lipofectamine 2000 Search Results


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  • 99
    Thermo Fisher lipofectamine lf 2000 reagent
    Lipofectamine Lf 2000 Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine lf 2000 reagent/product/Thermo Fisher
    Average 99 stars, based on 5 article reviews
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    85
    Thermo Fisher lipofectamine lf 2000
    Colony survival assay in A549 cells following transfection with <t>Lipofectamine</t> 2000 ( A ) or PEI-PCL-PEG (PPP) micelleplexes ( B ). Untreated (open circle), negative control (filled triangles) or ERCC1-XPF (filled squares) siRNA transfected cells were treated with increasing doses of cisplatin for 2 h, and cell viability was determined by a clonogenic assay. Results are represented as mean ± SD. IC 50 values were calculated using Compusyn software.
    Lipofectamine Lf 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipofectamine ltx
    SR-BI interacts with Src in macrophages, inducing Src phosphorylation and membrane recruitment of Src. A: WT macrophage cell lysates were immunoprecipitated with either anti-SR-BI or anti-LRP1 antibodies and protein A/G magnetic beads. Immunoprecipitated GULP or SR-BI as a control was then detected by Western blotting. B: WT macrophage cell lysates were immunoprecipitated with either anti-SR-BI or anti-Src antibodies and protein A/G magnetic beads. Immunoprecipitated Src or SR-BI was then detected by Western blotting. C: SR-BI plasmid was transfected for the indicated times into WT or SR-BI −/− macrophages using <t>Lipofectamine</t> <t>LTX</t> and Plus reagent. Plasma membrane proteins were isolated as described in the Materials and Methods and the SR-BI, pSrc (Tyr 416), and Na + /K + ATPase levels were detected by Western blotting. D: Efferocytosis was measured by flow cytometry from WT and SR-BI −/− macrophages transfected with pCMV6-mSR-BI plasmid or sham as described in the Materials and Methods. A–C: Data are representative of three experiments.
    Lipofectamine Ltx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 30153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Genechem lipofectamine 2000
    SR-BI interacts with Src in macrophages, inducing Src phosphorylation and membrane recruitment of Src. A: WT macrophage cell lysates were immunoprecipitated with either anti-SR-BI or anti-LRP1 antibodies and protein A/G magnetic beads. Immunoprecipitated GULP or SR-BI as a control was then detected by Western blotting. B: WT macrophage cell lysates were immunoprecipitated with either anti-SR-BI or anti-Src antibodies and protein A/G magnetic beads. Immunoprecipitated Src or SR-BI was then detected by Western blotting. C: SR-BI plasmid was transfected for the indicated times into WT or SR-BI −/− macrophages using <t>Lipofectamine</t> <t>LTX</t> and Plus reagent. Plasma membrane proteins were isolated as described in the Materials and Methods and the SR-BI, pSrc (Tyr 416), and Na + /K + ATPase levels were detected by Western blotting. D: Efferocytosis was measured by flow cytometry from WT and SR-BI −/− macrophages transfected with pCMV6-mSR-BI plasmid or sham as described in the Materials and Methods. A–C: Data are representative of three experiments.
    Lipofectamine 2000, supplied by Genechem, used in various techniques. Bioz Stars score: 90/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche lipofectamine 2000
    Silencing of a virus reporter protein in tick cells by siRNA treatment. a REE/CTVM28 cells were incubated with siRNA against RLuc in the presence or absence of <t>Lipofectamine</t> 2000 (Lipo) and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y- axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Virus + siRNA + Lipo) and siRNA alone followed by SFV (Virus + siRNA only). The values shown are means of three replicate cultures and the error bars are standard deviations of the mean. * Significant decrease from the virus only control. b Nine tick cell lines were incubated with siRNA targeting RLuc in the presence of a transfection reagent and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y -axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Lipo + siRNA + virus) and siRNA with Xtreme followed by SFV (Xtreme + siRNA + virus). The values shown are means of four replicate cultures and the error bars are standard deviations of the mean. The scale used for each y -axis reflects the range of luciferase levels generated in the particular tick cell line. * Significant decrease from the virus only control
    Lipofectamine 2000, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 486 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific lipofectamine
    Uptake efficiency of uncoated (grey) and <t>lipofectamine</t> coated (red) WGM resonators for different cell types as determined by the internalization bioassay. (a) Primary human macrophages, epithelial (Hela), fibroblast (NIH 3T3) and HEK 293 cells. (b) Cells from the nervous system, including primary mouse astrocytes and neuronal cell lines N7 and SH-SY5Y. Unless indicated otherwise, uptake efficiencies were determined after 4 h of incubation. Error bars represent standard error of the mean. Two sample t-test is used to evaluate statistical significance (****p
    Lipofectamine, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore lipofectamine 2000
    The HIV-1 inhibitory effect on A(H1N1)pdm09 replication is dependent on the IFITM3 expression. HeLa cells were transfected with 0.4(Scr) using <t>lipofectamine</t> 2000 and, after 6 h, cells were infected by A(H1N1)pdm09 (MOI 5) for 1 h. After that, cells were exposed to HIV-1 (10 ng/mL p24 Ag) for 24 h after influenza infection. ( A ) Monolayers were lysed with Laemmli buffer and submitted to blotting assay for the proteins IFITM3 and β-actin, as an housekeeping. Top panel: cells not exposed to HIV-1, and bottom panel: cells exposed to HIV-1. ( B ) The A(H1N1)pdm09 levels in culture supernatants were quantified by qRT-PCR (y-axis) and the immunoblotting bands were analyzed by densitometry using the ImageJ software 1.6.0 and n-fold changes of AU is scored as black squares (z-axis). Panel A is representative of three independent experiments. * P
    Lipofectamine 2000, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1351 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene lipofectamine 2000
    The HIV-1 inhibitory effect on A(H1N1)pdm09 replication is dependent on the IFITM3 expression. HeLa cells were transfected with 0.4(Scr) using <t>lipofectamine</t> 2000 and, after 6 h, cells were infected by A(H1N1)pdm09 (MOI 5) for 1 h. After that, cells were exposed to HIV-1 (10 ng/mL p24 Ag) for 24 h after influenza infection. ( A ) Monolayers were lysed with Laemmli buffer and submitted to blotting assay for the proteins IFITM3 and β-actin, as an housekeeping. Top panel: cells not exposed to HIV-1, and bottom panel: cells exposed to HIV-1. ( B ) The A(H1N1)pdm09 levels in culture supernatants were quantified by qRT-PCR (y-axis) and the immunoblotting bands were analyzed by densitometry using the ImageJ software 1.6.0 and n-fold changes of AU is scored as black squares (z-axis). Panel A is representative of three independent experiments. * P
    Lipofectamine 2000, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega lipofectamine
    p300 enhances p73-induced apoptosis. (A and B) The N-terminal deletion of p73 lacks apoptosis-promoting potential. As indicated, plasmids encoding no protein as a control (C; 3 μg), p73α (1.0 μg), ΔNp73α (1.0 μg), and/or p300 (2.0 μg), together with the pGFP plasmid (200 ng), were introduced into H1299 cells (10 5 /60-mm-diameter dish) using <t>LipofectAmine</t> (GIBCO-BRL); 32 h posttransfection, cells (100) expressing GFP were counted under a fluorescence microscope using a blind approach, with morphologically round cells (arrows) identified as apoptotic (A). (B) Statistical data (with standard error indicated by a bar above each column) from three independent assays. (C and D) Caspase 3 activation by wild-type but not N-terminus-deleted p73α is enhanced by p300. Transient transfection as described for panel A was conducted. Cells were harvested for caspase 3 activity analysis as described in Materials and Methods. (C) Representative result of the fluorescence graph of AMC released from Ac-DEVD-AMC substrates for one set of experiments with plasmids encoding no protein (curve B), ΔNp73α (curve C), ΔNp73α plus p300 (curve D), p73α (curve E), or p73α plus p300 (curve F). Curve A is a water control. Caspase 3 activity is presented in arbitrary units (A.U.) after normalization by a factor of 10 5 . (D) Statistical values for caspase 3 activity in triplicate assays. (E) DNA fragmentation analysis of p73-induced apoptosis in H1299 cells was conducted as described in Materials and Methods; 10 μg of DNA was analyzed by electrophoresis on 2% agarose. Each lane represents the result for three 60-mm-diameter plates of cells. MW, molecular weight markers.
    Lipofectamine, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Horizon Discovery lipofectamine 2000
