (A) Cy3-labeled 210 dvssj1 dsRNA is binding to WCR midgut cells (Top panel), but 21-bp siRNA of dvssj1 is not observed (middle panel). Commercial Cy3 dye was used as a control, which showed no binding also. (B) Summary of the fluorescent intensity of midgut cells. Corrected Total Cell Fluorescence (CTCF) was calculated in ImageJ. DsRNA treatment (n = 11) is significantly different (P

    Journal: PLoS ONE

    Article Title: Molecular characterization of the insecticidal activity of double-stranded RNA targeting the smooth septate junction of western corn rootworm (Diabrotica virgifera virgifera)

    doi: 10.1371/journal.pone.0210491

    Figure Lengend Snippet: RNA binding to midgut cells of western corn rootworm (WCR). (A) Cy3-labeled 210 dvssj1 dsRNA is binding to WCR midgut cells (Top panel), but 21-bp siRNA of dvssj1 is not observed (middle panel). Commercial Cy3 dye was used as a control, which showed no binding also. (B) Summary of the fluorescent intensity of midgut cells. Corrected Total Cell Fluorescence (CTCF) was calculated in ImageJ. DsRNA treatment (n = 11) is significantly different (P

    Article Snippet: For Cy3-labelling of dsRNA, 25% of CTP and UTP were replaced with Cy3-CTP and Cy3-UTP (GE Healthcare, UK).

    Techniques: RNA Binding Assay, Western Blot, Labeling, Binding Assay, Fluorescence

    Knockdown of Mfn1/Mfn2 and Drp1/Fis1 reduces mitochondrial fusion and fission in DM cybrid, respectively. siRNA transfection was performed to knockdown target gene expression. Scramble dsRNA (scr) was used as siRNA negative control. (a) Abundance of dynamic proteins Mfn1, Mfn2, Drp1, and Fis1 was determined using Western blotting. β -Actin served as loading control. (b) Mitochondrial morphology was visualized by transfecting cox4-DsRed (red fluorescence). An enlarged segment of each image was shown by a lower right square. (c) The MicroP algorithm categorized mitochondrial morphology into six types: small globe (blue), large globe (yellow), simple tube (green), twisted tube (orange), donut (red), and branching tube (purple). N = 75–400 mitochondria from 15–30 cells and three independent experiments.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: The Causal Role of Mitochondrial Dynamics in Regulating Insulin Resistance in Diabetes: Link through Mitochondrial Reactive Oxygen Species

    doi: 10.1155/2018/7514383

    Figure Lengend Snippet: Knockdown of Mfn1/Mfn2 and Drp1/Fis1 reduces mitochondrial fusion and fission in DM cybrid, respectively. siRNA transfection was performed to knockdown target gene expression. Scramble dsRNA (scr) was used as siRNA negative control. (a) Abundance of dynamic proteins Mfn1, Mfn2, Drp1, and Fis1 was determined using Western blotting. β -Actin served as loading control. (b) Mitochondrial morphology was visualized by transfecting cox4-DsRed (red fluorescence). An enlarged segment of each image was shown by a lower right square. (c) The MicroP algorithm categorized mitochondrial morphology into six types: small globe (blue), large globe (yellow), simple tube (green), twisted tube (orange), donut (red), and branching tube (purple). N = 75–400 mitochondria from 15–30 cells and three independent experiments.

    Article Snippet: Mock control of gene overexpression and siRNA was GFP-expression vector (OriGene-ORIPS100010, Origene Technologies, Inc., Rockville, MD, USA) and dsRNA with scramble sequence (sc-37007; Santa Cruz Biotechnology, Santa Cruz, CA), respectively.

