lipofectamine Search Results


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  • 99
    Thermo Fisher lipofectamine lf 2000 reagent
    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 <t>Lipofectamine</t> 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
    Lipofectamine Lf 2000 Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine lf 2000 reagent/product/Thermo Fisher
    Average 99 stars, based on 539 article reviews
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    85
    Thermo Fisher lipofectamine lf 2000
    Colony survival assay in A549 cells following transfection with <t>Lipofectamine</t> 2000 ( A ) or PEI-PCL-PEG (PPP) micelleplexes ( B ). Untreated (open circle), negative control (filled triangles) or ERCC1-XPF (filled squares) siRNA transfected cells were treated with increasing doses of cisplatin for 2 h, and cell viability was determined by a clonogenic assay. Results are represented as mean ± SD. IC 50 values were calculated using Compusyn software.
    Lipofectamine Lf 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 27 article reviews
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    99
    Thermo Fisher lipofectamine ltx
    Cellular reprogramming with single lipofection and monolayer cardiac differentiation of iPSCs. (A): Outline of reprogramming of skin fibroblasts into iPSCs using lipofection with <t>Lipofectamine</t> <t>LTX</t> in 24-well plate scale. (B): The number of iPSC-like colonies
    Lipofectamine Ltx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 30165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare lipofectamine
    Cellular reprogramming with single lipofection and monolayer cardiac differentiation of iPSCs. (A): Outline of reprogramming of skin fibroblasts into iPSCs using lipofection with <t>Lipofectamine</t> <t>LTX</t> in 24-well plate scale. (B): The number of iPSC-like colonies
    Lipofectamine, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore lipofectamine 2000
    The HIV-1 inhibitory effect on A(H1N1)pdm09 replication is dependent on the IFITM3 expression. HeLa cells were transfected with 0.4(Scr) using <t>lipofectamine</t> 2000 and, after 6 h, cells were infected by A(H1N1)pdm09 (MOI 5) for 1 h. After that, cells were exposed to HIV-1 (10 ng/mL p24 Ag) for 24 h after influenza infection. ( A ) Monolayers were lysed with Laemmli buffer and submitted to blotting assay for the proteins IFITM3 and β-actin, as an housekeeping. Top panel: cells not exposed to HIV-1, and bottom panel: cells exposed to HIV-1. ( B ) The A(H1N1)pdm09 levels in culture supernatants were quantified by qRT-PCR (y-axis) and the immunoblotting bands were analyzed by densitometry using the ImageJ software 1.6.0 and n-fold changes of AU is scored as black squares (z-axis). Panel A is representative of three independent experiments. * P
    Lipofectamine 2000, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1351 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    NEN Life Science lipofectamine
    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, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim lipofectamine
    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, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine/product/Boehringer Mannheim
    Average 92 stars, based on 32 article reviews
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    92
    Promega lipofectamine
    p300 enhances p73-induced apoptosis. (A and B) The N-terminal deletion of p73 lacks apoptosis-promoting potential. As indicated, plasmids encoding no protein as a control (C; 3 μg), p73α (1.0 μg), ΔNp73α (1.0 μg), and/or p300 (2.0 μg), together with the pGFP plasmid (200 ng), were introduced into H1299 cells (10 5 /60-mm-diameter dish) using <t>LipofectAmine</t> (GIBCO-BRL); 32 h posttransfection, cells (100) expressing GFP were counted under a fluorescence microscope using a blind approach, with morphologically round cells (arrows) identified as apoptotic (A). (B) Statistical data (with standard error indicated by a bar above each column) from three independent assays. (C and D) Caspase 3 activation by