    p300 enhances p73-induced apoptosis. (A and B) The N-terminal deletion of p73 lacks apoptosis-promoting potential. As indicated, plasmids encoding no protein as a control (C; 3 μg), p73α (1.0 μg), ΔNp73α (1.0 μg), and/or p300 (2.0 μg), together with the pGFP plasmid (200 ng), were introduced into H1299 cells (10 5 /60-mm-diameter dish) using <t>LipofectAmine</t> (GIBCO-BRL); 32 h posttransfection, cells (100) expressing GFP were counted under a fluorescence microscope using a blind approach, with morphologically round cells (arrows) identified as apoptotic (A). (B) Statistical data (with standard error indicated by a bar above each column) from three independent assays. (C and D) Caspase 3 activation by wild-type but not N-terminus-deleted p73α is enhanced by p300. Transient transfection as described for panel A was conducted. Cells were harvested for caspase 3 activity analysis as described in Materials and Methods. (C) Representative result of the fluorescence graph of AMC released from Ac-DEVD-AMC substrates for one set of experiments with plasmids encoding no protein (curve B), ΔNp73α (curve C), ΔNp73α plus p300 (curve D), p73α (curve E), or p73α plus p300 (curve F). Curve A is a water control. Caspase 3 activity is presented in arbitrary units (A.U.) after normalization by a factor of 10 5 . (D) Statistical values for caspase 3 activity in triplicate assays. (E) DNA fragmentation analysis of p73-induced apoptosis in H1299 cells was conducted as described in Materials and Methods; 10 μg of DNA was analyzed by electrophoresis on 2% agarose. Each lane represents the result for three 60-mm-diameter plates of cells. MW, molecular weight markers.
    Lipofectamine 2000, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 93/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenePharma Company lipofectamine 2000
    EDHB sensitivity of AKR1C1/C2-depleted KYSE 180 cells A. The efficiency of AKR1C1/C2 knockdown using different AKR1C1/C2 siRNAs was determined by Western blot analysis. B. KYSE 180 cells were transfected with various concentrations of siAKR1C1/C2 537 using Lipofectamine 2000. Knockdown efficiency was determined by Western blot analysis. C, D. A total of 50 pmol of siAKR1C1/C2 537 (C) or siAKR1C1/C2 630 (D) was transfected using <t>Lipofectamine</t> 2000 for 3 days. During this time, KYSE 180 cells were treated with 40 μg/ml EDHB. The MTT assay was performed after EDHB treatment for 1 or 2 days, and the ratio of inhibition was determined relative to untreated cells (mean + S.E.M. for triplicate samples).
    Lipofectamine 2000, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 92/100, based on 218 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher lipofectamine 2000 kit
    Properties of N-BLR. a ISH of the tissue microarray (described in Additional file 3 : Figure S5) shows differential expression of N-BLR in colon cancer (Adenocarcinoma) and normal colon (Normal tissue). Hematoxylin and eosin (H E) staining of matched tissues was added to distinguish tissue morphology. Increasing magnifications were provide to evaluate the distribution of N-BLR in the nucleus and in the cytoplasm of cells (5X, 20X, and 60X). b Image analysis of ISH was conducted to measure the expression levels of N-BLR in the different tissues. Adenocarcinoma and metastatic colon cancer tissues expressed higher levels of N-BLR compared with normal colon tissue. There were not significant differences between normal tissue and benign/polyp and colitis tissues. c ISH data on cytoplasmic/nuclear localization of N-BLR. The full arrows point to cytoplasm and the dashed arrows to nucleus. Those two cellular compartments were identified using H E staining. The H E staining and ISH for N-BLR were done on serial sections; therefore, perfect overlapping of tissue morphology did not occur between the two images that show the same tissue area. d PARP-1 expression following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. Profiling was carried out at 96 and 120 h of siRNA transfection. e left Expression of survivin, c-IAP-1, XIAP after 96 h following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. right Quantification of survivin, c-IAP-1, XIAP in Colo320 cells. f Activity of Caspase 3/7, Caspase 8, and Caspase 9 following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. Profiling was carried out after 96 and 120 h (siR = N-BLR siRNA 1 + 3 pool; Ctr = scramble control siRNA; N = <t>lipofectamine</t> only; GAPDH was used as loading control). (Student’s t-test; * p
    Lipofectamine 2000 Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 2133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Beijing TransGen Biotech lipofectamine 2000
    Properties of N-BLR. a ISH of the tissue microarray (described in Additional file 3 : Figure S5) shows differential expression of N-BLR in colon cancer (Adenocarcinoma) and normal colon (Normal tissue). Hematoxylin and eosin (H E) staining of matched tissues was added to distinguish tissue morphology. Increasing magnifications were provide to evaluate the distribution of N-BLR in the nucleus and in the cytoplasm of cells (5X, 20X, and 60X). b Image analysis of ISH was conducted to measure the expression levels of N-BLR in the different tissues. Adenocarcinoma and metastatic colon cancer tissues expressed higher levels of N-BLR compared with normal colon tissue. There were not significant differences between normal tissue and benign/polyp and colitis tissues. c ISH data on cytoplasmic/nuclear localization of N-BLR. The full arrows point to cytoplasm and the dashed arrows to nucleus. Those two cellular compartments were identified using H E staining. The H E staining and ISH for N-BLR were done on serial sections; therefore, perfect overlapping of tissue morphology did not occur between the two images that show the same tissue area. d PARP-1 expression following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. Profiling was carried out at 96 and 120 h of siRNA transfection. e left Expression of survivin, c-IAP-1, XIAP after 96 h following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. right Quantification of survivin, c-IAP-1, XIAP in Colo320 cells. f Activity of Caspase 3/7, Caspase 8, and Caspase 9 following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. Profiling was carried out after 96 and 120 h (siR = N-BLR siRNA 1 + 3 pool; Ctr = scramble control siRNA; N = <t>lipofectamine</t> only; GAPDH was used as loading control). (Student’s t-test; * p
    Lipofectamine 2000, supplied by Beijing TransGen Biotech, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen lipofectamine 2000
    The MCMV M35 protein inhibits signaling of multiple pattern recognition receptors. (A) NIH3T3 fibroblasts were co-transfected with a reporter plasmid containing firefly luciferase under the control of the murine IFNβ promoter (IFNβ-Luc) together with a Renilla luciferase normalization control (pRL-TK) and influenza NS1 or MCMV ORFs known or predicted to code for tegument or immediate-early proteins or their corresponding empty vector (pcDNA and pDEST40, respectively). At 24 hours post transfection, cells were treated with medium or stimulated by infection with Newcastle disease virus (NDV). 21 hours p.i. cells were lysed for analysis of luciferase activity. Luciferase fold induction was calculated based on firefly luciferase values normalized to Renilla luciferase from stimulated samples divided by corresponding values from unstimulated samples. Data set is combined from one to four independent experiments and represented as mean ± SD. (B) NIH3T3 fibroblasts were co-transfected with the IFNβ-Luc and pRL-TK luciferase plasmids described in (A) as well as V5-tagged M35 or pcDNA and luciferase assay was performed following stimulation as for (A). Data is combined from three independent experiments and shown as mean ± SD. (C) NIH3T3 fibroblasts were co-transfected as described in (B) and cells stimulated with 10 μg/ml of poly(I:C) in the presence of <t>Lipofectamine</t> 2000 or Lipofectamine 2000 alone for 6 hours before lysis for analysis by luciferase assay. Data is combined from three independent experiments and shown as mean ± SD. (D) 293T cells were co-transfected with expression plasmids for either cGAS (stimulated) or GFP (unstimulated) together with mCherry-STING, the IFNβ-Luc and pRL-TK luciferase plasmids, and either pcDNA, myc-tagged KSHV ORF36 or V5-tagged M35. At 20 hours post transfection, cells were lysed and luciferase production was analyzed. Data is combined from four independent experiments and shown as mean ± SD. (E) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of 3 μg/ml cGAMP or left unstimulated. RNA was extracted at indicated time points and IFNβ induction was measured by quantitative RT-PCR and expressed as IFNβ induction normalized to the housekeeping gene Rpl8. Data is shown as mean ± SD and combined from two independent experiments. (F) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of indicated amounts of cGAMP and supernatant was collected 16 hours later and analyzed by IFNβ ELISA. Data is shown as mean ± SD and representative of two independent experiments. (G) Immortalized BMDM stably expressing myc-tagged M35 or the corresponding empty vector (pMSCV) were stimulated by addition of 10 ng/ml LPS or 1 μM CpG-B 1826 and supernatant collected at 16 hours post stimulation for analysis by TNFα ELISA. Data is combined from three independent experiments and shown as mean ± SD.***p
    Lipofectamine 2000, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Colony survival assay in A549 cells following transfection with Lipofectamine 2000 ( A ) or PEI-PCL-PEG (PPP) micelleplexes ( B ). Untreated (open circle), negative control (filled triangles) or ERCC1-XPF (filled squares) siRNA transfected cells were treated with increasing doses of cisplatin for 2 h, and cell viability was determined by a clonogenic assay. Results are represented as mean ± SD. IC 50 values were calculated using Compusyn software.

