lipofectamine 2000 Search Results


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  • 99
    Thermo Fisher lipofectamine lf 2000
    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 <t>lipofectamine</t> 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.
    Lipofectamine Lf 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 65900 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine ltx
    The importance of syntaxin cleavage in botulinum-triggered cell demise. (a) Immunoblot showing that, in the presence of <t>Lipofectamine</t> <t>LTX,</t> the botulinum protease type C cleaves both syntaxin 1 and SNAP25 in N2A cells, whereas the type A protease cleaves SNAP25 only. Type C protease removes the last eight residues of SNAP25 (SNAP25Δ8), whereas type A removes the last nine (SNAP25Δ9). (b) The bar chart showing N2A cell demise following a 40 h treatment with the indicated proteases. Type A-induced cleavage of SNAP25, even in the presence of the type D protease, is insufficient to drive the cytotoxic effects. In contrast, cleavage of syntaxin by type C protease has a strong effect on cell survival (** p
    Lipofectamine Ltx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22487 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genechem lipofectamine 2000
    The importance of syntaxin cleavage in botulinum-triggered cell demise. (a) Immunoblot showing that, in the presence of <t>Lipofectamine</t> <t>LTX,</t> the botulinum protease type C cleaves both syntaxin 1 and SNAP25 in N2A cells, whereas the type A protease cleaves SNAP25 only. Type C protease removes the last eight residues of SNAP25 (SNAP25Δ8), whereas type A removes the last nine (SNAP25Δ9). (b) The bar chart showing N2A cell demise following a 40 h treatment with the indicated proteases. Type A-induced cleavage of SNAP25, even in the presence of the type D protease, is insufficient to drive the cytotoxic effects. In contrast, cleavage of syntaxin by type C protease has a strong effect on cell survival (** p
    Lipofectamine 2000, supplied by Genechem, used in various techniques. Bioz Stars score: 97/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 348 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 99/100, based on 1064 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 93/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 93/100, based on 316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Horizon Discovery lipofectamine 2000
    Transfection of FAK siRNA in osteoblasts. (A) Western blot analysis of FAK and Pyk2 expression and activation (phosphorylation of Y397, Y402) in osteoblasts treated with <t>lipofectamine</t> only (Lipo Only), 50 nM scrambled control siRNA (Scramble), or 50 nM
    Lipofectamine 2000, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 95/100, based on 217 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Beijing TransGen Biotech lipofectamine 2000
    Transfection of FAK siRNA in osteoblasts. (A) Western blot analysis of FAK and Pyk2 expression and activation (phosphorylation of Y397, Y402) in osteoblasts treated with <t>lipofectamine</t> only (Lipo Only), 50 nM scrambled control siRNA (Scramble), or 50 nM
    Lipofectamine 2000, supplied by Beijing TransGen Biotech, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 97/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc 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 Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime lipofectamine 2000
    pEGFP-C1 encapsulated by different vectors for gene delivery to hBMSCs. The expression of enhanced green fluorescent protein (EGFP) in hBMSCs transfected with complexes such as scAb GD2 -PEG-g-PEI-SPION/pDNA (a), <t>lipofectamine/pDNA</t> (b), and PEG-g-PEI-SPION/pDNA (c) was observed using an inverted fluorescence microscope and was quantified by flow cytometry (d). The transfection efficiency of scAb GD2 -PEG-g-PEI-SPION/pDNA was significantly higher than those of PEG-g-PEI-SPION/pDNA, scAb GD2 -PEG-g-PEI-SPION/pDNA + free Ab GD2 , and scAb IgG2a -PEG-g-PEI-SPION/pDNA at the same N/P ratio. # P
    Lipofectamine 2000, supplied by Beyotime, used in various techniques. Bioz Stars score: 96/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co lipofectamine 2000
    pEGFP-C1 encapsulated by different vectors for gene delivery to hBMSCs. The expression of enhanced green fluorescent protein (EGFP) in hBMSCs transfected with complexes such as scAb GD2 -PEG-g-PEI-SPION/pDNA (a), <t>lipofectamine/pDNA</t> (b), and PEG-g-PEI-SPION/pDNA (c) was observed using an inverted fluorescence microscope and was quantified by flow cytometry (d). The transfection efficiency of scAb GD2 -PEG-g-PEI-SPION/pDNA was significantly higher than those of PEG-g-PEI-SPION/pDNA, scAb GD2 -PEG-g-PEI-SPION/pDNA + free Ab GD2 , and scAb IgG2a -PEG-g-PEI-SPION/pDNA at the same N/P ratio. # P
    Lipofectamine 2000, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Wisent Corporation lipofectamine 2000
    pEGFP-C1 encapsulated by different vectors for gene delivery to hBMSCs. The expression of enhanced green fluorescent protein (EGFP) in hBMSCs transfected with complexes such as scAb GD2 -PEG-g-PEI-SPION/pDNA (a), <t>lipofectamine/pDNA</t> (b), and PEG-g-PEI-SPION/pDNA (c) was observed using an inverted fluorescence microscope and was quantified by flow cytometry (d). The transfection efficiency of scAb GD2 -PEG-g-PEI-SPION/pDNA was significantly higher than those of PEG-g-PEI-SPION/pDNA, scAb GD2 -PEG-g-PEI-SPION/pDNA + free Ab GD2 , and scAb IgG2a -PEG-g-PEI-SPION/pDNA at the same N/P ratio. # P
    Lipofectamine 2000, supplied by Wisent Corporation, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Introgen lipofectamine 2000
    pEGFP-C1 encapsulated by different vectors for gene delivery to hBMSCs. The expression of enhanced green fluorescent protein (EGFP) in hBMSCs transfected with complexes such as scAb GD2 -PEG-g-PEI-SPION/pDNA (a), <t>lipofectamine/pDNA</t> (b), and PEG-g-PEI-SPION/pDNA (c) was observed using an inverted fluorescence microscope and was quantified by flow cytometry (d). The transfection efficiency of scAb GD2 -PEG-g-PEI-SPION/pDNA was significantly higher than those of PEG-g-PEI-SPION/pDNA, scAb GD2 -PEG-g-PEI-SPION/pDNA + free Ab GD2 , and scAb IgG2a -PEG-g-PEI-SPION/pDNA at the same N/P ratio. # P
    Lipofectamine 2000, supplied by Introgen, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reagent lipofectamine
    Primary human fetal hepatocytes were transfected with EGFP that was cloned into the hTert vector. Following transfection with the hTert vector using <t>lipofectamine</t> as a transfection reagent, human fetal hepatocytes were highly proliferative. However, these
    Reagent Lipofectamine, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 260 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine 3000
    Upregulation of hepatic CDCA5 expression in HCC cell lines and patients with HCC and knockdown of CDCA5 in Huh7 and Hep3B. (A) Expression of CDCA5 mRNA in 4 human HCC cell lines and 2 normal human hepatic cells was evaluated by qRT-PCR. (B) Western blots show the CDCA5 protein level compared to Tubulin in HCC cell lines and normal hepatic cells. (C) Representative images of immunohistochemistry staining of liver sections of HCC tissues (n=81) and paired adjacent normal tissues (n=81). Original magnification, ×200. Upper panel, HCC tissues; Lower panel, adjacent normal tissues. (D and E) Western blots and qRT-PCR showing the effect of RNAi on CDCA5 expression. There different siCDCA5s at 50 nM were transfected into human HCC cell lines Huh7 and Hep3B using Lipofectamine 3000. *p
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    Thermo Fisher lipofectamine rnaimax
    Sca-1+/CD31− CSCs hTERT CM protects HL-1 cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Sca-1+/CD31− CSCs hTERT were cultured in six-well plates at a density of 5 × 10 4 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes or NC siRNA duplexes using <t>Lipofectamine</t> <t>RNAiMAX.</t> After a 48 h transfection, MCP-1 mRNA expression was assessed by real-time PCR ( A ); The average value of MCP-1 mRNA was normalized to that of GAPDH for each sample. The data represent the mean ± SD from three experiments. Sca-1+/CD31− CSCs hTERT CM ( B ) after transfection of NC siRNA or MCP-1 siRNA were subjected to densitometry and are presented as fold changes for MCP-1 (indicated by number 6) and IL-6 (indicated by number 15) ( C ), taking MCP-1 and IL-6 levels in NC siRNA-transfected Sca-1+/CD31− CSCs hTERT as a one-fold value, each in triplicate, * p
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    Polyplus Transfection lipofectamine 2000
    The Sesn2 response to an energetic stress is regulated by Akt. ( a ) LNCaP cells were either treated with <t>lipofectamine</t> (Lipo) or transfected with siCt or sip53 for 48 h. Then, 20 mM 2-DG was added for 15 h (2-DG15) or 24 h
    Lipofectamine 2000, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    scFv NLDC-145 targets tumor antigen into DCs in vivo. A schematic representation of expression vectors. scFv NLDC-145 -HER2, scFv NLDC-145 -neu, pcDNA3.1-HER2, or pcDNA3.1-neu encode under the control of a CMV promoter, all the fusion proteins consisting of an signal peptide, amino acid residues 22 to 561 of human HER2 or amino acid residues 22 to 582 of rat neu, and COOH-terminal His tag. The control plasmids pcDNA3.1-HER2 and pcDNA3.1-neu lack the NLDC-145 domains. B 293T cells grown in 100-mm dishes were transfected with various expression vectors using Lipofectamine 2000 (invitrogen). Immunoblot analysis of supernatants from 293T cells transfected with scFv NLDC-145 -HER2, scFv NLDC-145 -neu, pcDNA3.1-HER2 and pcDNA3.1-neu (lane 1, 2, 3 and 4). Vaccine proteins were probed with mouse anti-His tag mAb followed by HRP-conjugated secondary anti-mouse antibody. C left, scFv NLDC-145 -EGFP plasmid and controls as indicated were injected i.m. into mice, spleens were removed 48, 60, and 72 h thereafter, and stained with PE-conjugated anti-CD11c antibody. The GFP fluorescence in splenocytes was measured by flow cytometry. Representative results from one animal of each group 60 h post-injection. right, the expression of EGFP (MW,~25 kDa) and scFv NLDC-145 -EGFP (MW,~55 kDa) fusion protein in injected muscle tissues detected by western blotting using anti- EGFP antibody 24 h after plasmid injection with GAPDH (MW,42 kDa) as loading control. Lane 1, mice treated with pcDNA3.1 control vaccine; lane 2, mice treated with EGFP-encoding vaccine; lane 3, mice treated with scFv NLDC-145 -EGFP vaccine; D percentages of GFP + DC in total DC ( left panel ); Percentages of GFP + in CD11c-negative splenocytes ( right panel ). Bars, SE. *, P