    Techniques: Transfection, Expressing, Negative Control, Western Blot, Fluorescence

    Epigenetic modifications post dsRNA transfection. (A)Chromatin remodeling after S1, S5 or S9 dsRNA transfection. Significant reduction in MNase digestion 72 hours after S1 and S5 transfection while increase in the accessibilty after S9 transfection in HeLa cells. Cells were transfected with the respective dsRNAs followed by MNase-PCR 72 hours post transfection. S1:S1 dsRNA transfected cells, S5-dsRNA: S5 dsRNA transfected cells, S9-dsRNA: S9 dsRNA transfected cells, Control-dsRNA: Control dsRNA transfected cells (*) P

    Journal: PLoS ONE

    Article Title: Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region

    doi: 10.1371/journal.pone.0128416

    Figure Lengend Snippet: Epigenetic modifications post dsRNA transfection. (A)Chromatin remodeling after S1, S5 or S9 dsRNA transfection. Significant reduction in MNase digestion 72 hours after S1 and S5 transfection while increase in the accessibilty after S9 transfection in HeLa cells. Cells were transfected with the respective dsRNAs followed by MNase-PCR 72 hours post transfection. S1:S1 dsRNA transfected cells, S5-dsRNA: S5 dsRNA transfected cells, S9-dsRNA: S9 dsRNA transfected cells, Control-dsRNA: Control dsRNA transfected cells (*) P

    Article Snippet: Twenty four hours later transfection was carried out with dsRNAs (Eurofins, MWG Germany) or ODNs (IDT, USA) at 100nM concentration using OligofectamineTransfection Reagent (Life Technologies) as per the manufacturer's protocol.

    Techniques: Transfection, Polymerase Chain Reaction

    Screening of dsRNAs targeting HPV18 pRNAs. (A)Schematic representation of HPV18 LCR and the position of different dsRNA target sites. (B) C-4 II cells transfected with seven dsRNAs (100nM). C) S1 and S5 dsRNA transfection in HeLa cells (D) C-4 I cells transfected with S1 and S5 dsRNAs. Cells were transfected with the respective dsRNA and RNA was isolated 72 hours post transfection and analyzed by Real-Time RT-PCR. Expression ratio was calculated with respect to 18S rRNA, POLR2A, PPIA and β-actin by REST software and normalized to that of Control dsRNA. E6: Expression of E6 oncogene upon dsRNA transfection. E7: Expression of E7 oncogene upon dsRNA transfection. Control-dsRNA: Control dsRNA transfected cells, (*) P

    Journal: PLoS ONE

    Article Title: Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region

    doi: 10.1371/journal.pone.0128416

    Figure Lengend Snippet: Screening of dsRNAs targeting HPV18 pRNAs. (A)Schematic representation of HPV18 LCR and the position of different dsRNA target sites. (B) C-4 II cells transfected with seven dsRNAs (100nM). C) S1 and S5 dsRNA transfection in HeLa cells (D) C-4 I cells transfected with S1 and S5 dsRNAs. Cells were transfected with the respective dsRNA and RNA was isolated 72 hours post transfection and analyzed by Real-Time RT-PCR. Expression ratio was calculated with respect to 18S rRNA, POLR2A, PPIA and β-actin by REST software and normalized to that of Control dsRNA. E6: Expression of E6 oncogene upon dsRNA transfection. E7: Expression of E7 oncogene upon dsRNA transfection. Control-dsRNA: Control dsRNA transfected cells, (*) P

    Article Snippet: Twenty four hours later transfection was carried out with dsRNAs (Eurofins, MWG Germany) or ODNs (IDT, USA) at 100nM concentration using OligofectamineTransfection Reagent (Life Technologies) as per the manufacturer's protocol.

    Techniques: Transfection, Isolation, Quantitative RT-PCR, Expressing, Software

    pRNA mediated gene activation. (A) Screening of three dsRNAs (100nM) targeting non-CpG sites in the pRNA of HPV18 in HeLa cells. RNA from the transfected cells was analyzed by Real time PCR 72 hours post transfection. Expression ratio was calculated with respect to 18S, POLR2A, PPIA andβ-actin by REST software. S8-dsRNA: S8 dsRNA transfected cells, S9-dsRNA: S9 dsRNA transfected cells, S10-dsRNA: S10 dsRNA transfected cells. Control: Control dsRNA transfected cells. (*) P