    Journal: Molecules

    Article Title: Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy

    doi: 10.3390/molecules25081994

    Figure Lengend Snippet: Colony survival assay in A549 cells following transfection with Lipofectamine 2000 ( A ) or PEI-PCL-PEG (PPP) micelleplexes ( B ). Untreated (open circle), negative control (filled triangles) or ERCC1-XPF (filled squares) siRNA transfected cells were treated with increasing doses of cisplatin for 2 h, and cell viability was determined by a clonogenic assay. Results are represented as mean ± SD. IC 50 values were calculated using Compusyn software.

    Article Snippet: Negative controls consisted of untreated cells or cells transfected with control scrambled siRNA while positive control cells were transfected with Lipofectamine 2000 (Life Technologies) lipoplexes, which were prepared as described above.

    Techniques: Clonogenic Cell Survival Assay, Transfection, Negative Control, Clonogenic Assay, Software

    ERCC1 and XPF gene knockdown efficiency was validated in lung adenocarcinoma cells (A549) after 72 h following double transfection of Lipofectamine 2000 (LF) or micelleplexes (PPP) with 100 pmol ERCC1-XPF siRNA or negative control (NC) siRNA. ERCC1 and XPF expression was normalized with GAPDH expression and quantified by real time PCR. Data points indicate mean ± SD. (n = 6).

    Journal: Molecules

    Article Title: Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy

    doi: 10.3390/molecules25081994

    Figure Lengend Snippet: ERCC1 and XPF gene knockdown efficiency was validated in lung adenocarcinoma cells (A549) after 72 h following double transfection of Lipofectamine 2000 (LF) or micelleplexes (PPP) with 100 pmol ERCC1-XPF siRNA or negative control (NC) siRNA. ERCC1 and XPF expression was normalized with GAPDH expression and quantified by real time PCR. Data points indicate mean ± SD. (n = 6).

    Article Snippet: Negative controls consisted of untreated cells or cells transfected with control scrambled siRNA while positive control cells were transfected with Lipofectamine 2000 (Life Technologies) lipoplexes, which were prepared as described above.

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

    Western Blot analysis of excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum group F (XPF) protein levels within A549 cells following ( A ) single transfection of micelleplexes (PPP) loaded with 50 pmol small interfering RNA (siRNA) after 72 h and ( B ) double transfection of micelleplexes (PPP) loaded with 100 pmol siRNA after 72h. Lipofectamine 2000 (LF) lipoplexes were prepared according to manufacturer’s protocol with 50 pmol (A) and 100 pmol (B) siRNA (n = 2).

    Journal: Molecules

    Article Title: Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy

    doi: 10.3390/molecules25081994

    Figure Lengend Snippet: Western Blot analysis of excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum group F (XPF) protein levels within A549 cells following ( A ) single transfection of micelleplexes (PPP) loaded with 50 pmol small interfering RNA (siRNA) after 72 h and ( B ) double transfection of micelleplexes (PPP) loaded with 100 pmol siRNA after 72h. Lipofectamine 2000 (LF) lipoplexes were prepared according to manufacturer’s protocol with 50 pmol (A) and 100 pmol (B) siRNA (n = 2).

    Article Snippet: Negative controls consisted of untreated cells or cells transfected with control scrambled siRNA while positive control cells were transfected with Lipofectamine 2000 (Life Technologies) lipoplexes, which were prepared as described above.

    Techniques: Western Blot, Transfection, Small Interfering RNA

    Dependency of the DTS effect on mitosis. % of EGFP-expressing cells ( bars ; left y-axis) and MFI of positive cells ( lines ; right y-axis) after transfection of HeLa cells ( left panel ) or A431 cells ( right panel ) with plasmids with DTS (pCMV/EGFP; dark grey bar and asterix ) versus plasmids without DTS (pCMV/EGFP_noDTS; light grey bar and open circle ). Cells were transfected at a dose of 1 μg DNA/well in a 24-well plate with lipofectamine for 4 h in the presence of serum, washed and analyzed at the indicated timepoints after onset of transfection. As a model for non-dividing cells, cells were arrested at the S-phase by treatment with 15 μM aphidicolin starting 24 h prior to transfection and continuously throughout the experiment. Data are represented as mean ± SD of three independent measurements.

    Journal: Pharmaceutical Research

    Article Title: DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery

    doi: 10.1007/s11095-011-0407-8

    Figure Lengend Snippet: Dependency of the DTS effect on mitosis. % of EGFP-expressing cells ( bars ; left y-axis) and MFI of positive cells ( lines ; right y-axis) after transfection of HeLa cells ( left panel ) or A431 cells ( right panel ) with plasmids with DTS (pCMV/EGFP; dark grey bar and asterix ) versus plasmids without DTS (pCMV/EGFP_noDTS; light grey bar and open circle ). Cells were transfected at a dose of 1 μg DNA/well in a 24-well plate with lipofectamine for 4 h in the presence of serum, washed and analyzed at the indicated timepoints after onset of transfection. As a model for non-dividing cells, cells were arrested at the S-phase by treatment with 15 μM aphidicolin starting 24 h prior to transfection and continuously throughout the experiment. Data are represented as mean ± SD of three independent measurements.

    Article Snippet: TOPO TA Cloning Kit, TOP10 E. Coli bacteria, propidium iodide (PI), Lipofectamine™2000 (lipofectamine), Opti-MEM® I Reduced Serum Medium (optimem), UltraPure™ Buffer-Saturated Phenol (TE-saturated phenol) and UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) were purchased from Invitrogen (Oregon, USA).

    Techniques: Expressing, Transfection

    Dependency of the DTS effect on delivery strategies. % of EGFP-expressing cells ( a ) and mean expression per cell ( b ) after transfection with lipofectamine ( left ), 22 kDa linear pEI ( middle ) or after electroporation (right) of plasmids with DTS (pCMV/EGFP; dark grey bar ) versus plasmids without DTS (pCMV/EGFP_noDTS; light grey bar ). Cells were transfected at a dose of 1 μg DNA/well in a 24-well plate with lipofectamine or pEI for 4 h in the presence of serum, washed and analyzed after 48 h or electroporated at a dose of 2 μg/2.25·10 5 cells and analyzed after 24 h. Data are represented as mean ± SD of three independent measurements. MFI: mean fluorescence intensity.