    Journal: BMC Immunology

    Article Title: DNA vaccines targeting the encoded antigens to dendritic cells induce potent antitumor immunity in mice

    doi: 10.1186/1471-2172-14-39

    Figure Lengend Snippet: scFv NLDC-145 targets tumor antigen into DCs in vivo. A schematic representation of expression vectors. scFv NLDC-145 -HER2, scFv NLDC-145 -neu, pcDNA3.1-HER2, or pcDNA3.1-neu encode under the control of a CMV promoter, all the fusion proteins consisting of an signal peptide, amino acid residues 22 to 561 of human HER2 or amino acid residues 22 to 582 of rat neu, and COOH-terminal His tag. The control plasmids pcDNA3.1-HER2 and pcDNA3.1-neu lack the NLDC-145 domains. B 293T cells grown in 100-mm dishes were transfected with various expression vectors using Lipofectamine 2000 (invitrogen). Immunoblot analysis of supernatants from 293T cells transfected with scFv NLDC-145 -HER2, scFv NLDC-145 -neu, pcDNA3.1-HER2 and pcDNA3.1-neu (lane 1, 2, 3 and 4). Vaccine proteins were probed with mouse anti-His tag mAb followed by HRP-conjugated secondary anti-mouse antibody. C left, scFv NLDC-145 -EGFP plasmid and controls as indicated were injected i.m. into mice, spleens were removed 48, 60, and 72 h thereafter, and stained with PE-conjugated anti-CD11c antibody. The GFP fluorescence in splenocytes was measured by flow cytometry. Representative results from one animal of each group 60 h post-injection. right, the expression of EGFP (MW,~25 kDa) and scFv NLDC-145 -EGFP (MW,~55 kDa) fusion protein in injected muscle tissues detected by western blotting using anti- EGFP antibody 24 h after plasmid injection with GAPDH (MW,42 kDa) as loading control. Lane 1, mice treated with pcDNA3.1 control vaccine; lane 2, mice treated with EGFP-encoding vaccine; lane 3, mice treated with scFv NLDC-145 -EGFP vaccine; D percentages of GFP + DC in total DC ( left panel ); Percentages of GFP + in CD11c-negative splenocytes ( right panel ). Bars, SE. *, P

    Article Snippet: Expression of protein encoded by DNA vaccines The different pcDNA3.1 constructs were transiently transfected in 293T cells using Lipofectamine 2000 according to the manual instruction (Invitrogen).

    Techniques: In Vivo, Expressing, Transfection, Plasmid Preparation, Injection, Mouse Assay, Staining, Fluorescence, Flow Cytometry, Cytometry, Western Blot

    RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using lipofectamine reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p

    Journal: Nutrition & Metabolism

    Article Title: Mice subjected to aP2-Cre mediated ablation of microsomal triglyceride transfer protein are resistant to high fat diet induced obesity

    doi: 10.1186/s12986-016-0061-6

    Figure Lengend Snippet: RNAi-based MTP gene silencing and MTP overexpression in differentiated 3T3-L1 cells modulates adipogenesis. Cells were partially differentiated in DMEM containing 10 % FBS until D4. On day 4, cells were transfected with scrambled siRNA or siRNA for MTP and replated. Three days postransfection (day 7), cells were collected and assessed for MTP expression ( a ), MTP activity ( b ), total cell protein content ( c ), and lipid droplet formation ( d ). In a different experiment, cells were transfected using lipofectamine reagent and 1 μg of cDNA of empty vector ( pCMV6 ) or human MTP (hMTP-Flag or pRC-hMTP) for 24 h and 48 h transfection. Cells were harvested in homogenization buffer to determine MTP activity ( e ), intracellular triglycerides ( f ), and total cell protein ( g ). Results shown are representative of two independent experiments. Values are mean of triplicates ± SD. *, p

    Article Snippet: Cell pellets were resuspended in 200 μl of OptiMEM (Gibco, Life Technologies) and incubated at 37 °C with equal volume of lipofectamine complexes (RNAiMax; Life Technologies) containing siRNA (100 nM) or plasmid DNA (1 μg/ml).

    Techniques: Over Expression, Transfection, Expressing, Activity Assay, Plasmid Preparation, Homogenization

    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

    DNA-PKcs is an important but not exclusive target of KU-0060648 in HCC cells Same amount (5 × 10 4 cells/dish) of stable HepG2 cells expressing scramble control shRNA (“Scr shRNA”), DNA-PKcs shRNA (Seq-1) or DNA-PKcs shRNA (Seq-2), as well as the parental HepG2 cells (“No shRNA”), treated with KU-0060648 (“KU”, 300 nM) or vehicle (PBS) for applied time, were cultured for indicated time. Expressions of DNA-PKcs and tubulin (loading control) were tested by Western blotting A . Cell growth was tested by MTT assay B. Cell apoptosis was evaluated by Histone DNA ELISA assay C. Above experiments were also performed in stable HepG2 cells expressing dominant negative (T2609A) DNA-PKcs (dn-DNA-PKcs) or the empty vector (pSV2 neo) D-F. Primary human HCC cells (line-1/-2), transfected with scramble control (“Scr siRNA”) or DNA-PKcs siRNA (200 nM each), were treated with PBS or KU-0060648 (“KU”, 300 nM). After 72h culture, DNA-PKcs and tubulin expressions were tested by Western blotting G. Cell proliferation was tested by MTT assay H. Stable HepG2 cells expressing the empty vector (“Vector”) or wild-type DNA-PKcs (wt-DNA-PKcs-Flag) were subjected to Western blotting assay I. and cell proliferation assay J. and K. “Transfection” stands for Lipofectamine 2000 reagent alone. DNA-PKcs expression (vs. Tubulin) was quantified. Experiments in this figure were repeated three times, with similar results obtained. n=5 for each repeat. Bars stand for mean ± SD * p

    Journal: Oncotarget

    Article Title: KU-0060648 inhibits hepatocellular carcinoma cells through DNA-PKcs-dependent and DNA-PKcs-independent mechanisms

    doi: 10.18632/oncotarget.7742

    Figure Lengend Snippet: DNA-PKcs is an important but not exclusive target of KU-0060648 in HCC cells Same amount (5 × 10 4 cells/dish) of stable HepG2 cells expressing scramble control shRNA (“Scr shRNA”), DNA-PKcs shRNA (Seq-1) or DNA-PKcs shRNA (Seq-2), as well as the parental HepG2 cells (“No shRNA”), treated with KU-0060648 (“KU”, 300 nM) or vehicle (PBS) for applied time, were cultured for indicated time. Expressions of DNA-PKcs and tubulin (loading control) were tested by Western blotting A . Cell growth was tested by MTT assay B. Cell apoptosis was evaluated by Histone DNA ELISA assay C. Above experiments were also performed in stable HepG2 cells expressing dominant negative (T2609A) DNA-PKcs (dn-DNA-PKcs) or the empty vector (pSV2 neo) D-F. Primary human HCC cells (line-1/-2), transfected with scramble control (“Scr siRNA”) or DNA-PKcs siRNA (200 nM each), were treated with PBS or KU-0060648 (“KU”, 300 nM). After 72h culture, DNA-PKcs and tubulin expressions were tested by Western blotting G. Cell proliferation was tested by MTT assay H. Stable HepG2 cells expressing the empty vector (“Vector”) or wild-type DNA-PKcs (wt-DNA-PKcs-Flag) were subjected to Western blotting assay I. and cell proliferation assay J. and K. “Transfection” stands for Lipofectamine 2000 reagent alone. DNA-PKcs expression (vs. Tubulin) was quantified. Experiments in this figure were repeated three times, with similar results obtained. n=5 for each repeat. Bars stand for mean ± SD * p

    Article Snippet: The T2609A DNA-PKcs pSV2 neo Flag plasmid [ ] or the wild-type (wt-) DNA-PKcs pSV2 neo Flag plasmid (gift from Dr. Li He at Kunming Medical University) [ ] was transfected into HepG2 cells with the Lipofectamine 2000 protocol (Invitrogen) [ ].