    Journal: PLoS ONE

    Article Title: Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region

    doi: 10.1371/journal.pone.0128416

    Figure Lengend Snippet: pRNA mediated gene activation. (A) Screening of three dsRNAs (100nM) targeting non-CpG sites in the pRNA of HPV18 in HeLa cells. RNA from the transfected cells was analyzed by Real time PCR 72 hours post transfection. Expression ratio was calculated with respect to 18S, POLR2A, PPIA andβ-actin by REST software. S8-dsRNA: S8 dsRNA transfected cells, S9-dsRNA: S9 dsRNA transfected cells, S10-dsRNA: S10 dsRNA transfected cells. Control: Control dsRNA transfected cells. (*) P

    Article Snippet: Twenty four hours later transfection was carried out with dsRNAs (Eurofins, MWG Germany) or ODNs (IDT, USA) at 100nM concentration using OligofectamineTransfection Reagent (Life Technologies) as per the manufacturer's protocol.

    Techniques: Activation Assay, Transfection, Real-time Polymerase Chain Reaction, Expressing, Software

    pRNA expression in HeLa cells. Cells were transfected with S1 or S5 dsRNAs (100nM) and analyzed for pRNA expression 72 hours post transfection. Analysis was done with Real Time PCR using pRNA specific primers. S1 and S5 transfection showed significant (p

    Journal: PLoS ONE

    Article Title: Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region

    doi: 10.1371/journal.pone.0128416

    Figure Lengend Snippet: pRNA expression in HeLa cells. Cells were transfected with S1 or S5 dsRNAs (100nM) and analyzed for pRNA expression 72 hours post transfection. Analysis was done with Real Time PCR using pRNA specific primers. S1 and S5 transfection showed significant (p

    Article Snippet: Twenty four hours later transfection was carried out with dsRNAs (Eurofins, MWG Germany) or ODNs (IDT, USA) at 100nM concentration using OligofectamineTransfection Reagent (Life Technologies) as per the manufacturer's protocol.

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction

    zic-1 inhibits Wnt signaling to promote head outgrowth. (A) Single and double-RNAi as indicated to examine interactions between zic-1 and beta-catenin-1 . Total concentrations of dsRNA were normalized by control dsRNA so that the absolute amount of each utilized dsRNA was equivalent across treatments. zic-1(RNAi);beta-catenin-1(RNAi) animals all regenerated heads after decapitation suggesting that beta-catenin-1 inhibition is required for the zic-1 's head-promoting function. (B) Scoring percent of anterior regeneration blastema with 0, 1, or 2 photoreceptors (PR) (n > 9 animals). (C) qPCR analysis of zic-1 expression versus gapdh in animals treated with dsRNA as indicated. zic-1 dsRNA reduced zic-1 mRNA levels (p

    Journal: PLoS Genetics

    Article Title: zic-1 Expression in Planarian Neoblasts after Injury Controls Anterior Pole Regeneration

    doi: 10.1371/journal.pgen.1004452

    Figure Lengend Snippet: zic-1 inhibits Wnt signaling to promote head outgrowth. (A) Single and double-RNAi as indicated to examine interactions between zic-1 and beta-catenin-1 . Total concentrations of dsRNA were normalized by control dsRNA so that the absolute amount of each utilized dsRNA was equivalent across treatments. zic-1(RNAi);beta-catenin-1(RNAi) animals all regenerated heads after decapitation suggesting that beta-catenin-1 inhibition is required for the zic-1 's head-promoting function. (B) Scoring percent of anterior regeneration blastema with 0, 1, or 2 photoreceptors (PR) (n > 9 animals). (C) qPCR analysis of zic-1 expression versus gapdh in animals treated with dsRNA as indicated. zic-1 dsRNA reduced zic-1 mRNA levels (p

    Article Snippet: For RNAi by injection, dsRNA was synthesized from in vitro transcription reactions (Promega) using PCR templates with flanking T7 sequences (Denville), purified by phenol extraction and ethanol precipitation, resuspended in RNase-free water and annealed at 65°C for 10 minutes, 37°C for 20–30 minutes, then on ice for at least 10 minutes. dsRNA corresponding to C. elegans unc-22 , a gene not present in the planarian genome, served as a negative control.