    Journal: Pharmaceutical Research

    Article Title: DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery

    doi: 10.1007/s11095-011-0407-8

    Figure Lengend Snippet: Dependency of the DTS effect on delivery strategies. % of EGFP-expressing cells ( a ) and mean expression per cell ( b ) after transfection with lipofectamine ( left ), 22 kDa linear pEI ( middle ) or after electroporation (right) of plasmids with DTS (pCMV/EGFP; dark grey bar ) versus plasmids without DTS (pCMV/EGFP_noDTS; light grey bar ). Cells were transfected at a dose of 1 μg DNA/well in a 24-well plate with lipofectamine or pEI for 4 h in the presence of serum, washed and analyzed after 48 h or electroporated at a dose of 2 μg/2.25·10 5 cells and analyzed after 24 h. Data are represented as mean ± SD of three independent measurements. MFI: mean fluorescence intensity.

    Article Snippet: TOPO TA Cloning Kit, TOP10 E. Coli bacteria, propidium iodide (PI), Lipofectamine™2000 (lipofectamine), Opti-MEM® I Reduced Serum Medium (optimem), UltraPure™ Buffer-Saturated Phenol (TE-saturated phenol) and UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) were purchased from Invitrogen (Oregon, USA).

    Techniques: Expressing, Transfection, Electroporation, Fluorescence

    The effect of various DTS on transfection efficiency. % EGFP-expressing cells after transfection with SV40-based DTS ( a ), NFκB DTS ( b ) and a GRE DTS ( c ). HeLa cells were transfected with the indicated plasmids and lipofectamine for 4 h in the presence of serum, washed and analyzed 6 h after the onset of transfection. Cells were non-stimulated ( light grey bars ) or stimulated by incubation with 25 ng/ml TNF-α during time of transfection ( dark grey bars ). Data are presented as mean ± SD ( n = 3). One-way ANOVA followed by post hoc analysis with Bonferroni correction was performed on the expression data of each plasmid. None of the plasmids differed significantly from plasmid noDTS either in the presence or absence of TNF-α.

    Journal: Pharmaceutical Research

    Article Title: DNA Nuclear Targeting Sequences for Non-Viral Gene Delivery

    doi: 10.1007/s11095-011-0407-8

    Figure Lengend Snippet: The effect of various DTS on transfection efficiency. % EGFP-expressing cells after transfection with SV40-based DTS ( a ), NFκB DTS ( b ) and a GRE DTS ( c ). HeLa cells were transfected with the indicated plasmids and lipofectamine for 4 h in the presence of serum, washed and analyzed 6 h after the onset of transfection. Cells were non-stimulated ( light grey bars ) or stimulated by incubation with 25 ng/ml TNF-α during time of transfection ( dark grey bars ). Data are presented as mean ± SD ( n = 3). One-way ANOVA followed by post hoc analysis with Bonferroni correction was performed on the expression data of each plasmid. None of the plasmids differed significantly from plasmid noDTS either in the presence or absence of TNF-α.

    Article Snippet: TOPO TA Cloning Kit, TOP10 E. Coli bacteria, propidium iodide (PI), Lipofectamine™2000 (lipofectamine), Opti-MEM® I Reduced Serum Medium (optimem), UltraPure™ Buffer-Saturated Phenol (TE-saturated phenol) and UltraPure™ Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) were purchased from Invitrogen (Oregon, USA).

    Techniques: Transfection, Expressing, Incubation, Plasmid Preparation

    SR-BI interacts with Src in macrophages, inducing Src phosphorylation and membrane recruitment of Src. A: WT macrophage cell lysates were immunoprecipitated with either anti-SR-BI or anti-LRP1 antibodies and protein A/G magnetic beads. Immunoprecipitated GULP or SR-BI as a control was then detected by Western blotting. B: WT macrophage cell lysates were immunoprecipitated with either anti-SR-BI or anti-Src antibodies and protein A/G magnetic beads. Immunoprecipitated Src or SR-BI was then detected by Western blotting. C: SR-BI plasmid was transfected for the indicated times into WT or SR-BI −/− macrophages using Lipofectamine LTX and Plus reagent. Plasma membrane proteins were isolated as described in the Materials and Methods and the SR-BI, pSrc (Tyr 416), and Na + /K + ATPase levels were detected by Western blotting. D: Efferocytosis was measured by flow cytometry from WT and SR-BI −/− macrophages transfected with pCMV6-mSR-BI plasmid or sham as described in the Materials and Methods. A–C: Data are representative of three experiments.

    Journal: Journal of Lipid Research

    Article Title: Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis [S]

    doi: 10.1194/jlr.M056689

    Figure Lengend Snippet: SR-BI interacts with Src in macrophages, inducing Src phosphorylation and membrane recruitment of Src. A: WT macrophage cell lysates were immunoprecipitated with either anti-SR-BI or anti-LRP1 antibodies and protein A/G magnetic beads. Immunoprecipitated GULP or SR-BI as a control was then detected by Western blotting. B: WT macrophage cell lysates were immunoprecipitated with either anti-SR-BI or anti-Src antibodies and protein A/G magnetic beads. Immunoprecipitated Src or SR-BI was then detected by Western blotting. C: SR-BI plasmid was transfected for the indicated times into WT or SR-BI −/− macrophages using Lipofectamine LTX and Plus reagent. Plasma membrane proteins were isolated as described in the Materials and Methods and the SR-BI, pSrc (Tyr 416), and Na + /K + ATPase levels were detected by Western blotting. D: Efferocytosis was measured by flow cytometry from WT and SR-BI −/− macrophages transfected with pCMV6-mSR-BI plasmid or sham as described in the Materials and Methods. A–C: Data are representative of three experiments.

    Article Snippet: For transient transfections, Lipofectamine LTX and Plus reagent (Invitrogen) with pCMV6-mSR-BI or scramble pCMV6 plasmid (OriGene Technologies) were applied to macrophages.

    Techniques: Immunoprecipitation, Magnetic Beads, Western Blot, Plasmid Preparation, Transfection, Isolation, Flow Cytometry, Cytometry

    Silencing of a virus reporter protein in tick cells by siRNA treatment. a REE/CTVM28 cells were incubated with siRNA against RLuc in the presence or absence of Lipofectamine 2000 (Lipo) and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y- axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Virus + siRNA + Lipo) and siRNA alone followed by SFV (Virus + siRNA only). The values shown are means of three replicate cultures and the error bars are standard deviations of the mean. * Significant decrease from the virus only control. b Nine tick cell lines were incubated with siRNA targeting RLuc in the presence of a transfection reagent and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y -axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Lipo + siRNA + virus) and siRNA with Xtreme followed by SFV (Xtreme + siRNA + virus). The values shown are means of four replicate cultures and the error bars are standard deviations of the mean. The scale used for each y -axis reflects the range of luciferase levels generated in the particular tick cell line. * Significant decrease from the virus only control

    Journal: Experimental & Applied Acarology

    Article Title: Gene silencing in tick cell lines using small interfering or long double-stranded RNA

    doi: 10.1007/s10493-012-9598-x

    Figure Lengend Snippet: Silencing of a virus reporter protein in tick cells by siRNA treatment. a REE/CTVM28 cells were incubated with siRNA against RLuc in the presence or absence of Lipofectamine 2000 (Lipo) and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y- axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Virus + siRNA + Lipo) and siRNA alone followed by SFV (Virus + siRNA only). The values shown are means of three replicate cultures and the error bars are standard deviations of the mean. * Significant decrease from the virus only control. b Nine tick cell lines were incubated with siRNA targeting RLuc in the presence of a transfection reagent and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y -axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Lipo + siRNA + virus) and siRNA with Xtreme followed by SFV (Xtreme + siRNA + virus). The values shown are means of four replicate cultures and the error bars are standard deviations of the mean. The scale used for each y -axis reflects the range of luciferase levels generated in the particular tick cell line. * Significant decrease from the virus only control

    Article Snippet: While it was not feasible to test every commercially available transfection reagent, six commonly used reagents were selected; the most effective were Lipofectamine 2000 and Roche XtremeGene (Xtreme).