    Techniques: Expressing, shRNA, Cell Culture, Western Blot, MTT Assay, Enzyme-linked Immunosorbent Assay, Dominant Negative Mutation, Plasmid Preparation, Transfection, Proliferation Assay

    Cytotoxicity of Lipofectamine TM 2000 or HPhA in HL-7702 cells. The data represent mean±SD ( n =4). * P

    Journal: International Journal of Medical Sciences

    Article Title: Inhibition of HCV 5?-NTR and Core Expression by a Small Hairpin RNA Delivered by a Histone Gene Carrier, HPhA

    doi: 10.7150/ijms.5632

    Figure Lengend Snippet: Cytotoxicity of Lipofectamine TM 2000 or HPhA in HL-7702 cells. The data represent mean±SD ( n =4). * P

    Article Snippet: The following day, the plasmids pCMV/T7 NTRCΔ-luc, 1 μg, and shRNA expression plasmids (psh-72, psh-274, psh-365, psh-1019 and scrambled control), 1 μg, were simultaneously introduced into HL-7702 cells by 3 μg HphA and 3 μL Lipofectamine TM 2000 (Invitrogen), respectively .

    Techniques:

    Overexpression of microRNA-128 inhibits cell growth of U87 glioma cells. A: Real-time PCR analysis of microRNA-128 in U87 cells after transfection with pre-mi128 or negative control for 48 h. Pre-miR-128 and negative control (NC) were transfected into U87 using Lipofectamine 2000 (Invitrogen). B: Cell growth curve of U87 cells after treatment with negative control or miR-128 as above. Values represent means ± standard deviation (SD) for three wells. * means p value ≤0.05, all compared with control.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: MicroRNA-128 increases glioma cell radio-sensitivity by suppressing senescent evasion through oncogene Bmi-1

    doi:

    Figure Lengend Snippet: Overexpression of microRNA-128 inhibits cell growth of U87 glioma cells. A: Real-time PCR analysis of microRNA-128 in U87 cells after transfection with pre-mi128 or negative control for 48 h. Pre-miR-128 and negative control (NC) were transfected into U87 using Lipofectamine 2000 (Invitrogen). B: Cell growth curve of U87 cells after treatment with negative control or miR-128 as above. Values represent means ± standard deviation (SD) for three wells. * means p value ≤0.05, all compared with control.

    Article Snippet: Transfection of microRNA128 was carried out using Lipofectamine 2000 agent (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Over Expression, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Standard Deviation

    Luciferase (lux) expression after bactofection of cystic fibrosis tracheal epithelial (CFTE29o—) cells with invasive E. coli . CFTE29o— cells were transfected with invasive E. coli BM4570 carrying the eukaryotic expression plasmid pCIKLux at MOI 50-5000. After 48 h of infection cells were harvested and lux activity assayed. Bacteria-mediated expression was compared to cells transfected with pCIKLux complexed to Lipofectamine 2000 (Lipo/pCIKLux) or untransfected cells. Data are expressed as mean±s.e.m. ( n = 5 per group, ** P

    Journal: Gene therapy

    Article Title: Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli

    doi: 10.1038/sj.gt.3303090

    Figure Lengend Snippet: Luciferase (lux) expression after bactofection of cystic fibrosis tracheal epithelial (CFTE29o—) cells with invasive E. coli . CFTE29o— cells were transfected with invasive E. coli BM4570 carrying the eukaryotic expression plasmid pCIKLux at MOI 50-5000. After 48 h of infection cells were harvested and lux activity assayed. Bacteria-mediated expression was compared to cells transfected with pCIKLux complexed to Lipofectamine 2000 (Lipo/pCIKLux) or untransfected cells. Data are expressed as mean±s.e.m. ( n = 5 per group, ** P

    Article Snippet: However, lux activity even at the highest MOI was significantly ( P < 0.05) lower than after Lipofectamine 2000-mediated (Invitrogen Ltd., Paisley, UK) gene transfer, despite the fact that a similar number of plasmid molecules was added to the cell preparation in each case.

    Techniques: Luciferase, Expressing, Transfection, Plasmid Preparation, Infection, Activity Assay

    Transfection efficiency in vitro (H1299/LUC cells) of oligospermines polyplexes at a N/P ratio of 2 on the mRNA level measured by qRT-PCR and compared to lipofectamine (LF). Hs_GAPDH primers were used to quantify hGAPDH gene expression. Hs_β-actin primers were employed as an internal standard to evaluate the relative gene expression of the two genes and to normalize the changes in hGAPDH expression. Calibration curves of hGAPDH and β-actin were plotted by serial dilution of cDNA from untreated cells. Polyplexes made of GADPH siRNA and linear tetraspermine showed the best knockdown compared to dendritic tetraspermine (54.6 vs 75.1% residual GAPDH expression) and linear bisspermine polyplexes (no knockdown).

    Journal: Biomacromolecules

    Article Title: Influence of Oligospermines Architecture on Their Suitability for siRNA Delivery

    doi: 10.1021/bm401849d

    Figure Lengend Snippet: Transfection efficiency in vitro (H1299/LUC cells) of oligospermines polyplexes at a N/P ratio of 2 on the mRNA level measured by qRT-PCR and compared to lipofectamine (LF). Hs_GAPDH primers were used to quantify hGAPDH gene expression. Hs_β-actin primers were employed as an internal standard to evaluate the relative gene expression of the two genes and to normalize the changes in hGAPDH expression. Calibration curves of hGAPDH and β-actin were plotted by serial dilution of cDNA from untreated cells. Polyplexes made of GADPH siRNA and linear tetraspermine showed the best knockdown compared to dendritic tetraspermine (54.6 vs 75.1% residual GAPDH expression) and linear bisspermine polyplexes (no knockdown).

    Article Snippet: Lipofectamine 2000 (LF), SYBR Gold dye, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Life Technologies (Grand Island, NY).

    Techniques: Transfection, In Vitro, Quantitative RT-PCR, Expressing, Serial Dilution

    Silencing efficiency of firefly luciferase expression in H1299/LUC cells by oligospermine polyplexes with FLUC siRNA or nonspecific control siRNA at a N/P ratio of 2 after 48 h of transfection compared to lipofectamine (LF). The relative gene silencing was normalized to blank untreated cells. Results are the mean value of triplicate measurements ± SD.

    Journal: Biomacromolecules

    Article Title: Influence of Oligospermines Architecture on Their Suitability for siRNA Delivery

    doi: 10.1021/bm401849d

    Figure Lengend Snippet: Silencing efficiency of firefly luciferase expression in H1299/LUC cells by oligospermine polyplexes with FLUC siRNA or nonspecific control siRNA at a N/P ratio of 2 after 48 h of transfection compared to lipofectamine (LF). The relative gene silencing was normalized to blank untreated cells. Results are the mean value of triplicate measurements ± SD.

    Article Snippet: Lipofectamine 2000 (LF), SYBR Gold dye, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Life Technologies (Grand Island, NY).

    Techniques: Luciferase, Expressing, Transfection

    Cytotoxicity profiles of oligospermine polymers obtained by MTT assays and compared to polyethylenimine (PEI) and lipofectamine (LF). Percentage of viability of H1299/LUC cells is shown as a function of increasing polymer concentration after 24 h of polymer incubation. The table shows the IC 50 concentrations of the polymers in milligrams per milliliter.

    Journal: Biomacromolecules

    Article Title: Influence of Oligospermines Architecture on Their Suitability for siRNA Delivery

    doi: 10.1021/bm401849d

    Figure Lengend Snippet: Cytotoxicity profiles of oligospermine polymers obtained by MTT assays and compared to polyethylenimine (PEI) and lipofectamine (LF). Percentage of viability of H1299/LUC cells is shown as a function of increasing polymer concentration after 24 h of polymer incubation. The table shows the IC 50 concentrations of the polymers in milligrams per milliliter.

    Article Snippet: Lipofectamine 2000 (LF), SYBR Gold dye, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Life Technologies (Grand Island, NY).