wild-type but not N-terminus-deleted p73α is enhanced by p300. Transient transfection as described for panel A was conducted. Cells were harvested for caspase 3 activity analysis as described in Materials and Methods. (C) Representative result of the fluorescence graph of AMC released from Ac-DEVD-AMC substrates for one set of experiments with plasmids encoding no protein (curve B), ΔNp73α (curve C), ΔNp73α plus p300 (curve D), p73α (curve E), or p73α plus p300 (curve F). Curve A is a water control. Caspase 3 activity is presented in arbitrary units (A.U.) after normalization by a factor of 10 5 . (D) Statistical values for caspase 3 activity in triplicate assays. (E) DNA fragmentation analysis of p73-induced apoptosis in H1299 cells was conducted as described in Materials and Methods; 10 μg of DNA was analyzed by electrophoresis on 2% agarose. Each lane represents the result for three 60-mm-diameter plates of cells. MW, molecular weight markers.
    Lipofectamine, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine/product/Promega
    Average 92 stars, based on 358 article reviews
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    lipofectamine - by Bioz Stars, 2020-07
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    92
    Avantor 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 Avantor, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    InvivoGen lipofectamine 2000
    The MCMV M35 protein inhibits signaling of multiple pattern recognition receptors. (A) NIH3T3 fibroblasts were co-transfected with a reporter plasmid containing firefly luciferase under the control of the murine IFNβ promoter (IFNβ-Luc) together with a Renilla luciferase normalization control (pRL-TK) and influenza NS1 or MCMV ORFs known or predicted to code for tegument or immediate-early proteins or their corresponding empty vector (pcDNA and pDEST40, respectively). At 24 hours post transfection, cells were treated with medium or stimulated by infection with Newcastle disease virus (NDV). 21 hours p.i. cells were lysed for analysis of luciferase activity. Luciferase fold induction was calculated based on firefly luciferase values normalized to Renilla luciferase from stimulated samples divided by corresponding values from unstimulated samples. Data set is combined from one to four independent experiments and represented as mean ± SD. (B) NIH3T3 fibroblasts were co-transfected with the IFNβ-Luc and pRL-TK luciferase plasmids described in (A) as well as V5-tagged M35 or pcDNA and luciferase assay was performed following stimulation as for (A). Data is combined from three independent experiments and shown as mean ± SD. (C) NIH3T3 fibroblasts were co-transfected as described in (B) and cells stimulated with 10 μg/ml of poly(I:C) in the presence of <t>Lipofectamine</t> 2000 or Lipofectamine 2000 alone for 6 hours before lysis for analysis by luciferase assay. Data is combined from three independent experiments and shown as mean ± SD. (D) 293T cells were co-transfected with expression plasmids for either cGAS (stimulated) or GFP (unstimulated) together with mCherry-STING, the IFNβ-Luc and pRL-TK luciferase plasmids, and either pcDNA, myc-tagged KSHV ORF36 or V5-tagged M35. At 20 hours post transfection, cells were lysed and luciferase production was analyzed. Data is combined from four independent experiments and shown as mean ± SD. (E) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of 3 μg/ml cGAMP or left unstimulated. RNA was extracted at indicated time points and IFNβ induction was measured by quantitative RT-PCR and expressed as IFNβ induction normalized to the housekeeping gene Rpl8. Data is shown as mean ± SD and combined from two independent experiments. (F) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of indicated amounts of cGAMP and supernatant was collected 16 hours later and analyzed by IFNβ ELISA. Data is shown as mean ± SD and representative of two independent experiments. (G) Immortalized BMDM stably expressing myc-tagged M35 or the corresponding empty vector (pMSCV) were stimulated by addition of 10 ng/ml LPS or 1 μM CpG-B 1826 and supernatant collected at 16 hours post stimulation for analysis by TNFα ELISA. Data is combined from three independent experiments and shown as mean ± SD.***p