    Techniques: Incubation, Infection, Expressing, Luciferase, Transfection, Generated

    Efficiency of subolesin mRNA transcript knockdown in IDE8 and ISE6 cells. IDE8 ( upper ) and ISE6 ( lower ) cells were transfected (with or without a transfection reagent) with dsRNA targeting subolesin or negative control dsRNA targeting eGFP and incubated for 96 h. Data for all replicates are included to illustrate the level of variability encountered between individual cultures. Total RNA was extracted and equal amounts of RNA were used to make cDNA. Transcripts were measured by real-time PCR and C t values for subolesin mRNA were then normalised against tick β-actin mRNA for each replicate. Subolesin mRNA expression levels (y-axis) are shown in arbitrary units. –ve dsRNA targeting eGFP, dsRNA only dsRNA targeting subolesin, lipo Lipofectamine 2000 + dsRNA targeting subolesin, x Xtreme + dsRNA targeting subolesin

    Journal: Experimental & Applied Acarology

    Article Title: Gene silencing in tick cell lines using small interfering or long double-stranded RNA

    doi: 10.1007/s10493-012-9598-x

    Figure Lengend Snippet: Efficiency of subolesin mRNA transcript knockdown in IDE8 and ISE6 cells. IDE8 ( upper ) and ISE6 ( lower ) cells were transfected (with or without a transfection reagent) with dsRNA targeting subolesin or negative control dsRNA targeting eGFP and incubated for 96 h. Data for all replicates are included to illustrate the level of variability encountered between individual cultures. Total RNA was extracted and equal amounts of RNA were used to make cDNA. Transcripts were measured by real-time PCR and C t values for subolesin mRNA were then normalised against tick β-actin mRNA for each replicate. Subolesin mRNA expression levels (y-axis) are shown in arbitrary units. –ve dsRNA targeting eGFP, dsRNA only dsRNA targeting subolesin, lipo Lipofectamine 2000 + dsRNA targeting subolesin, x Xtreme + dsRNA targeting subolesin

    Article Snippet: While it was not feasible to test every commercially available transfection reagent, six commonly used reagents were selected; the most effective were Lipofectamine 2000 and Roche XtremeGene (Xtreme).

    Techniques: Transfection, Negative Control, Incubation, Real-time Polymerase Chain Reaction, Expressing

    Silencing of a foreign virus reporter protein by long dsRNA treatment. Nine tick cell lines were incubated with long dsRNA targeting RLuc in the presence or absence of a transfection reagent and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y -axis) were then measured 48 h later in cultures with SFV alone (Virus only), dsRNA followed by SFV (Virus + dsRNA), dsRNA with Lipofectamine 2000 followed by SFV (Lipo + dsRNA + virus) and dsRNA with Xtreme followed by SFV (Xtreme + dsRNA + virus). The values shown are means of four replicate cultures and the error bars are standard deviations of the mean. The scale used for each y -axis reflects the range of luciferase levels generated in the particular tick cell line. * Significant decrease from the virus only control

    Journal: Experimental & Applied Acarology

    Article Title: Gene silencing in tick cell lines using small interfering or long double-stranded RNA

    doi: 10.1007/s10493-012-9598-x

    Figure Lengend Snippet: Silencing of a foreign virus reporter protein by long dsRNA treatment. Nine tick cell lines were incubated with long dsRNA targeting RLuc in the presence or absence of a transfection reagent and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y -axis) were then measured 48 h later in cultures with SFV alone (Virus only), dsRNA followed by SFV (Virus + dsRNA), dsRNA with Lipofectamine 2000 followed by SFV (Lipo + dsRNA + virus) and dsRNA with Xtreme followed by SFV (Xtreme + dsRNA + virus). The values shown are means of four replicate cultures and the error bars are standard deviations of the mean. The scale used for each y -axis reflects the range of luciferase levels generated in the particular tick cell line. * Significant decrease from the virus only control

    Article Snippet: While it was not feasible to test every commercially available transfection reagent, six commonly used reagents were selected; the most effective were Lipofectamine 2000 and Roche XtremeGene (Xtreme).

    Techniques: Incubation, Transfection, Infection, Expressing, Luciferase, Generated

    Images of tick cells 24 h after addition of fluorescent siRNA ( a – h ) or dsRNA ( i – l ) in the presence or absence of a transfection reagent. a – d photomicrographs of BDE/CTVM16 cells taken at ×100 magnification with simultaneous normal transmitted light and UV reflected light to facilitate counting of fluorescent ( green siRNA) and non-fluorescent cells. e – h confocal images of IRE/CTVM19 cells taken at ×630 magnification; cell nuclei are stained blue , while the siRNAs are green . i – l confocal images of IDE8 cells taken at ×630 magnification; cell nuclei are stained blue , while the long dsRNAs are green . a , e , i show untreated control cells; b , f , j show cells to which siRNA or dsRNA alone was added; c , g , k show cells to which siRNA or long dsRNA mixed with Lipofectamine 2000 was added; d , h , l show cells to which siRNA or long dsRNA mixed with Xtreme was added. (Color figure online)

    Journal: Experimental & Applied Acarology

    Article Title: Gene silencing in tick cell lines using small interfering or long double-stranded RNA

    doi: 10.1007/s10493-012-9598-x

    Figure Lengend Snippet: Images of tick cells 24 h after addition of fluorescent siRNA ( a – h ) or dsRNA ( i – l ) in the presence or absence of a transfection reagent. a – d photomicrographs of BDE/CTVM16 cells taken at ×100 magnification with simultaneous normal transmitted light and UV reflected light to facilitate counting of fluorescent ( green siRNA) and non-fluorescent cells. e – h confocal images of IRE/CTVM19 cells taken at ×630 magnification; cell nuclei are stained blue , while the siRNAs are green . i – l confocal images of IDE8 cells taken at ×630 magnification; cell nuclei are stained blue , while the long dsRNAs are green . a , e , i show untreated control cells; b , f , j show cells to which siRNA or dsRNA alone was added; c , g , k show cells to which siRNA or long dsRNA mixed with Lipofectamine 2000 was added; d , h , l show cells to which siRNA or long dsRNA mixed with Xtreme was added. (Color figure online)

    Article Snippet: While it was not feasible to test every commercially available transfection reagent, six commonly used reagents were selected; the most effective were Lipofectamine 2000 and Roche XtremeGene (Xtreme).

    Techniques: Transfection, Staining

    Uptake efficiency of uncoated (grey) and lipofectamine coated (red) WGM resonators for different cell types as determined by the internalization bioassay. (a) Primary human macrophages, epithelial (Hela), fibroblast (NIH 3T3) and HEK 293 cells. (b) Cells from the nervous system, including primary mouse astrocytes and neuronal cell lines N7 and SH-SY5Y. Unless indicated otherwise, uptake efficiencies were determined after 4 h of incubation. Error bars represent standard error of the mean. Two sample t-test is used to evaluate statistical significance (****p

    Journal: Scientific Reports

    Article Title: Lasing in Live Mitotic and Non-Phagocytic Cells by Efficient Delivery of Microresonators

    doi: 10.1038/srep40877

    Figure Lengend Snippet: Uptake efficiency of uncoated (grey) and lipofectamine coated (red) WGM resonators for different cell types as determined by the internalization bioassay. (a) Primary human macrophages, epithelial (Hela), fibroblast (NIH 3T3) and HEK 293 cells. (b) Cells from the nervous system, including primary mouse astrocytes and neuronal cell lines N7 and SH-SY5Y. Unless indicated otherwise, uptake efficiencies were determined after 4 h of incubation. Error bars represent standard error of the mean. Two sample t-test is used to evaluate statistical significance (****p

    Article Snippet: Preparation of lipofectamine and biotin coated resonators PS-DVB microspheres that were internally stained with a green fluorescent dye (15 μm, Fischer Scientific) were submersed in NHS-LC-LC-Biotin (10 mg/ml in DMSO) for 30 min to achieve non-covalent biotin coating.