    Techniques: Inhibition, Real-time Polymerase Chain Reaction, Expressing

    zic-1 controls utilization of foxD+smedwi-1+ progenitors for notum expression and anterior pole formation. (A) Measurement of expression changes in X1 neoblasts due to treatment with zic-1 versus control dsRNA. Trunk fragments were macerated 5 minutes (0 days), 24 hours (1 day) and 48 hours (2 days) after head and tail amputation, and X1 cells were isolated from 3 independent biological replicates of 10 worms each. qPCR examined putative expression changes in a cohort of 15 transcription factors and signaling molecules that mark neoblast subpopulations induced by regeneration. Cartoon depicts experimental design. Heat map shows log 2 fold-changes in gene expression for the indicated genes in zic-1(RNAi) X1 neoblasts versus controls averaged from three biological replicates for each condition. Blue color indicates downregulation and yellow indicates upregulation, due to zic-1 RNAi. (B–D) Double FISH to detect expression of zic-1 , foxD , smedwi-1 and/or notum with nuclei counterstained with Hoechst (gray). (B) In situ hybridization probing to detect notum (magenta) and smedwi-1 (green) to detect notum+smedwi-1+ cells. (B, top) Representative image of notum+smedwi-1 + cells, (B, bottom) quantification of notum+smedwi-1+ cells. zic-1(RNAi) reduced numbers of notum+ neoblasts by 48 hours and smedwi-1+ cells were more numerous in anterior than posterior. (C) foxD (magenta) expression in smedwi-1+ (green) neoblasts 24 hours after injury was not eliminated due to zic-1 RNAi. Bottom, quantification of foxD+smedwi-1+ cells from control and zic-1(RNAi) animals fixed 24 hours after head and tail amputation and probed by double FISH. foxD+smedwi-1+ cells were produced in equal numbers at anterior- versus posterior-facing wound sites and were not reduced by zic-1 inhibition (control animals, 29±1 foxD+smedwi-1+ cells in anterior- versus 32±4 in posterior-facing wounds; zic-1 RNAi animals, 19±5 foxD+smedwi-1+ cells in anterior- versus 26±6 in posterior-facing wounds, n = 3 worms each). (D) foxD (green) is expressed in a subpopulation of zic-1+ cells (magenta, 3.4±1.8% of 420 zic-1 + cells were foxD +, n = 5 worms) by 24 hours. Yellow arrow indicates double positive cell (also inset), and white arrows indicate zic-1+ cells. Cartoons show surgeries and zoomed areas. Error bars standard deviations, asterisks p-value

    Journal: PLoS Genetics

    Article Title: zic-1 Expression in Planarian Neoblasts after Injury Controls Anterior Pole Regeneration

    doi: 10.1371/journal.pgen.1004452

    Figure Lengend Snippet: zic-1 controls utilization of foxD+smedwi-1+ progenitors for notum expression and anterior pole formation. (A) Measurement of expression changes in X1 neoblasts due to treatment with zic-1 versus control dsRNA. Trunk fragments were macerated 5 minutes (0 days), 24 hours (1 day) and 48 hours (2 days) after head and tail amputation, and X1 cells were isolated from 3 independent biological replicates of 10 worms each. qPCR examined putative expression changes in a cohort of 15 transcription factors and signaling molecules that mark neoblast subpopulations induced by regeneration. Cartoon depicts experimental design. Heat map shows log 2 fold-changes in gene expression for the indicated genes in zic-1(RNAi) X1 neoblasts versus controls averaged from three biological replicates for each condition. Blue color indicates downregulation and yellow indicates upregulation, due to zic-1 RNAi. (B–D) Double FISH to detect expression of zic-1 , foxD , smedwi-1 and/or notum with nuclei counterstained with Hoechst (gray). (B) In situ hybridization probing to detect notum (magenta) and smedwi-1 (green) to detect notum+smedwi-1+ cells. (B, top) Representative image of notum+smedwi-1 + cells, (B, bottom) quantification of notum+smedwi-1+ cells. zic-1(RNAi) reduced numbers of notum+ neoblasts by 48 hours and smedwi-1+ cells were more numerous in anterior than posterior. (C) foxD (magenta) expression in smedwi-1+ (green) neoblasts 24 hours after injury was not eliminated due to zic-1 RNAi. Bottom, quantification of foxD+smedwi-1+ cells from control and zic-1(RNAi) animals fixed 24 hours after head and tail amputation and probed by double FISH. foxD+smedwi-1+ cells were produced in equal numbers at anterior- versus posterior-facing wound sites and were not reduced by zic-1 inhibition (control animals, 29±1 foxD+smedwi-1+ cells in anterior- versus 32±4 in posterior-facing wounds; zic-1 RNAi animals, 19±5 foxD+smedwi-1+ cells in anterior- versus 26±6 in posterior-facing wounds, n = 3 worms each). (D) foxD (green) is expressed in a subpopulation of zic-1+ cells (magenta, 3.4±1.8% of 420 zic-1 + cells were foxD +, n = 5 worms) by 24 hours. Yellow arrow indicates double positive cell (also inset), and white arrows indicate zic-1+ cells. Cartoons show surgeries and zoomed areas. Error bars standard deviations, asterisks p-value