    Techniques: MTT Assay, Concentration Assay, Incubation

    Flow cytometric analysis of RFP expressing PAM212 cells that were stained with either MitoTracker or Sytox red nucleic acid stain. Cells were transfected with either 18-3-18 GL-NPs or Lipofectamine Plus reagent. a A comparison between the proportion of live and dead PAM212 cells represented by either MitoTracker or Sytox red fluorescent signal. b The density plots show representative data from one of three separate experiments

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Flow cytometric analysis of RFP expressing PAM212 cells that were stained with either MitoTracker or Sytox red nucleic acid stain. Cells were transfected with either 18-3-18 GL-NPs or Lipofectamine Plus reagent. a A comparison between the proportion of live and dead PAM212 cells represented by either MitoTracker or Sytox red fluorescent signal. b The density plots show representative data from one of three separate experiments

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Flow Cytometry, Expressing, Staining, Transfection

    Evaluating SSC intensity as a parameter of NPs internalization into the cells using flow cytometry. a The SSC intensity of 18-3-18 GL-NPs treated cells was higher than Lipofectamine Plus treated and untreated cells (R1). b RFP expression in SSC high (R3) and low cell population (R4). Each dot plot represents 10,000 events

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Evaluating SSC intensity as a parameter of NPs internalization into the cells using flow cytometry. a The SSC intensity of 18-3-18 GL-NPs treated cells was higher than Lipofectamine Plus treated and untreated cells (R1). b RFP expression in SSC high (R3) and low cell population (R4). Each dot plot represents 10,000 events

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Flow Cytometry, Cytometry, Expressing

    RFP expression in PAM212 cells with different status of cell membrane integrity. a The status of cell membrane integrity was determined based on the intensity of Sytox red DNA stain using flow cytometry; R1 highly-porated cells, R2 partially-porated cells, R3 intact cells, R4 debris and cell fragments. b Stacked bar graph presents the membrane status of cells expressing RFP after transfection by Lipofectamine Plus or 18-3-18 GL-NPs

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: RFP expression in PAM212 cells with different status of cell membrane integrity. a The status of cell membrane integrity was determined based on the intensity of Sytox red DNA stain using flow cytometry; R1 highly-porated cells, R2 partially-porated cells, R3 intact cells, R4 debris and cell fragments. b Stacked bar graph presents the membrane status of cells expressing RFP after transfection by Lipofectamine Plus or 18-3-18 GL-NPs

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry, Transfection

    Effect of GL-NPs on the viability of PAM212 cells measured by MitoTracker staining and flow cytometry. Results are expressed as the mean of cell viability index ± standard deviation compared to the untreated control (as 100 %). Asterisks represent LSD post hoc statistical significance compared to Lipofectamine Plus (P

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Effect of GL-NPs on the viability of PAM212 cells measured by MitoTracker staining and flow cytometry. Results are expressed as the mean of cell viability index ± standard deviation compared to the untreated control (as 100 %). Asterisks represent LSD post hoc statistical significance compared to Lipofectamine Plus (P

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Staining, Flow Cytometry, Cytometry, Standard Deviation

    RFP expression in PAM212 cells, transfected GL-NPs and Lipofectamine Plus reagent measured by flow cytometry. Results are expressed as the mean percentage of RFP positive cells ± standard deviation. Results are expressed as mean measurements ± SD (n = 4). Asterisks represent LSD post hoc statistical significance compared to Lipofectamine Plus (P

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: RFP expression in PAM212 cells, transfected GL-NPs and Lipofectamine Plus reagent measured by flow cytometry. Results are expressed as the mean percentage of RFP positive cells ± standard deviation. Results are expressed as mean measurements ± SD (n = 4). Asterisks represent LSD post hoc statistical significance compared to Lipofectamine Plus (P

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Standard Deviation

    Median fluorescence intensity (MFI) of RFP positive PAM212 cells transfected by Lipofectamine Plus or 18-3-18 GL-NPs. MFI for RFP in cells transfected by 18-3-18 GL-NPs was 1.6-fold higher than MFI for RFP in Lipofectamine Plus treated cells. The black and red peaks represent control negative and test respectively

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Median fluorescence intensity (MFI) of RFP positive PAM212 cells transfected by Lipofectamine Plus or 18-3-18 GL-NPs. MFI for RFP in cells transfected by 18-3-18 GL-NPs was 1.6-fold higher than MFI for RFP in Lipofectamine Plus treated cells. The black and red peaks represent control negative and test respectively

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Fluorescence, Transfection

    Confocal microscopic images of PAM212 cells treated with pDNA complexed to Lipofectamine Plus or GL-NPs, prepared using gemini surfactant series 12, 16, and 18. The expression of the tdTomato RFP is shown in red and nuclei were stained with DRAQ-5 and are shown in blue . Magnification ×20

    Journal: Journal of Nanobiotechnology

    Article Title: A flow cytometric approach to study the mechanism of gene delivery to cells by gemini-lipid nanoparticles: an implication for cell membrane nanoporation

    doi: 10.1186/s12951-015-0125-1

    Figure Lengend Snippet: Confocal microscopic images of PAM212 cells treated with pDNA complexed to Lipofectamine Plus or GL-NPs, prepared using gemini surfactant series 12, 16, and 18. The expression of the tdTomato RFP is shown in red and nuclei were stained with DRAQ-5 and are shown in blue . Magnification ×20

    Article Snippet: The commercially available Lipofectamine Plus (Life Technologies Inc. Rockville, MD, USA) was used as a positive control in each transfection experiment and prepared according to the manufacturer’s instructions.

    Techniques: Expressing, Staining

    Analysis of AAT Expression after Transfection with Liposomes or Lipofectamine 2000 Containing AAT-Encoding mRNA Concentration of AAT protein in cell supernatants after transfection with AAT mRNA encapsulated in liposomes or Lipofectamine 2000 for 24 or 72 hr. Data are shown as mean ± SEM (n = 5). **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Cationic Nanoliposomes Meet mRNA: Efficient Delivery of Modified mRNA Using Hemocompatible and Stable Vectors for Therapeutic Applications

    doi: 10.1016/j.omtn.2017.07.013

    Figure Lengend Snippet: Analysis of AAT Expression after Transfection with Liposomes or Lipofectamine 2000 Containing AAT-Encoding mRNA Concentration of AAT protein in cell supernatants after transfection with AAT mRNA encapsulated in liposomes or Lipofectamine 2000 for 24 or 72 hr. Data are shown as mean ± SEM (n = 5). **p

    Article Snippet: For the Lipofectamine 2000 transfection, 1,000 μL of Opti-MEM I (Invitrogen), 2 μL of Lipofectamine 2000 (Invitrogen), and 1 μg of AAT mRNA were mixed.

    Techniques: Expressing, Transfection, Concentration Assay

    RT-PCR determined the effects of pcDNA3- MTA1 , control-siRNA and  MTA1 -siRNA transfection on  MTA1  expression at the mRNA level. pcDNA3- MTA1  and  MTA1 -siRNA were transfected into the cell line with Lipofectamine 2000. Total RNA was extracted and RT-PCR was performed. pcDNA3- MTA1  transfection increased  MTA1  mRNA levels, while  MTA1 -siRNA decreased  MTA1  mRNA levels.  MTA1 , metastasis-associated gene 1.

    Journal: Oncology Letters

    Article Title: Metastasis-associated gene 1 promotes invasion and migration potential of laryngeal squamous cell carcinoma cells

    doi: 10.3892/ol.2013.1729

    Figure Lengend Snippet: RT-PCR determined the effects of pcDNA3- MTA1 , control-siRNA and MTA1 -siRNA transfection on MTA1 expression at the mRNA level. pcDNA3- MTA1 and MTA1 -siRNA were transfected into the cell line with Lipofectamine 2000. Total RNA was extracted and RT-PCR was performed. pcDNA3- MTA1 transfection increased MTA1 mRNA levels, while MTA1 -siRNA decreased MTA1 mRNA levels. MTA1 , metastasis-associated gene 1.

    Article Snippet: Reagents Lipofectamine 2000 was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). siRNA sequence was chemically synthesized by Jikai Co. (Shanghai, China).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Expressing

    Western blot analysis of  MTA1  expression after transfection with pcDNA3- MTA1  and pSilencer3.1- MTA1 -siRNA. pcDNA3- MTA1  and  MTA1 -siRNA were transfected into the cell line with Lipofectamine 2000. Whole protein was extracted and loaded for SDS-PAGE separation. The protein was transferred to a nitrocellulose membrane and pcDNA3- MTA1  increased  MTA1  expression and  MTA1 -siRNA greatly decreased  MTA1  levels.  MTA1 , metastasis-associated gene 1.

    Journal: Oncology Letters

    Article Title: Metastasis-associated gene 1 promotes invasion and migration potential of laryngeal squamous cell carcinoma cells

    doi: 10.3892/ol.2013.1729

    Figure Lengend Snippet: Western blot analysis of MTA1 expression after transfection with pcDNA3- MTA1 and pSilencer3.1- MTA1 -siRNA. pcDNA3- MTA1 and MTA1 -siRNA were transfected into the cell line with Lipofectamine 2000. Whole protein was extracted and loaded for SDS-PAGE separation. The protein was transferred to a nitrocellulose membrane and pcDNA3- MTA1 increased MTA1 expression and MTA1 -siRNA greatly decreased MTA1 levels. MTA1 , metastasis-associated gene 1.

    Article Snippet: Reagents Lipofectamine 2000 was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). siRNA sequence was chemically synthesized by Jikai Co. (Shanghai, China).

    Techniques: Western Blot, Expressing, Transfection, SDS Page

    Induction of IRF-10, IRF-4, IRF-8, cIRF-3, IRF-1, IRF-2, and c- rel expression by dsRNA (dsR). Fibroblasts cultivated for 2 weeks after explantation from chicken embryos were treated with pI · pC (10 μg/ml) (lanes 1 to 6) by using LipofectAMIN 2000 (LF) as described in Materials and Methods. Parallel cultures were simultaneously treated with cycloheximide (CHX; 10 μg/ml) (lanes 7 to 9). The control cells were treated with cycloheximide (lanes 10 to 12) from Sigma or LipofectAMIN 2000 (lanes 13 to 15) alone. RNA was isolated from these cells at the time points indicated. Total RNA (10 μg per lane) was subjected to Northern analysis and hybridized with probes described in Materials and Methods. The intensity of the rRNA staining with ethidium bromide is shown at the bottom (rRNA).