    Techniques: Incubation

    The HIV-1 inhibitory effect on A(H1N1)pdm09 replication is dependent on the IFITM3 expression. HeLa cells were transfected with 0.4(Scr) using lipofectamine 2000 and, after 6 h, cells were infected by A(H1N1)pdm09 (MOI 5) for 1 h. After that, cells were exposed to HIV-1 (10 ng/mL p24 Ag) for 24 h after influenza infection. ( A ) Monolayers were lysed with Laemmli buffer and submitted to blotting assay for the proteins IFITM3 and β-actin, as an housekeeping. Top panel: cells not exposed to HIV-1, and bottom panel: cells exposed to HIV-1. ( B ) The A(H1N1)pdm09 levels in culture supernatants were quantified by qRT-PCR (y-axis) and the immunoblotting bands were analyzed by densitometry using the ImageJ software 1.6.0 and n-fold changes of AU is scored as black squares (z-axis). Panel A is representative of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: HIV-1 and Its gp120 Inhibits the Influenza A(H1N1)pdm09 Life Cycle in an IFITM3-Dependent Fashion

    doi: 10.1371/journal.pone.0101056

    Figure Lengend Snippet: The HIV-1 inhibitory effect on A(H1N1)pdm09 replication is dependent on the IFITM3 expression. HeLa cells were transfected with 0.4(Scr) using lipofectamine 2000 and, after 6 h, cells were infected by A(H1N1)pdm09 (MOI 5) for 1 h. After that, cells were exposed to HIV-1 (10 ng/mL p24 Ag) for 24 h after influenza infection. ( A ) Monolayers were lysed with Laemmli buffer and submitted to blotting assay for the proteins IFITM3 and β-actin, as an housekeeping. Top panel: cells not exposed to HIV-1, and bottom panel: cells exposed to HIV-1. ( B ) The A(H1N1)pdm09 levels in culture supernatants were quantified by qRT-PCR (y-axis) and the immunoblotting bands were analyzed by densitometry using the ImageJ software 1.6.0 and n-fold changes of AU is scored as black squares (z-axis). Panel A is representative of three independent experiments. * P

    Article Snippet: Cells were transfected with siRNA for IFITM3 (Life Technologies) or its scramble sequence at a concentration of 0.4 nM in OPTI-MEM, using lipofectamine 2000 (Sigma).

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

    p300 enhances p73-induced apoptosis. (A and B) The N-terminal deletion of p73 lacks apoptosis-promoting potential. As indicated, plasmids encoding no protein as a control (C; 3 μg), p73α (1.0 μg), ΔNp73α (1.0 μg), and/or p300 (2.0 μg), together with the pGFP plasmid (200 ng), were introduced into H1299 cells (10 5 /60-mm-diameter dish) using LipofectAmine (GIBCO-BRL); 32 h posttransfection, cells (100) expressing GFP were counted under a fluorescence microscope using a blind approach, with morphologically round cells (arrows) identified as apoptotic (A). (B) Statistical data (with standard error indicated by a bar above each column) from three independent assays. (C and D) Caspase 3 activation by wild-type but not N-terminus-deleted p73α is enhanced by p300. Transient transfection as described for panel A was conducted. Cells were harvested for caspase 3 activity analysis as described in Materials and Methods. (C) Representative result of the fluorescence graph of AMC released from Ac-DEVD-AMC substrates for one set of experiments with plasmids encoding no protein (curve B), ΔNp73α (curve C), ΔNp73α plus p300 (curve D), p73α (curve E), or p73α plus p300 (curve F). Curve A is a water control. Caspase 3 activity is presented in arbitrary units (A.U.) after normalization by a factor of 10 5 . (D) Statistical values for caspase 3 activity in triplicate assays. (E) DNA fragmentation analysis of p73-induced apoptosis in H1299 cells was conducted as described in Materials and Methods; 10 μg of DNA was analyzed by electrophoresis on 2% agarose. Each lane represents the result for three 60-mm-diameter plates of cells. MW, molecular weight markers.

    Journal: Molecular and Cellular Biology

    Article Title: The N-Terminal Domain of p73 Interacts with the CH1 Domain of p300/CREB Binding Protein and Mediates Transcriptional Activation and Apoptosis

    doi:

    Figure Lengend Snippet: p300 enhances p73-induced apoptosis. (A and B) The N-terminal deletion of p73 lacks apoptosis-promoting potential. As indicated, plasmids encoding no protein as a control (C; 3 μg), p73α (1.0 μg), ΔNp73α (1.0 μg), and/or p300 (2.0 μg), together with the pGFP plasmid (200 ng), were introduced into H1299 cells (10 5 /60-mm-diameter dish) using LipofectAmine (GIBCO-BRL); 32 h posttransfection, cells (100) expressing GFP were counted under a fluorescence microscope using a blind approach, with morphologically round cells (arrows) identified as apoptotic (A). (B) Statistical data (with standard error indicated by a bar above each column) from three independent assays. (C and D) Caspase 3 activation by wild-type but not N-terminus-deleted p73α is enhanced by p300. Transient transfection as described for panel A was conducted. Cells were harvested for caspase 3 activity analysis as described in Materials and Methods. (C) Representative result of the fluorescence graph of AMC released from Ac-DEVD-AMC substrates for one set of experiments with plasmids encoding no protein (curve B), ΔNp73α (curve C), ΔNp73α plus p300 (curve D), p73α (curve E), or p73α plus p300 (curve F). Curve A is a water control. Caspase 3 activity is presented in arbitrary units (A.U.) after normalization by a factor of 10 5 . (D) Statistical values for caspase 3 activity in triplicate assays. (E) DNA fragmentation analysis of p73-induced apoptosis in H1299 cells was conducted as described in Materials and Methods; 10 μg of DNA was analyzed by electrophoresis on 2% agarose. Each lane represents the result for three 60-mm-diameter plates of cells. MW, molecular weight markers.

    Article Snippet: H1299 cells (50% confluent) were transfected with plasmids as indicated in the legend to Fig. , using LipofectAmine (Promega) as described above.

    Techniques: Plasmid Preparation, Expressing, Fluorescence, Microscopy, Activation Assay, Transfection, Activity Assay, Electrophoresis, Molecular Weight

    EDHB sensitivity of AKR1C1/C2-depleted KYSE 180 cells A. The efficiency of AKR1C1/C2 knockdown using different AKR1C1/C2 siRNAs was determined by Western blot analysis. B. KYSE 180 cells were transfected with various concentrations of siAKR1C1/C2 537 using Lipofectamine 2000. Knockdown efficiency was determined by Western blot analysis. C, D. A total of 50 pmol of siAKR1C1/C2 537 (C) or siAKR1C1/C2 630 (D) was transfected using Lipofectamine 2000 for 3 days. During this time, KYSE 180 cells were treated with 40 μg/ml EDHB. The MTT assay was performed after EDHB treatment for 1 or 2 days, and the ratio of inhibition was determined relative to untreated cells (mean + S.E.M. for triplicate samples).