    Article Snippet: For RNAi by injection, dsRNA was synthesized from in vitro transcription reactions (Promega) using PCR templates with flanking T7 sequences (Denville), purified by phenol extraction and ethanol precipitation, resuspended in RNase-free water and annealed at 65°C for 10 minutes, 37°C for 20–30 minutes, then on ice for at least 10 minutes. dsRNA corresponding to C. elegans unc-22 , a gene not present in the planarian genome, served as a negative control.

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Fluorescence In Situ Hybridization, In Situ Hybridization, Produced, Inhibition

    G3BP1 antagonizes RNF125-mediated degradation of RIG-I. a , b Dose-dependent effects of G3BP1 on the expression of RIG-I or IRF3. HEK293T cells (4 × 10 5 ) were transfected with the G3BP1 (0, 1, and 2 μg) and the RIG-I or IRF3 (2 μg) plasmids for 24 h. Then the cell lysates were subjected to immunoblotting with the indicated antibodies. For the HA-RIG-I, HA-G3BP1 and HA-IRF3, band intensities were determined by Image J software. c Effects of inhibitors on G3BP1-mediated stabilization of RIG-I. HEK293T cells (4 × 10 5 ) were transfected with the indicated plasmids for 18 h and then cells were treated with the indicated inhibitors for 6 h before immunoblotting analysis. For the Flag-RIG-I, band intensities were determined by Image J software. d , e Effects of G3BP1 on ubiquitination of RIG-I mediated by RNF125 or RNF125 (C72/75 A) mutant. HEK293T cells (2 × 10 6 ) were transfected with RIG-I (10 μg), G3BP1 (3 μg), HA-Ub (2 μg), and RNF125 or RNF125 (C72/75 A) (5 μg) for 24 h. Co-IP and immunoblotting analysis were performed with the indicated antibodies. f Effects of G3BP1 knockdown on RNF125-mediated ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were transfected with RIG-I (10 μg), HA-Ub (2 μg), and RNF125 (5 μg) for 24 h. Then Co-IP and immunoblotting were performed with the indicated antibodies. g Effects of G3BP1 on K48-linked ubiquitination of RIG-I mediated by RNF125. The experiments were similarly to those described in d . h Effects of G3BP1 knockdown on K48-linked ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. i Effects of G3BP1 knockout on K48-linked ubiquitination of RIG-I. The experiments were similarly to those described in h . j G3BP1 binds to 5´ppp-dsRNA and enhances the binding of RIG-I to 5´ppp-dsRNA. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were first incubated with biotinylated-5´ppp-dsRNA and streptavidin-Sepharose. Then conjugated proteins were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Co-IP Co-immunoprecipitation, αF anti-Flag, Coni control RNAi, KO knockout, WT wild-type.