    Journal: Molecular and Cellular Biology

    Article Title: A Novel Interferon Regulatory Factor (IRF), IRF-10, Has a Unique Role in Immune Defense and Is Induced by the v-Rel Oncoprotein

    doi: 10.1128/MCB.22.11.3942-3957.2002

    Figure Lengend Snippet: Induction of IRF-10, IRF-4, IRF-8, cIRF-3, IRF-1, IRF-2, and c- rel expression by dsRNA (dsR). Fibroblasts cultivated for 2 weeks after explantation from chicken embryos were treated with pI · pC (10 μg/ml) (lanes 1 to 6) by using LipofectAMIN 2000 (LF) as described in Materials and Methods. Parallel cultures were simultaneously treated with cycloheximide (CHX; 10 μg/ml) (lanes 7 to 9). The control cells were treated with cycloheximide (lanes 10 to 12) from Sigma or LipofectAMIN 2000 (lanes 13 to 15) alone. RNA was isolated from these cells at the time points indicated. Total RNA (10 μg per lane) was subjected to Northern analysis and hybridized with probes described in Materials and Methods. The intensity of the rRNA staining with ethidium bromide is shown at the bottom (rRNA).

    Article Snippet: The cells were washed with Opti-MEM I medium (Gibco BRL Life Technologies) and overlaid with 10 μg of pI · pC (Sigma Chemical Co.) per ml and 10 μl of LipofectAMIN 2000 (Gibco BRL Life Technologies) per ml in 8 ml of Opti-MEM I per plate.

    Techniques: Expressing, Isolation, Northern Blot, Staining

    TBP-1 and TBPIP physically associate in mammalian cells. Flag-tagged TBP-1 and Xpress-tagged TBPIP cDNA vectors were cotransfected into CV-1 cells using Lipofectamine 2000. Forty-eight hours after transfection, WCL was prepared as described, and 500 μg

    Journal: Endocrinology

    Article Title: Tat-Binding Protein-1 (TBP-1), an ATPase of 19S Regulatory Particles of the 26S Proteasome, Enhances Androgen Receptor Function in Cooperation with TBP-1-Interacting Protein/Hop2

    doi: 10.1210/en.2008-1122

    Figure Lengend Snippet: TBP-1 and TBPIP physically associate in mammalian cells. Flag-tagged TBP-1 and Xpress-tagged TBPIP cDNA vectors were cotransfected into CV-1 cells using Lipofectamine 2000. Forty-eight hours after transfection, WCL was prepared as described, and 500 μg

    Article Snippet: CV-1 cells split into 60-mm dishes were transfected with pKCR2AR in the absence or presence of pcDNA4HisMaxCTBPIP, pKCR2TBP-1, or pKCR2K233H using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol, and WCL was prepared and subjected to Western blot analysis.

    Techniques: Transfection

    In vitro transfection and expression results of Agm-CBA and Arg-CBA complexed with pDNA at weight ratios (pDNA:polymer) varying from 1:6 to 1:96. Notes: The fluorescence intensity of FITC-labeled secondary antibodies connected to FANCF was observed by fluorescence microscopy (100×, scale bar 100 μm), and transfection efficiency was determined by flow cytometry 48 hours after gene transfection. The percentage marked as black represents the expression of FANCF, and the percentage marked as blue represents the silencing efficiency of FNACF (1 – expression of FANCF, transfection efficiency). Nonfluorescent MCF7 cell images were recorded under natural light as cell-density control. Abbreviations: Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; pDNA, plasmid DNA; FITC, fluorescein isothiocyanate; Lipo, Lipofectamine 2000; PEI, polyethylenimine.

    Journal: International Journal of Nanomedicine

    Article Title: Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery

    doi: 10.2147/IJN.S115773

    Figure Lengend Snippet: In vitro transfection and expression results of Agm-CBA and Arg-CBA complexed with pDNA at weight ratios (pDNA:polymer) varying from 1:6 to 1:96. Notes: The fluorescence intensity of FITC-labeled secondary antibodies connected to FANCF was observed by fluorescence microscopy (100×, scale bar 100 μm), and transfection efficiency was determined by flow cytometry 48 hours after gene transfection. The percentage marked as black represents the expression of FANCF, and the percentage marked as blue represents the silencing efficiency of FNACF (1 – expression of FANCF, transfection efficiency). Nonfluorescent MCF7 cell images were recorded under natural light as cell-density control. Abbreviations: Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; pDNA, plasmid DNA; FITC, fluorescein isothiocyanate; Lipo, Lipofectamine 2000; PEI, polyethylenimine.

    Article Snippet: Lipofectamine 2000 (Lipo) reagent, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, DNase I, trypsin, and N ,N ′-cystamine bisacrylamide (CBA) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: In Vitro, Transfection, Expressing, Crocin Bleaching Assay, Fluorescence, Labeling, Microscopy, Flow Cytometry, Cytometry, Plasmid Preparation

    Intracellular trafficking of different complexes (pDNA/Agm-CBA and pDNA/Arg-CBA) at the weight ratio (pDNA:polymer) of 1:48. Notes: Trafficking at 5-minute, 30-minute, 1-hour, 2-hour, 3-hour, and 4-hour time intervals. Scale bar 100 μm. The fluorescence signals were collected with three channels: pDNA stained with Hoechst 33342, blue; DiO-labeled cell membrane, green; nucleolus stained with the nucleolus tracker, green; nucleus stained with the nuclear tracker, red. Only merged images of three channels are shown. Abbreviations: pDNA, plasmid DNA; Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; FITC, fluorescein isothiocyanate; Lipo, Lipofectamine 2000; PEI, polyethylenimine.

    Journal: International Journal of Nanomedicine

    Article Title: Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery

    doi: 10.2147/IJN.S115773

    Figure Lengend Snippet: Intracellular trafficking of different complexes (pDNA/Agm-CBA and pDNA/Arg-CBA) at the weight ratio (pDNA:polymer) of 1:48. Notes: Trafficking at 5-minute, 30-minute, 1-hour, 2-hour, 3-hour, and 4-hour time intervals. Scale bar 100 μm. The fluorescence signals were collected with three channels: pDNA stained with Hoechst 33342, blue; DiO-labeled cell membrane, green; nucleolus stained with the nucleolus tracker, green; nucleus stained with the nuclear tracker, red. Only merged images of three channels are shown. Abbreviations: pDNA, plasmid DNA; Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; FITC, fluorescein isothiocyanate; Lipo, Lipofectamine 2000; PEI, polyethylenimine.

    Article Snippet: Lipofectamine 2000 (Lipo) reagent, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, DNase I, trypsin, and N ,N ′-cystamine bisacrylamide (CBA) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Crocin Bleaching Assay, Fluorescence, Staining, Labeling, Plasmid Preparation

    Atomic force microscopy images of Gua-SS-PAA complexes and naked pDNA in dry form or in buffer system. Notes: Agm-CBA and Arg-CBA were complexed with pDNA at a weight ratio (pDNA:polymer) of 1:48; each image represents a 2×2 μm scan. Dry, complexes measured in a dry state; Buffer, complexes measured in HEPES buffer solution. Abbreviations: Gua, guanidino; SS, disulfide; PAA, poly(amidoamine); pDNA, plasmid DNA; Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; Lipo, Lipofectamine 2000; PEI, polyethylenimine.

    Journal: International Journal of Nanomedicine

    Article Title: Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery

    doi: 10.2147/IJN.S115773

    Figure Lengend Snippet: Atomic force microscopy images of Gua-SS-PAA complexes and naked pDNA in dry form or in buffer system. Notes: Agm-CBA and Arg-CBA were complexed with pDNA at a weight ratio (pDNA:polymer) of 1:48; each image represents a 2×2 μm scan. Dry, complexes measured in a dry state; Buffer, complexes measured in HEPES buffer solution. Abbreviations: Gua, guanidino; SS, disulfide; PAA, poly(amidoamine); pDNA, plasmid DNA; Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; Lipo, Lipofectamine 2000; PEI, polyethylenimine.

    Article Snippet: Lipofectamine 2000 (Lipo) reagent, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, DNase I, trypsin, and N ,N ′-cystamine bisacrylamide (CBA) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Microscopy, Crocin Bleaching Assay, Plasmid Preparation

    In vitro cytotoxicity of Agm-CBA and Arg-CBA complexed with pDNA at weight ratios (pDNA:polymer) varying from 1:6 to 1:96. Notes: MCF7 cells were incubated with the desired amount of complexes for 24 hours. Results reported as mean ± standard deviation for three individual measurements. Abbreviations: Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; pDNA, plasmid DNA; PEI, polyethylenimine; Lipo, Lipofectamine 2000.

    Journal: International Journal of Nanomedicine

    Article Title: Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery

    doi: 10.2147/IJN.S115773

    Figure Lengend Snippet: In vitro cytotoxicity of Agm-CBA and Arg-CBA complexed with pDNA at weight ratios (pDNA:polymer) varying from 1:6 to 1:96. Notes: MCF7 cells were incubated with the desired amount of complexes for 24 hours. Results reported as mean ± standard deviation for three individual measurements. Abbreviations: Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; pDNA, plasmid DNA; PEI, polyethylenimine; Lipo, Lipofectamine 2000.