    Journal: Oncotarget

    Article Title: The roles of AKR1C1 and AKR1C2 in ethyl-3,4-dihydroxybenzoate induced esophageal squamous cell carcinoma cell death

    doi: 10.18632/oncotarget.7775

    Figure Lengend Snippet: EDHB sensitivity of AKR1C1/C2-depleted KYSE 180 cells A. The efficiency of AKR1C1/C2 knockdown using different AKR1C1/C2 siRNAs was determined by Western blot analysis. B. KYSE 180 cells were transfected with various concentrations of siAKR1C1/C2 537 using Lipofectamine 2000. Knockdown efficiency was determined by Western blot analysis. C, D. A total of 50 pmol of siAKR1C1/C2 537 (C) or siAKR1C1/C2 630 (D) was transfected using Lipofectamine 2000 for 3 days. During this time, KYSE 180 cells were treated with 40 μg/ml EDHB. The MTT assay was performed after EDHB treatment for 1 or 2 days, and the ratio of inhibition was determined relative to untreated cells (mean + S.E.M. for triplicate samples).

    Article Snippet: After 12–16 h of culture, 5 μl of Lipofectamine 2000 and 5 μl of duplexed siRNA (sequences: 5′-UCCAGUGUCU GUAAAGCCATT-3′, 5′-UGGCUUUACAGACACUG GATT-3′, 5′-GGCCGUGGAGAAGUGUAAATT-3′, 5′-UUUACACUUCUCCACGGCCTT-3′, 5′-GGAGAUG AUCCUCAACAAGTT-3′, and 5′-CUUGUUGAGG AUCAUCUCCTT-3′; GenePharma, Shanghai, China) were separately pre-incubated in 150 μl of Opti-MEM for 5 min.

    Techniques: Western Blot, Transfection, MTT Assay, Inhibition

    Properties of N-BLR. a ISH of the tissue microarray (described in Additional file 3 : Figure S5) shows differential expression of N-BLR in colon cancer (Adenocarcinoma) and normal colon (Normal tissue). Hematoxylin and eosin (H E) staining of matched tissues was added to distinguish tissue morphology. Increasing magnifications were provide to evaluate the distribution of N-BLR in the nucleus and in the cytoplasm of cells (5X, 20X, and 60X). b Image analysis of ISH was conducted to measure the expression levels of N-BLR in the different tissues. Adenocarcinoma and metastatic colon cancer tissues expressed higher levels of N-BLR compared with normal colon tissue. There were not significant differences between normal tissue and benign/polyp and colitis tissues. c ISH data on cytoplasmic/nuclear localization of N-BLR. The full arrows point to cytoplasm and the dashed arrows to nucleus. Those two cellular compartments were identified using H E staining. The H E staining and ISH for N-BLR were done on serial sections; therefore, perfect overlapping of tissue morphology did not occur between the two images that show the same tissue area. d PARP-1 expression following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. Profiling was carried out at 96 and 120 h of siRNA transfection. e left Expression of survivin, c-IAP-1, XIAP after 96 h following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. right Quantification of survivin, c-IAP-1, XIAP in Colo320 cells. f Activity of Caspase 3/7, Caspase 8, and Caspase 9 following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. Profiling was carried out after 96 and 120 h (siR = N-BLR siRNA 1 + 3 pool; Ctr = scramble control siRNA; N = lipofectamine only; GAPDH was used as loading control). (Student’s t-test; * p

    Journal: Genome Biology

    Article Title: N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration

    doi: 10.1186/s13059-017-1224-0

    Figure Lengend Snippet: Properties of N-BLR. a ISH of the tissue microarray (described in Additional file 3 : Figure S5) shows differential expression of N-BLR in colon cancer (Adenocarcinoma) and normal colon (Normal tissue). Hematoxylin and eosin (H E) staining of matched tissues was added to distinguish tissue morphology. Increasing magnifications were provide to evaluate the distribution of N-BLR in the nucleus and in the cytoplasm of cells (5X, 20X, and 60X). b Image analysis of ISH was conducted to measure the expression levels of N-BLR in the different tissues. Adenocarcinoma and metastatic colon cancer tissues expressed higher levels of N-BLR compared with normal colon tissue. There were not significant differences between normal tissue and benign/polyp and colitis tissues. c ISH data on cytoplasmic/nuclear localization of N-BLR. The full arrows point to cytoplasm and the dashed arrows to nucleus. Those two cellular compartments were identified using H E staining. The H E staining and ISH for N-BLR were done on serial sections; therefore, perfect overlapping of tissue morphology did not occur between the two images that show the same tissue area. d PARP-1 expression following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. Profiling was carried out at 96 and 120 h of siRNA transfection. e left Expression of survivin, c-IAP-1, XIAP after 96 h following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. right Quantification of survivin, c-IAP-1, XIAP in Colo320 cells. f Activity of Caspase 3/7, Caspase 8, and Caspase 9 following transfection of Colo320 and SW620 cells with siRNAs (N-BLR siRNA1 + 3 pool) against N-BLR. Profiling was carried out after 96 and 120 h (siR = N-BLR siRNA 1 + 3 pool; Ctr = scramble control siRNA; N = lipofectamine only; GAPDH was used as loading control). (Student’s t-test; * p

    Article Snippet: Transfections were performed using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer’s instructions.

    Techniques: In Situ Hybridization, Microarray, Expressing, Staining, Transfection, Activity Assay

    The MCMV M35 protein inhibits signaling of multiple pattern recognition receptors. (A) NIH3T3 fibroblasts were co-transfected with a reporter plasmid containing firefly luciferase under the control of the murine IFNβ promoter (IFNβ-Luc) together with a Renilla luciferase normalization control (pRL-TK) and influenza NS1 or MCMV ORFs known or predicted to code for tegument or immediate-early proteins or their corresponding empty vector (pcDNA and pDEST40, respectively). At 24 hours post transfection, cells were treated with medium or stimulated by infection with Newcastle disease virus (NDV). 21 hours p.i. cells were lysed for analysis of luciferase activity. Luciferase fold induction was calculated based on firefly luciferase values normalized to Renilla luciferase from stimulated samples divided by corresponding values from unstimulated samples. Data set is combined from one to four independent experiments and represented as mean ± SD. (B) NIH3T3 fibroblasts were co-transfected with the IFNβ-Luc and pRL-TK luciferase plasmids described in (A) as well as V5-tagged M35 or pcDNA and luciferase assay was performed following stimulation as for (A). Data is combined from three independent experiments and shown as mean ± SD. (C) NIH3T3 fibroblasts were co-transfected as described in (B) and cells stimulated with 10 μg/ml of poly(I:C) in the presence of Lipofectamine 2000 or Lipofectamine 2000 alone for 6 hours before lysis for analysis by luciferase assay. Data is combined from three independent experiments and shown as mean ± SD. (D) 293T cells were co-transfected with expression plasmids for either cGAS (stimulated) or GFP (unstimulated) together with mCherry-STING, the IFNβ-Luc and pRL-TK luciferase plasmids, and either pcDNA, myc-tagged KSHV ORF36 or V5-tagged M35. At 20 hours post transfection, cells were lysed and luciferase production was analyzed. Data is combined from four independent experiments and shown as mean ± SD. (E) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of 3 μg/ml cGAMP or left unstimulated. RNA was extracted at indicated time points and IFNβ induction was measured by quantitative RT-PCR and expressed as IFNβ induction normalized to the housekeeping gene Rpl8. Data is shown as mean ± SD and combined from two independent experiments. (F) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of indicated amounts of cGAMP and supernatant was collected 16 hours later and analyzed by IFNβ ELISA. Data is shown as mean ± SD and representative of two independent experiments. (G) Immortalized BMDM stably expressing myc-tagged M35 or the corresponding empty vector (pMSCV) were stimulated by addition of 10 ng/ml LPS or 1 μM CpG-B 1826 and supernatant collected at 16 hours post stimulation for analysis by TNFα ELISA. Data is combined from three independent experiments and shown as mean ± SD.***p