    Journal: Cell Death & Disease

    Article Title: G3BP1 inhibits RNA virus replication by positively regulating RIG-I-mediated cellular antiviral response

    doi: 10.1038/s41419-019-2178-9

    Figure Lengend Snippet: G3BP1 antagonizes RNF125-mediated degradation of RIG-I. a , b Dose-dependent effects of G3BP1 on the expression of RIG-I or IRF3. HEK293T cells (4 × 10 5 ) were transfected with the G3BP1 (0, 1, and 2 μg) and the RIG-I or IRF3 (2 μg) plasmids for 24 h. Then the cell lysates were subjected to immunoblotting with the indicated antibodies. For the HA-RIG-I, HA-G3BP1 and HA-IRF3, band intensities were determined by Image J software. c Effects of inhibitors on G3BP1-mediated stabilization of RIG-I. HEK293T cells (4 × 10 5 ) were transfected with the indicated plasmids for 18 h and then cells were treated with the indicated inhibitors for 6 h before immunoblotting analysis. For the Flag-RIG-I, band intensities were determined by Image J software. d , e Effects of G3BP1 on ubiquitination of RIG-I mediated by RNF125 or RNF125 (C72/75 A) mutant. HEK293T cells (2 × 10 6 ) were transfected with RIG-I (10 μg), G3BP1 (3 μg), HA-Ub (2 μg), and RNF125 or RNF125 (C72/75 A) (5 μg) for 24 h. Co-IP and immunoblotting analysis were performed with the indicated antibodies. f Effects of G3BP1 knockdown on RNF125-mediated ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were transfected with RIG-I (10 μg), HA-Ub (2 μg), and RNF125 (5 μg) for 24 h. Then Co-IP and immunoblotting were performed with the indicated antibodies. g Effects of G3BP1 on K48-linked ubiquitination of RIG-I mediated by RNF125. The experiments were similarly to those described in d . h Effects of G3BP1 knockdown on K48-linked ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. i Effects of G3BP1 knockout on K48-linked ubiquitination of RIG-I. The experiments were similarly to those described in h . j G3BP1 binds to 5´ppp-dsRNA and enhances the binding of RIG-I to 5´ppp-dsRNA. HEK293T cells (2 × 10 6 ) were transfected with the indicated plasmids (5 μg each). Cell lysates were first incubated with biotinylated-5´ppp-dsRNA and streptavidin-Sepharose. Then conjugated proteins were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Co-IP Co-immunoprecipitation, αF anti-Flag, Coni control RNAi, KO knockout, WT wild-type.

    Article Snippet: Reagents, virus, and cells Antibodies against Myc-tag, G3BP1, RIG-I, p-P65, P65, p-IRF3, IRF3, p-IκBα, and IκBα were obtained from Cell Signaling Technology; anti-Flag, anti-HA, and anti-β-actin were from Sigma; horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were from Thermo; HRP-conjugated anti-goat IgG was from Zhong Shan Jin Qiao; MG132 and 3-MA were from Sigma; RNF125 and K48-Ub were from Abcam; Ub was from Santa Cruz Biotechnology; 5´ppp-dsRNA was from InvivoGen; EZ-link Psoralen-PEG3-Biotin and Streptavidin agarose resin were from Thermo Fisher.

    Techniques: Expressing, Transfection, Software, Mutagenesis, Co-Immunoprecipitation Assay, Infection, Knock-Out, Binding Assay, Incubation, Immunoprecipitation

    Smurf2 is required for stability and functional localization of Mad2. (a) Smurf2 silencing rapidly down-regulates Mad2 protein but not mRNA. HeLa cells in asynchronous culture were transfected with Smurf2 siRNA or nonspecific dsRNAs (siNS) and harvested at the indicated times after transfection for Western blotting and RT-PCR. (b) Smurf2 silencing results in increased polyubiquitination of Mad2 followed by proteasomal degradation. HeLa cells were transfected with a Mad2 expression plasmid and Smurf2 siRNA #1 or control dsRNA. Cells were then treated with 2 μM MG132 for 4 h and analyzed by immunoprecipitation (IP) with Mad2 antibody followed by Western blotting using the indicated antibodies. (c) Smurf2 physically interacts with Mad2. Untransfected HeLa cells were released from synchronization at S phase with double-thymidine treatment and were harvested at the indicated times for immunoprecipitation followed by Western blotting. Ig, Ig heavy chain; NS, nonspecific band. (d) Ectopic expression of Mad2 is unable to restore the spindle assembly checkpoint in Smurf2-depleted cells. Western blotting for mitotic regulators in HeLa cells transfected with the indicated plasmid and siRNA. Cells were synchronized with thymidine treatment, released into a nocodazole-containing medium, and harvested at the indicated times after release (see Fig. 4 b and Materials and methods). (e) Ectopically expressed Mad2 localizes at centromeres in nocodazole-treated control cells but not in Smurf2-depleted cells. Immunofluorescence microscopy with anti-Mad2 and ACAs 10 h after release from the thymidine block. The close-up images are shown with threefold higher magnifications. Bars, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