    Article Snippet: Lipofectamine 2000 (Lipo) reagent, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, DNase I, trypsin, and N ,N ′-cystamine bisacrylamide (CBA) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: In Vitro, Crocin Bleaching Assay, Incubation, Standard Deviation, Plasmid Preparation

    Encapsulation efficiency of Agm-CBA and Arg-CBA at weight ratios (pDNA:polymer) varying from 1:0.5 to 1:96. Note: Results reported as mean ± standard deviation for three individual measurements. Abbreviations: Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; pDNA, plasmid DNA; Lipo, Lipofectamine 2000; PEI, polyethylenimine.

    Journal: International Journal of Nanomedicine

    Article Title: Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery

    doi: 10.2147/IJN.S115773

    Figure Lengend Snippet: Encapsulation efficiency of Agm-CBA and Arg-CBA at weight ratios (pDNA:polymer) varying from 1:0.5 to 1:96. Note: Results reported as mean ± standard deviation for three individual measurements. Abbreviations: Agm, agmatine; CBA, N , N ′-cystamine bisacrylamide; Arg, arginine; pDNA, plasmid DNA; Lipo, Lipofectamine 2000; PEI, polyethylenimine.

    Article Snippet: Lipofectamine 2000 (Lipo) reagent, Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, DNase I, trypsin, and N ,N ′-cystamine bisacrylamide (CBA) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Crocin Bleaching Assay, Standard Deviation, Plasmid Preparation

    DsRed expression in HFLS transfected with bubble liposomes and ultrasound exposure compared with Lipofectamine 2000. pDNA (pDsRed-Express-N1) and BL were mixed together with culture medium and added to the HFLS. The cells were immediately exposed to US (frequency, 2 MHz; duty, 50%; intensity, 2.5 W/cm 2 ; US exposure time, 10 sec.). The cells were washed and cultured for 2 days and then treated cells were examined by a fluorescence microscope original magnification X200. Transfection of pDNA by LF2000 was also performed. BL: bubble liposomes; US: ultrasound exposure. Fluorescence, (a–d); phase contrast, (e–h).

    Journal: Journal of Drug Delivery

    Article Title: Local Gene Delivery System by Bubble Liposomes and Ultrasound Exposure into Joint Synovium

    doi: 10.1155/2011/203986

    Figure Lengend Snippet: DsRed expression in HFLS transfected with bubble liposomes and ultrasound exposure compared with Lipofectamine 2000. pDNA (pDsRed-Express-N1) and BL were mixed together with culture medium and added to the HFLS. The cells were immediately exposed to US (frequency, 2 MHz; duty, 50%; intensity, 2.5 W/cm 2 ; US exposure time, 10 sec.). The cells were washed and cultured for 2 days and then treated cells were examined by a fluorescence microscope original magnification X200. Transfection of pDNA by LF2000 was also performed. BL: bubble liposomes; US: ultrasound exposure. Fluorescence, (a–d); phase contrast, (e–h).

    Article Snippet: Next, 1.25 μ g Lipofectamine 2000 (LF2000) (Invitrogen Japan K.K., Tokyo, Japan) was diluted in Opti-MEM.

    Techniques: Expressing, Transfection, Size-exclusion Chromatography, Cell Culture, Fluorescence, Microscopy

    Luciferase expression in HFLS transfected with bubble liposomes and ultrasound exposure compared with Lipofectamine 2000. pDNA (pCMV-Luc) and BL were mixed together with culture medium and added to the HFLS. The cells were immediately exposed to US (frequency, 2 MHz; duty, 50%; intensity, 2.5 W/cm 2 ; US exposure time, 10 sec.). The cells were washed and cultured for 2 days, and then luciferase activity was determined as described in Section 2 . The transfection of pDNA by LF2000 was also performed according to the manufacturers' instructions. All data are shown as the mean ± SD ( n = 4). ** P

    Journal: Journal of Drug Delivery

    Article Title: Local Gene Delivery System by Bubble Liposomes and Ultrasound Exposure into Joint Synovium

    doi: 10.1155/2011/203986

    Figure Lengend Snippet: Luciferase expression in HFLS transfected with bubble liposomes and ultrasound exposure compared with Lipofectamine 2000. pDNA (pCMV-Luc) and BL were mixed together with culture medium and added to the HFLS. The cells were immediately exposed to US (frequency, 2 MHz; duty, 50%; intensity, 2.5 W/cm 2 ; US exposure time, 10 sec.). The cells were washed and cultured for 2 days, and then luciferase activity was determined as described in Section 2 . The transfection of pDNA by LF2000 was also performed according to the manufacturers' instructions. All data are shown as the mean ± SD ( n = 4). ** P

    Article Snippet: Next, 1.25 μ g Lipofectamine 2000 (LF2000) (Invitrogen Japan K.K., Tokyo, Japan) was diluted in Opti-MEM.

    Techniques: Luciferase, Expressing, Transfection, Size-exclusion Chromatography, Cell Culture, Activity Assay

    The importance of syntaxin cleavage in botulinum-triggered cell demise. (a) Immunoblot showing that, in the presence of Lipofectamine LTX, the botulinum protease type C cleaves both syntaxin 1 and SNAP25 in N2A cells, whereas the type A protease cleaves SNAP25 only. Type C protease removes the last eight residues of SNAP25 (SNAP25Δ8), whereas type A removes the last nine (SNAP25Δ9). (b) The bar chart showing N2A cell demise following a 40 h treatment with the indicated proteases. Type A-induced cleavage of SNAP25, even in the presence of the type D protease, is insufficient to drive the cytotoxic effects. In contrast, cleavage of syntaxin by type C protease has a strong effect on cell survival (** p

    Journal: Journal of Neurochemistry

    Article Title: Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells

    doi: 10.1111/jnc.12645

    Figure Lengend Snippet: The importance of syntaxin cleavage in botulinum-triggered cell demise. (a) Immunoblot showing that, in the presence of Lipofectamine LTX, the botulinum protease type C cleaves both syntaxin 1 and SNAP25 in N2A cells, whereas the type A protease cleaves SNAP25 only. Type C protease removes the last eight residues of SNAP25 (SNAP25Δ8), whereas type A removes the last nine (SNAP25Δ9). (b) The bar chart showing N2A cell demise following a 40 h treatment with the indicated proteases. Type A-induced cleavage of SNAP25, even in the presence of the type D protease, is insufficient to drive the cytotoxic effects. In contrast, cleavage of syntaxin by type C protease has a strong effect on cell survival (** p

    Article Snippet: Lipofectamine (Invitrogen), Lipofectamine LTX (Invitrogen), Transpass P (New England BioLabs; Hitchin, UK), and Fugene HD (Promega; Southampton, UK) were used as received from manufacturer.

    Techniques:

    Shorted soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) fragments can interact and cause cytotoxicity upon transduction. (a) Western immunoblotting of N2A cells, treated with 0.002 μg of type C protease for up to 24 h, reveals cleaved syntaxin fragment 1-253 (syntaxinΔ35). In this instance immunoblotting was performed using the HPC-1 anti-syntaxin antibody. (b) Schematic of a SNARE pull-down reaction (upper panel). The lower panel showing cleavage of Glutathione S -transferase (GST)-synaptobrevin 2 (GST-Syb2) by type D protease and the ability of this product to bind syntaxin fragment (201-245) and SNAP25 as seen in the Coomassie-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. Note SNAP25 is required for the syntaxin pull-down by GST-Syb2 and GST-Syb2 D product highlighting the ternary nature of the SNARE interaction. (c) GST-SNAP25 pull-down experiment reveals interaction of the indicated shortened synaptobrevin and syntaxin fragments, strictly in a ternary manner. Syntaxin fragment 1-226 incorporating the regulatory syntaxin head with a truncated SNARE motif was revealed by Coomassie staining, whereas short SNARE peptides, carrying FITC, were imaged in the Bio-Rad XRS imaging station. (d) Bar chart showing cell survival measured using CCK-8 assay. Intracellular delivery of syntaxin fragment (201-245) and synaptobrevin (25-52) peptide using Lipofectamine LTX causes a significant reduction in cell viability, whereas transduction of control peptides, complexin (31-59) and penetratin, at the same concentration does not lead to cell death (* p

    Journal: Journal of Neurochemistry

    Article Title: Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells

    doi: 10.1111/jnc.12645

    Figure Lengend Snippet: Shorted soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) fragments can interact and cause cytotoxicity upon transduction. (a) Western immunoblotting of N2A cells, treated with 0.002 μg of type C protease for up to 24 h, reveals cleaved syntaxin fragment 1-253 (syntaxinΔ35). In this instance immunoblotting was performed using the HPC-1 anti-syntaxin antibody. (b) Schematic of a SNARE pull-down reaction (upper panel). The lower panel showing cleavage of Glutathione S -transferase (GST)-synaptobrevin 2 (GST-Syb2) by type D protease and the ability of this product to bind syntaxin fragment (201-245) and SNAP25 as seen in the Coomassie-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. Note SNAP25 is required for the syntaxin pull-down by GST-Syb2 and GST-Syb2 D product highlighting the ternary nature of the SNARE interaction. (c) GST-SNAP25 pull-down experiment reveals interaction of the indicated shortened synaptobrevin and syntaxin fragments, strictly in a ternary manner. Syntaxin fragment 1-226 incorporating the regulatory syntaxin head with a truncated SNARE motif was revealed by Coomassie staining, whereas short SNARE peptides, carrying FITC, were imaged in the Bio-Rad XRS imaging station. (d) Bar chart showing cell survival measured using CCK-8 assay. Intracellular delivery of syntaxin fragment (201-245) and synaptobrevin (25-52) peptide using Lipofectamine LTX causes a significant reduction in cell viability, whereas transduction of control peptides, complexin (31-59) and penetratin, at the same concentration does not lead to cell death (* p

    Article Snippet: Lipofectamine (Invitrogen), Lipofectamine LTX (Invitrogen), Transpass P (New England BioLabs; Hitchin, UK), and Fugene HD (Promega; Southampton, UK) were used as received from manufacturer.