    Lipofectamine 2000, supplied by InvivoGen, used in various techniques. Bioz Stars score: 92/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    lipofectamine 2000 - by Bioz Stars, 2020-07
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    92
    Roche lipofectamine 2000
    Silencing of a virus reporter protein in tick cells by siRNA treatment. a REE/CTVM28 cells were incubated with siRNA against RLuc in the presence or absence of <t>Lipofectamine</t> 2000 (Lipo) and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y- axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Virus + siRNA + Lipo) and siRNA alone followed by SFV (Virus + siRNA only). The values shown are means of three replicate cultures and the error bars are standard deviations of the mean. * Significant decrease from the virus only control. b Nine tick cell lines were incubated with siRNA targeting RLuc in the presence of a transfection reagent and then infected 24 h later with SFV expressing RLuc . Luciferase levels ( y -axis) were then measured 48 h later in cultures with SFV alone (Virus only), siRNA with Lipofectamine 2000 followed by SFV (Lipo + siRNA + virus) and siRNA with Xtreme followed by SFV (Xtreme + siRNA + virus). The values shown are means of four replicate cultures and the error bars are standard deviations of the mean. The scale used for each y -axis reflects the range of luciferase levels generated in the particular tick cell line. * Significant decrease from the virus only control
    Lipofectamine 2000, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 486 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    SignaGen lipofectamine
    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, supplied by SignaGen, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene lipofectamine
    Antiestrogenic effects of chalcone and flavone require intact JNK signaling. Stable expressing MCF-7/Vec, MCF-7/DN-JNK1, and MCF-7/DN-JNK2 cells were transfected with an ERE-luciferase reporter construct using <t>Lipofectamine</t> for 6 hours followed by treatment
    Lipofectamine, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine/product/Stratagene
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    lipofectamine - by Bioz Stars, 2020-07
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    92
    TaKaRa lipofectamine
    Antiestrogenic effects of chalcone and flavone require intact JNK signaling. Stable expressing MCF-7/Vec, MCF-7/DN-JNK1, and MCF-7/DN-JNK2 cells were transfected with an ERE-luciferase reporter construct using <t>Lipofectamine</t> for 6 hours followed by treatment
    Lipofectamine, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine/product/TaKaRa
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    92
    Welgene inc lipofectamine
    Antiestrogenic effects of chalcone and flavone require intact JNK signaling. Stable expressing MCF-7/Vec, MCF-7/DN-JNK1, and MCF-7/DN-JNK2 cells were transfected with an ERE-luciferase reporter construct using <t>Lipofectamine</t> for 6 hours followed by treatment
    Lipofectamine, supplied by Welgene inc, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine/product/Welgene inc
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    lipofectamine - by Bioz Stars, 2020-07
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    99
    Thermo Fisher lipofectamine plus
    Effect of Aktl AO on Aktl mRNA expression in various human cancer cells. The cells were transfected without (−) or with 0.3 µM of Akt1 AO (+) using <t>Lipofectamine</t> Plus for 3 h and incubated for 6 h with fresh media containing 10% FBS. Total RNA was isolated and Akt1 mRNA expression was determined using RT-PCR. β-Actin was determined as a loading control. Representative data are shown from two independent cell culture experiments and each RT-PCR reaction was performed in duplicate.
    Lipofectamine Plus, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15416 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