    doi: 10.1083/jcb.200801049

    Figure Lengend Snippet: Smurf2 is required for stability and functional localization of Mad2. (a) Smurf2 silencing rapidly down-regulates Mad2 protein but not mRNA. HeLa cells in asynchronous culture were transfected with Smurf2 siRNA or nonspecific dsRNAs (siNS) and harvested at the indicated times after transfection for Western blotting and RT-PCR. (b) Smurf2 silencing results in increased polyubiquitination of Mad2 followed by proteasomal degradation. HeLa cells were transfected with a Mad2 expression plasmid and Smurf2 siRNA #1 or control dsRNA. Cells were then treated with 2 μM MG132 for 4 h and analyzed by immunoprecipitation (IP) with Mad2 antibody followed by Western blotting using the indicated antibodies. (c) Smurf2 physically interacts with Mad2. Untransfected HeLa cells were released from synchronization at S phase with double-thymidine treatment and were harvested at the indicated times for immunoprecipitation followed by Western blotting. Ig, Ig heavy chain; NS, nonspecific band. (d) Ectopic expression of Mad2 is unable to restore the spindle assembly checkpoint in Smurf2-depleted cells. Western blotting for mitotic regulators in HeLa cells transfected with the indicated plasmid and siRNA. Cells were synchronized with thymidine treatment, released into a nocodazole-containing medium, and harvested at the indicated times after release (see Fig. 4 b and Materials and methods). (e) Ectopically expressed Mad2 localizes at centromeres in nocodazole-treated control cells but not in Smurf2-depleted cells. Immunofluorescence microscopy with anti-Mad2 and ACAs 10 h after release from the thymidine block. The close-up images are shown with threefold higher magnifications. Bars, 10 μm.

    Article Snippet: The SMARTpool siRNAs against Smurf2 and Cdc20 and the control dsRNAs were obtained from Thermo Fisher Scientific.

    Techniques: Functional Assay, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Microscopy, Blocking Assay

    RNAi suppression of cold exposure-induced up-regulation of PaHsp70 expression and its effect on survival. Relative levels of Pahsp70 mRNA (A), PaHsp70 protein (B) and Pahsc70 mRNA (C) were measured in the fat bodies of male Pyrrhocoris apterus at different times of recovery after the cold exposure to −5°C for 5 d. The insects were either untreated (control) or injected two days prior to heat shock with: 2 µL of the injection buffer alone (blank); or 2 µL (2 µg) of Pahsp70 dsRNA. (D) An example of Western blotting. (E, F) Survival in blank-injected (E, n = 49) and Pahsp70 dsRNA-injected (F, n = 48) insects after the cold exposure. (G) Cross-tolerance was assesed by observing the survival in insects ( n = 48) that were pretreated with a mild heat shock (+41°C for 1 h) prior to the cold exposure to −5°C for 5 d. See Figs. 1 and 2 for more information.

    Journal: PLoS ONE

    Article Title: The 70 kDa Heat Shock Protein Assists during the Repair of Chilling Injury in the Insect, Pyrrhocoris apterus

    doi: 10.1371/journal.pone.0004546

    Figure Lengend Snippet: RNAi suppression of cold exposure-induced up-regulation of PaHsp70 expression and its effect on survival. Relative levels of Pahsp70 mRNA (A), PaHsp70 protein (B) and Pahsc70 mRNA (C) were measured in the fat bodies of male Pyrrhocoris apterus at different times of recovery after the cold exposure to −5°C for 5 d. The insects were either untreated (control) or injected two days prior to heat shock with: 2 µL of the injection buffer alone (blank); or 2 µL (2 µg) of Pahsp70 dsRNA. (D) An example of Western blotting. (E, F) Survival in blank-injected (E, n = 49) and Pahsp70 dsRNA-injected (F, n = 48) insects after the cold exposure. (G) Cross-tolerance was assesed by observing the survival in insects ( n = 48) that were pretreated with a mild heat shock (+41°C for 1 h) prior to the cold exposure to −5°C for 5 d. See Figs. 1 and 2 for more information.