    Techniques: Transduction, Western Blot, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Imaging, CCK-8 Assay, Concentration Assay

    Cytotoxicity of the botulinum C and D proteases. (a) Cell counting assay (CCK-8) was used to monitor neuroblastoma N2A cell viability 40 h after application of the proteases in the presence or absence of Lipofectamine LTX. Botulinum protease type C significantly impaired viability compared with a control condition (* p

    Journal: Journal of Neurochemistry

    Article Title: Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells

    doi: 10.1111/jnc.12645

    Figure Lengend Snippet: Cytotoxicity of the botulinum C and D proteases. (a) Cell counting assay (CCK-8) was used to monitor neuroblastoma N2A cell viability 40 h after application of the proteases in the presence or absence of Lipofectamine LTX. Botulinum protease type C significantly impaired viability compared with a control condition (* p

    Article Snippet: Lipofectamine (Invitrogen), Lipofectamine LTX (Invitrogen), Transpass P (New England BioLabs; Hitchin, UK), and Fugene HD (Promega; Southampton, UK) were used as received from manufacturer.

    Techniques: Cell Counting, CCK-8 Assay

    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

    Transfection of FAK siRNA in osteoblasts. (A) Western blot analysis of FAK and Pyk2 expression and activation (phosphorylation of Y397, Y402) in osteoblasts treated with lipofectamine only (Lipo Only), 50 nM scrambled control siRNA (Scramble), or 50 nM

    Journal: Journal of Bone and Mineral Research

    Article Title: Focal Adhesion Kinase Is Important for Fluid Shear Stress–Induced Mechanotransduction in Osteoblasts

    doi: 10.1359/JBMR.081102

    Figure Lengend Snippet: Transfection of FAK siRNA in osteoblasts. (A) Western blot analysis of FAK and Pyk2 expression and activation (phosphorylation of Y397, Y402) in osteoblasts treated with lipofectamine only (Lipo Only), 50 nM scrambled control siRNA (Scramble), or 50 nM

    Article Snippet: Two control transfections were performed: one with Lipofectamine 2000 alone and the other with a non targeting scramble siRNA control (Dharmacon).

    Techniques: Transfection, Western Blot, Expressing, Activation 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

    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

    pEGFP-C1 encapsulated by different vectors for gene delivery to hBMSCs. The expression of enhanced green fluorescent protein (EGFP) in hBMSCs transfected with complexes such as scAb GD2 -PEG-g-PEI-SPION/pDNA (a), lipofectamine/pDNA (b), and PEG-g-PEI-SPION/pDNA (c) was observed using an inverted fluorescence microscope and was quantified by flow cytometry (d). The transfection efficiency of scAb GD2 -PEG-g-PEI-SPION/pDNA was significantly higher than those of PEG-g-PEI-SPION/pDNA, scAb GD2 -PEG-g-PEI-SPION/pDNA + free Ab GD2 , and scAb IgG2a -PEG-g-PEI-SPION/pDNA at the same N/P ratio. # P

    Journal: PLoS ONE

    Article Title: An MRI-Visible Non-Viral Vector Bearing GD2 Single Chain Antibody for Targeted Gene Delivery to Human Bone Marrow Mesenchymal Stem Cells

    doi: 10.1371/journal.pone.0076612

    Figure Lengend Snippet: pEGFP-C1 encapsulated by different vectors for gene delivery to hBMSCs. The expression of enhanced green fluorescent protein (EGFP) in hBMSCs transfected with complexes such as scAb GD2 -PEG-g-PEI-SPION/pDNA (a), lipofectamine/pDNA (b), and PEG-g-PEI-SPION/pDNA (c) was observed using an inverted fluorescence microscope and was quantified by flow cytometry (d). The transfection efficiency of scAb GD2 -PEG-g-PEI-SPION/pDNA was significantly higher than those of PEG-g-PEI-SPION/pDNA, scAb GD2 -PEG-g-PEI-SPION/pDNA + free Ab GD2 , and scAb IgG2a -PEG-g-PEI-SPION/pDNA at the same N/P ratio. # P

    Article Snippet: Lipofectamine™ 2000 was used as a reference and was purchased from Beyotime Institute of Biotechnology (Guangzhou, China).

    Techniques: Expressing, Transfection, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    Primary human fetal hepatocytes were transfected with EGFP that was cloned into the hTert vector. Following transfection with the hTert vector using lipofectamine as a transfection reagent, human fetal hepatocytes were highly proliferative. However, these

    Journal: Journal of Clinical and Experimental Hepatology

    Article Title: Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

    doi: 10.1016/j.jceh.2014.08.001

    Figure Lengend Snippet: Primary human fetal hepatocytes were transfected with EGFP that was cloned into the hTert vector. Following transfection with the hTert vector using lipofectamine as a transfection reagent, human fetal hepatocytes were highly proliferative. However, these

    Article Snippet: The efficiency of the reagent GeneJammer (Stratagene) was about 40%–50%, but the best result could be obtained with the reagent Lipofectamine (Invitrogen, Karlsruhe).

    Techniques: Transfection, Clone Assay, Plasmid Preparation

    Comparison of the transfection efficiency of A) lipofectamine and B) effectene in primary human hepatocytes to obtain an immortalized cell line. Transfection using lipofectamine as a reagent yielded a higher number of EGFP-expressing cells, when compared

    Journal: Journal of Clinical and Experimental Hepatology

    Article Title: Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

    doi: 10.1016/j.jceh.2014.08.001

    Figure Lengend Snippet: Comparison of the transfection efficiency of A) lipofectamine and B) effectene in primary human hepatocytes to obtain an immortalized cell line. Transfection using lipofectamine as a reagent yielded a higher number of EGFP-expressing cells, when compared

    Article Snippet: The efficiency of the reagent GeneJammer (Stratagene) was about 40%–50%, but the best result could be obtained with the reagent Lipofectamine (Invitrogen, Karlsruhe).

    Techniques: Transfection, Expressing

    Comparison of intracellular uptake of plasmids in different formats. Second from left: I-plasmid labeled with Cy5, third: I-plasmid labeled with Cy5 complexed with lipofectamine, fourth: shRNA labeled with Cy5, fifth: shRNA labeled with Cy5 complexed with lipofectamine and the last: I-gel labeled with Cy5. Scale bar: 20 μm

    Journal: Nature Communications

    Article Title: A RNA producing DNA hydrogel as a platform for a high performance RNA interference system

    doi: 10.1038/s41467-018-06864-0

    Figure Lengend Snippet: Comparison of intracellular uptake of plasmids in different formats. Second from left: I-plasmid labeled with Cy5, third: I-plasmid labeled with Cy5 complexed with lipofectamine, fourth: shRNA labeled with Cy5, fifth: shRNA labeled with Cy5 complexed with lipofectamine and the last: I-gel labeled with Cy5. Scale bar: 20 μm

    Article Snippet: The MDCK expressing GFP (MDCK-GFP) cells were prepared by transfecting pEGFP-N1 vector into the cells with the use of Lipofectamine reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Plasmid Preparation, Labeling, shRNA

    Gene-silencing effect by I-gel i n a cellular level. a Fluorescence images of MDCK-GFP-expressing (MDCK-GFP) cells coincubated with I-plasmid, I-plasmid complexed with lipofectamine, and I-gel. Scale bar: 20 μm. b Fluorescence-activated cell sorter analysis of GFP-expressing cell line, MDCK-GFP cells after treatment of sample/polymerase complexes in serum-deficient medium. The number means the percentage of GFP-overexpressing cells sorted within a prefixed gate region as indicated by a bar. c , d The relative amounts of ( c ) GFP mRNA and ( d ) shRNA from GFP-expressing MDCK cells after incubation in various conditions. ( † concentration of these conditions were 102-fold increased in consideration of the template to RNA transcription rate of the I-gel) ( c : * P

    Journal: Nature Communications

    Article Title: A RNA producing DNA hydrogel as a platform for a high performance RNA interference system

    doi: 10.1038/s41467-018-06864-0

    Figure Lengend Snippet: Gene-silencing effect by I-gel i n a cellular level. a Fluorescence images of MDCK-GFP-expressing (MDCK-GFP) cells coincubated with I-plasmid, I-plasmid complexed with lipofectamine, and I-gel. Scale bar: 20 μm. b Fluorescence-activated cell sorter analysis of GFP-expressing cell line, MDCK-GFP cells after treatment of sample/polymerase complexes in serum-deficient medium. The number means the percentage of GFP-overexpressing cells sorted within a prefixed gate region as indicated by a bar. c , d The relative amounts of ( c ) GFP mRNA and ( d ) shRNA from GFP-expressing MDCK cells after incubation in various conditions. ( † concentration of these conditions were 102-fold increased in consideration of the template to RNA transcription rate of the I-gel) ( c : * P