    Journal: PLoS Pathogens

    Article Title: The murine cytomegalovirus M35 protein antagonizes type I IFN induction downstream of pattern recognition receptors by targeting NF-κB mediated transcription

    doi: 10.1371/journal.ppat.1006382

    Figure Lengend Snippet: The MCMV M35 protein inhibits signaling of multiple pattern recognition receptors. (A) NIH3T3 fibroblasts were co-transfected with a reporter plasmid containing firefly luciferase under the control of the murine IFNβ promoter (IFNβ-Luc) together with a Renilla luciferase normalization control (pRL-TK) and influenza NS1 or MCMV ORFs known or predicted to code for tegument or immediate-early proteins or their corresponding empty vector (pcDNA and pDEST40, respectively). At 24 hours post transfection, cells were treated with medium or stimulated by infection with Newcastle disease virus (NDV). 21 hours p.i. cells were lysed for analysis of luciferase activity. Luciferase fold induction was calculated based on firefly luciferase values normalized to Renilla luciferase from stimulated samples divided by corresponding values from unstimulated samples. Data set is combined from one to four independent experiments and represented as mean ± SD. (B) NIH3T3 fibroblasts were co-transfected with the IFNβ-Luc and pRL-TK luciferase plasmids described in (A) as well as V5-tagged M35 or pcDNA and luciferase assay was performed following stimulation as for (A). Data is combined from three independent experiments and shown as mean ± SD. (C) NIH3T3 fibroblasts were co-transfected as described in (B) and cells stimulated with 10 μg/ml of poly(I:C) in the presence of Lipofectamine 2000 or Lipofectamine 2000 alone for 6 hours before lysis for analysis by luciferase assay. Data is combined from three independent experiments and shown as mean ± SD. (D) 293T cells were co-transfected with expression plasmids for either cGAS (stimulated) or GFP (unstimulated) together with mCherry-STING, the IFNβ-Luc and pRL-TK luciferase plasmids, and either pcDNA, myc-tagged KSHV ORF36 or V5-tagged M35. At 20 hours post transfection, cells were lysed and luciferase production was analyzed. Data is combined from four independent experiments and shown as mean ± SD. (E) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of 3 μg/ml cGAMP or left unstimulated. RNA was extracted at indicated time points and IFNβ induction was measured by quantitative RT-PCR and expressed as IFNβ induction normalized to the housekeeping gene Rpl8. Data is shown as mean ± SD and combined from two independent experiments. (F) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of indicated amounts of cGAMP and supernatant was collected 16 hours later and analyzed by IFNβ ELISA. Data is shown as mean ± SD and representative of two independent experiments. (G) Immortalized BMDM stably expressing myc-tagged M35 or the corresponding empty vector (pMSCV) were stimulated by addition of 10 ng/ml LPS or 1 μM CpG-B 1826 and supernatant collected at 16 hours post stimulation for analysis by TNFα ELISA. Data is combined from three independent experiments and shown as mean ± SD.***p

    Article Snippet: 24 hours later, the medium was replaced with fresh medium and cells were mock-treated or stimulated by transfection of 10 μg/ml poly(I:C) (Invivogen) complexed with Lipofectamine 2000.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Infection, Activity Assay, Lysis, Expressing, Stable Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    M35 does not target the phosphorylation or nuclear translocation of key transcription factors. (A) Immortalized BMDM stably expressing LacZ-myc or M35-myc were stimulated by addition of 3 μg/ml cGAMP and cells lysed at indicated time points. Lysates were separated by SDS-PAGE and endogenous IRF3 and phospho-IRF3 were detected with specific antibodies. Expression of myc-tagged LacZ and M35 was verified with a myc-specific antibody. Immunoblot shown is representative of three independent experiments. (B) NIH3T3 fibroblasts stably expressing myc-tagged LacZ or M35 and eGFP-IRF3 were stimulated by transfection of 10 μg/ml poly(I:C) with Lipofectamine 2000. At indicated times post stimulation, cells were fixed for immunolabeling with a mouse anti-myc antibody and imaged by confocal microscopy (upper panel). Nuclei were stained with Hoechst. Scale bars represent 10 μm. Corresponding counts for cells showing nuclear IRF3 (lower panel) are represented as the percentage of total cells (with a minimum of 100 cells counted per timepoint). Data is shown as mean ± SD and combined from two independent experiments. (C) NIH3T3 fibroblasts stably expressing myc-tagged M35 or corresponding empty vector pQCXIH were stimulated by transfection of 10 μg/ml poly(I:C) in the presence of Lipofectamine 2000 and cells were lysed at indicated time points. Lysates were separated by SDS-PAGE and endogenous p65, phospho-p65 and tubulin were detected with specific antibodies. Expression of myc-tagged M35 was verified with a myc-specific antibody. Immunoblot shown is representative of three independent experiments. (D) NIH3T3 fibroblasts stably expressing myc-tagged M35 or corresponding empty vector pQCXIH were stimulated as for (B). At indicated times post stimulation, cells were fixed for immunolabeling with a mouse anti-myc antibody and a rabbit anti-p65 antibody and imaged by confocal microscopy (upper panel). Nuclei were stained with Hoechst. Scale bars represent 10 μm. Corresponding counts for cells showing nuclear p65 (lower panel) are represented as percentage of total cells (with a minimum of 300 cells counted per timepoint). Data is shown as mean ± SD and combined from two independent experiments.

    Journal: PLoS Pathogens

    Article Title: The murine cytomegalovirus M35 protein antagonizes type I IFN induction downstream of pattern recognition receptors by targeting NF-κB mediated transcription

    doi: 10.1371/journal.ppat.1006382

    Figure Lengend Snippet: M35 does not target the phosphorylation or nuclear translocation of key transcription factors. (A) Immortalized BMDM stably expressing LacZ-myc or M35-myc were stimulated by addition of 3 μg/ml cGAMP and cells lysed at indicated time points. Lysates were separated by SDS-PAGE and endogenous IRF3 and phospho-IRF3 were detected with specific antibodies. Expression of myc-tagged LacZ and M35 was verified with a myc-specific antibody. Immunoblot shown is representative of three independent experiments. (B) NIH3T3 fibroblasts stably expressing myc-tagged LacZ or M35 and eGFP-IRF3 were stimulated by transfection of 10 μg/ml poly(I:C) with Lipofectamine 2000. At indicated times post stimulation, cells were fixed for immunolabeling with a mouse anti-myc antibody and imaged by confocal microscopy (upper panel). Nuclei were stained with Hoechst. Scale bars represent 10 μm. Corresponding counts for cells showing nuclear IRF3 (lower panel) are represented as the percentage of total cells (with a minimum of 100 cells counted per timepoint). Data is shown as mean ± SD and combined from two independent experiments. (C) NIH3T3 fibroblasts stably expressing myc-tagged M35 or corresponding empty vector pQCXIH were stimulated by transfection of 10 μg/ml poly(I:C) in the presence of Lipofectamine 2000 and cells were lysed at indicated time points. Lysates were separated by SDS-PAGE and endogenous p65, phospho-p65 and tubulin were detected with specific antibodies. Expression of myc-tagged M35 was verified with a myc-specific antibody. Immunoblot shown is representative of three independent experiments. (D) NIH3T3 fibroblasts stably expressing myc-tagged M35 or corresponding empty vector pQCXIH were stimulated as for (B). At indicated times post stimulation, cells were fixed for immunolabeling with a mouse anti-myc antibody and a rabbit anti-p65 antibody and imaged by confocal microscopy (upper panel). Nuclei were stained with Hoechst. Scale bars represent 10 μm. Corresponding counts for cells showing nuclear p65 (lower panel) are represented as percentage of total cells (with a minimum of 300 cells counted per timepoint). Data is shown as mean ± SD and combined from two independent experiments.

    Article Snippet: 24 hours later, the medium was replaced with fresh medium and cells were mock-treated or stimulated by transfection of 10 μg/ml poly(I:C) (Invivogen) complexed with Lipofectamine 2000.

    Techniques: Translocation Assay, Stable Transfection, Expressing, SDS Page, Transfection, Immunolabeling, Confocal Microscopy, Staining, Plasmid Preparation