    Article Snippet: RNAi The cDNA prepared from heat shocked insects (+41°C/1 h) was used to amplify two complementary DNA templates for dsRNA synthesis (using TaKaRa ExTaq polymerase, annealing at 55°C, 35 cycles, Biometra T300 Thermocycler).

    Techniques: Expressing, Injection, Western Blot

    RNAi suppression of heat shock-induced up-regulation of PaHsp70 expression and its effect on survival. Relative levels of Pahsp70 mRNA (A), PaHsp70 protein (B) and Pahsc70 mRNA (C) were measured in the fat bodies of male Pyrrhocoris apterus at different times of recovery after the heat shock (+41°C for 1 h). The insects were either untreated (control) or injected two days prior to heat shock with: 2 µL of the injection buffer alone (blank); 2 µL (10 µg) of ß-galactosidase ( ß-gal ) dsRNA; or 2 µL (2 µg) of Pahsp70 dsRNA. Each column is a mean±S.E.M. of 3–4 independent samples (5 fat bodies per sample). The differences in mRNA levels were assessed by ANOVA followed by Tukey's multiple comparison test at p = 0.05 (columns flanked by different letters differ significantly). (D) An example of Western blotting. (E, F) Survival in blank-injected (E, n = 39) and Pahsp70 dsRNA-injected (F, n = 40) insects after a severe heat shock (+45°C for 3 h) were assessed during recovery at 25°C for 7 days. The fit insects were those showing normal, rapid and coordinated crawling; the injured insects displayed signs of heat injury, i.e. slow, uncoordinated crawling or movements of body appendages only; and the dead insect did not respond to stimulation with a fine paintbrush. See Fig. 1 for more information.

    Journal: PLoS ONE

    Article Title: The 70 kDa Heat Shock Protein Assists during the Repair of Chilling Injury in the Insect, Pyrrhocoris apterus

    doi: 10.1371/journal.pone.0004546

    Figure Lengend Snippet: RNAi suppression of heat shock-induced up-regulation of PaHsp70 expression and its effect on survival. Relative levels of Pahsp70 mRNA (A), PaHsp70 protein (B) and Pahsc70 mRNA (C) were measured in the fat bodies of male Pyrrhocoris apterus at different times of recovery after the heat shock (+41°C for 1 h). The insects were either untreated (control) or injected two days prior to heat shock with: 2 µL of the injection buffer alone (blank); 2 µL (10 µg) of ß-galactosidase ( ß-gal ) dsRNA; or 2 µL (2 µg) of Pahsp70 dsRNA. Each column is a mean±S.E.M. of 3–4 independent samples (5 fat bodies per sample). The differences in mRNA levels were assessed by ANOVA followed by Tukey's multiple comparison test at p = 0.05 (columns flanked by different letters differ significantly). (D) An example of Western blotting. (E, F) Survival in blank-injected (E, n = 39) and Pahsp70 dsRNA-injected (F, n = 40) insects after a severe heat shock (+45°C for 3 h) were assessed during recovery at 25°C for 7 days. The fit insects were those showing normal, rapid and coordinated crawling; the injured insects displayed signs of heat injury, i.e. slow, uncoordinated crawling or movements of body appendages only; and the dead insect did not respond to stimulation with a fine paintbrush. See Fig. 1 for more information.

    Article Snippet: RNAi The cDNA prepared from heat shocked insects (+41°C/1 h) was used to amplify two complementary DNA templates for dsRNA synthesis (using TaKaRa ExTaq polymerase, annealing at 55°C, 35 cycles, Biometra T300 Thermocycler).

    Techniques: Expressing, Injection, Western Blot