    Article Snippet: The MDCK expressing GFP (MDCK-GFP) cells were prepared by transfecting pEGFP-N1 vector into the cells with the use of Lipofectamine reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Fluorescence, Expressing, Plasmid Preparation, shRNA, Incubation, Concentration Assay

    DACH1 over-expression inhibits HSF-2-enhanced RANKL expression. A: SAKA-T-cells were transiently co-transfected with hRANKL promoter luciferase reporter plasmid (hRANKL P#3), DACH1, ΔDS expression plasmids, and HSF2-siRNA by lipofectamine method

    Journal:

    Article Title: DACH1 Negatively Regulates the Human RANK Ligand Gene Expression in Stromal/Preosteoblast Cells

    doi: 10.1002/jcb.21561

    Figure Lengend Snippet: DACH1 over-expression inhibits HSF-2-enhanced RANKL expression. A: SAKA-T-cells were transiently co-transfected with hRANKL promoter luciferase reporter plasmid (hRANKL P#3), DACH1, ΔDS expression plasmids, and HSF2-siRNA by lipofectamine method

    Article Snippet: A day after seeding, cells were co-transfected with hRANKL promoter-luciferase reporter plasmid (2 μg) and DACH1, ΔDS in the presence or absence of double-stranded siRNA (10 μM) against NCoR and HSF-2 (Santa Cruz Biotechnology, Inc., CA) by Lipofectamine method (Invitrogen, Carlsbad, CA).

    Techniques: Over Expression, Expressing, Transfection, Luciferase, Plasmid Preparation

    Upregulation of hepatic CDCA5 expression in HCC cell lines and patients with HCC and knockdown of CDCA5 in Huh7 and Hep3B. (A) Expression of CDCA5 mRNA in 4 human HCC cell lines and 2 normal human hepatic cells was evaluated by qRT-PCR. (B) Western blots show the CDCA5 protein level compared to Tubulin in HCC cell lines and normal hepatic cells. (C) Representative images of immunohistochemistry staining of liver sections of HCC tissues (n=81) and paired adjacent normal tissues (n=81). Original magnification, ×200. Upper panel, HCC tissues; Lower panel, adjacent normal tissues. (D and E) Western blots and qRT-PCR showing the effect of RNAi on CDCA5 expression. There different siCDCA5s at 50 nM were transfected into human HCC cell lines Huh7 and Hep3B using Lipofectamine 3000. *p

    Journal: Journal of Cancer

    Article Title: Aberrantly DNA Methylated-Differentially Expressed Genes and Pathways in Hepatocellular Carcinoma

    doi: 10.7150/jca.27832

    Figure Lengend Snippet: Upregulation of hepatic CDCA5 expression in HCC cell lines and patients with HCC and knockdown of CDCA5 in Huh7 and Hep3B. (A) Expression of CDCA5 mRNA in 4 human HCC cell lines and 2 normal human hepatic cells was evaluated by qRT-PCR. (B) Western blots show the CDCA5 protein level compared to Tubulin in HCC cell lines and normal hepatic cells. (C) Representative images of immunohistochemistry staining of liver sections of HCC tissues (n=81) and paired adjacent normal tissues (n=81). Original magnification, ×200. Upper panel, HCC tissues; Lower panel, adjacent normal tissues. (D and E) Western blots and qRT-PCR showing the effect of RNAi on CDCA5 expression. There different siCDCA5s at 50 nM were transfected into human HCC cell lines Huh7 and Hep3B using Lipofectamine 3000. *p

    Article Snippet: Hep3B and Huh7 cells were transfected with small interfering RNA (siRNA) specific for CDCA5 (siCDCA5-1: GCAGGGAGCTTACTAAGGA, siCDCA5-2: CGAGCATCCTCCCTGAAAT, siCDCA5-3: GTGGTGTGCTCCAAACTCA) or corresponding scramble siRNA as negative control (Ribobio, China) using Lipofectamine 3000 (Invitrogen, Shanghai, China) following the manufacturer's instructions.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining, Transfection

    Sca-1+/CD31− CSCs hTERT CM protects HL-1 cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Sca-1+/CD31− CSCs hTERT were cultured in six-well plates at a density of 5 × 10 4 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes or NC siRNA duplexes using Lipofectamine RNAiMAX. After a 48 h transfection, MCP-1 mRNA expression was assessed by real-time PCR ( A ); The average value of MCP-1 mRNA was normalized to that of GAPDH for each sample. The data represent the mean ± SD from three experiments. Sca-1+/CD31− CSCs hTERT CM ( B ) after transfection of NC siRNA or MCP-1 siRNA were subjected to densitometry and are presented as fold changes for MCP-1 (indicated by number 6) and IL-6 (indicated by number 15) ( C ), taking MCP-1 and IL-6 levels in NC siRNA-transfected Sca-1+/CD31− CSCs hTERT as a one-fold value, each in triplicate, * p

    Journal: International Journal of Molecular Sciences

    Article Title: Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

    doi: 10.3390/ijms17060800

    Figure Lengend Snippet: Sca-1+/CD31− CSCs hTERT CM protects HL-1 cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Sca-1+/CD31− CSCs hTERT were cultured in six-well plates at a density of 5 × 10 4 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes or NC siRNA duplexes using Lipofectamine RNAiMAX. After a 48 h transfection, MCP-1 mRNA expression was assessed by real-time PCR ( A ); The average value of MCP-1 mRNA was normalized to that of GAPDH for each sample. The data represent the mean ± SD from three experiments. Sca-1+/CD31− CSCs hTERT CM ( B ) after transfection of NC siRNA or MCP-1 siRNA were subjected to densitometry and are presented as fold changes for MCP-1 (indicated by number 6) and IL-6 (indicated by number 15) ( C ), taking MCP-1 and IL-6 levels in NC siRNA-transfected Sca-1+/CD31− CSCs hTERT as a one-fold value, each in triplicate, * p

    Article Snippet: Apoptosis Assay of HL-1 Cardiomyocytes Sca-1+/CD31− CSCshTERT were cultured on six-well plates at a density of 5 × 104 cells/well and transfected with 50 nM of MCP-1 siRNA duplexes (5′-CACAACCACCTCAAGCACT-3′) or NC siRNA (all from Bioneer) using Lipofectamine RNAiMAX (Invitrogen) for 48 h as suggested by the manufacturer.

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

    Intracellular amount of emodin in K562/ADM and K562 cells. Both cells were incubated with various concentrations of emodin (0.1–10 μM) at 37°C or 4°C for 2 h (A); K562/ADM cells were co-treated with 0, 1, 3, 10 μM of verapamil or 0, 2, 4, 10 μM of cyclosporine A and emodin (5 μM) for 2 h (B). RNAi of P-gp (C). A-: K562/ADM cells transfected with negative control siRNA; AL: K562/ADM cells transfected with Lipofectamine RNAiMAX Reagent; AP: K562/ADM cells transfected with P-gp siRNA; A: K562/ADM cells; S: K562 cells. After transfection with P-gp siRNA, intracellular amount of emodin (5 μM) was enhanced compared with other control groups (D). Data were represented as the mean ± S.D. of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein

    doi: 10.1371/journal.pone.0187971

    Figure Lengend Snippet: Intracellular amount of emodin in K562/ADM and K562 cells. Both cells were incubated with various concentrations of emodin (0.1–10 μM) at 37°C or 4°C for 2 h (A); K562/ADM cells were co-treated with 0, 1, 3, 10 μM of verapamil or 0, 2, 4, 10 μM of cyclosporine A and emodin (5 μM) for 2 h (B). RNAi of P-gp (C). A-: K562/ADM cells transfected with negative control siRNA; AL: K562/ADM cells transfected with Lipofectamine RNAiMAX Reagent; AP: K562/ADM cells transfected with P-gp siRNA; A: K562/ADM cells; S: K562 cells. After transfection with P-gp siRNA, intracellular amount of emodin (5 μM) was enhanced compared with other control groups (D). Data were represented as the mean ± S.D. of three independent experiments. * P

    Article Snippet: The Lipofectamine RNAiMAX Reagent was purchased from Invitrogen Trading Co., Ltd (Shanghai, China).

    Techniques: Incubation, Transfection, Negative Control

    The Sesn2 response to an energetic stress is regulated by Akt. ( a ) LNCaP cells were either treated with lipofectamine (Lipo) or transfected with siCt or sip53 for 48 h. Then, 20 mM 2-DG was added for 15 h (2-DG15) or 24 h

    Journal: Cell Death and Differentiation

    Article Title: Sestrin2 integrates Akt and mTOR signaling to protect cells against energetic stress-induced death

    doi: 10.1038/cdd.2012.157

    Figure Lengend Snippet: The Sesn2 response to an energetic stress is regulated by Akt. ( a ) LNCaP cells were either treated with lipofectamine (Lipo) or transfected with siCt or sip53 for 48 h. Then, 20 mM 2-DG was added for 15 h (2-DG15) or 24 h

    Article Snippet: LNCaP and MEFs cells were transfected with pCMV-Flag-Sesn2 or with pCMV-empty-vector plasmids using Lipofectamine 2000 or Jet PEI (Polyplus, Illkirch, France).

    Techniques: Transfection