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

    Journal: Molecules

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

    doi: 10.3390/molecules25081994

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

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

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

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

    Journal: Molecules

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

    doi: 10.3390/molecules25081994

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

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

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

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

    Journal: Molecules

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

    doi: 10.3390/molecules25081994

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

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

    Techniques: Western Blot, Transfection, Small Interfering RNA

    Cellular reprogramming with single lipofection and monolayer cardiac differentiation of iPSCs. (A): Outline of reprogramming of skin fibroblasts into iPSCs using lipofection with Lipofectamine LTX in 24-well plate scale. (B): The number of iPSC-like colonies

    Journal: Stem Cells Translational Medicine

    Article Title: Dual Optical Recordings for Action Potentials and Calcium Handling in Induced Pluripotent Stem Cell Models of Cardiac Arrhythmias Using Genetically Encoded Fluorescent Indicators

    doi: 10.5966/sctm.2014-0245

    Figure Lengend Snippet: Cellular reprogramming with single lipofection and monolayer cardiac differentiation of iPSCs. (A): Outline of reprogramming of skin fibroblasts into iPSCs using lipofection with Lipofectamine LTX in 24-well plate scale. (B): The number of iPSC-like colonies

    Article Snippet: Procedures using Xfect (Clontech, TaKaRa Bio, Shiga, Japan, ), Lipofectamine 2000, and Lipofectamine LTX (both from Life Technologies) were followed according to the manufacturer’s instructions. pGW1-YFP [ ] and pCXLE-enhanced green fluorescent protein (EGFP) (catalog no. 27082; Addgene, Cambridge, MA, ) were used to examine transfection efficiency.

    Techniques:

    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

    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

    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

    Antiestrogenic effects of chalcone and flavone require intact JNK signaling. Stable expressing MCF-7/Vec, MCF-7/DN-JNK1, and MCF-7/DN-JNK2 cells were transfected with an ERE-luciferase reporter construct using Lipofectamine for 6 hours followed by treatment

    Journal: The Journal of steroid biochemistry and molecular biology

    Article Title: Antiestrogenic Activity of Flavonoid Phytochemicals Mediated via the c-Jun N-terminal Protein Kinase Pathway

    doi: 10.1016/j.jsbmb.2012.05.004

    Figure Lengend Snippet: Antiestrogenic effects of chalcone and flavone require intact JNK signaling. Stable expressing MCF-7/Vec, MCF-7/DN-JNK1, and MCF-7/DN-JNK2 cells were transfected with an ERE-luciferase reporter construct using Lipofectamine for 6 hours followed by treatment

    Article Snippet: For experiments in stable Vec, DN-JNK1 or DN-JNK2 expressing MCF-7 cells were transfected with either pERE2-luciferase using Lipofectamine as above or with an AP-1(7×)-luciferase plasmid (Stratagene, La Jolla, CA) using FuGENE6 (Roche, Indianapolis, IN) according to manufacturer’s instructions.

    Techniques: Expressing, Transfection, Luciferase, Construct

    Effect of Aktl AO on Aktl mRNA expression in various human cancer cells. The cells were transfected without (−) or with 0.3 µM of Akt1 AO (+) using Lipofectamine Plus for 3 h and incubated for 6 h with fresh media containing 10% FBS. Total RNA was isolated and Akt1 mRNA expression was determined using RT-PCR. β-Actin was determined as a loading control. Representative data are shown from two independent cell culture experiments and each RT-PCR reaction was performed in duplicate.

    Journal: Journal of cellular biochemistry

    Article Title: Antitumor Activity of a Novel Antisense Oligonucleotide Against Akt1

    doi: 10.1002/jcb.22311

    Figure Lengend Snippet: Effect of Aktl AO on Aktl mRNA expression in various human cancer cells. The cells were transfected without (−) or with 0.3 µM of Akt1 AO (+) using Lipofectamine Plus for 3 h and incubated for 6 h with fresh media containing 10% FBS. Total RNA was isolated and Akt1 mRNA expression was determined using RT-PCR. β-Actin was determined as a loading control. Representative data are shown from two independent cell culture experiments and each RT-PCR reaction was performed in duplicate.

    Article Snippet: All media and reagents including Lipofectamine Plus and Lipofectamine 2000 used for transfection and M-MLV enzyme kit were obtained from Invitrogen (Carlsbad, CA).

    Techniques: Expressing, Transfection, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Cell Culture

    Effect of Akt1 AO on AKT1 protein expression in various human cancer cells. The cells were transfected without (−) or with 0.3 µM of Akt1 AO (+) using Lipofectamine Plus for 3 h and incubated for 24 h with fresh media containing 10% FBS. Cellular protein was isolated and AKT1 protein expression was determined by Western blot analysis. β-Actin was determined as a loading control. Representative data are shown from two independent cell culture experiments.

    Journal: Journal of cellular biochemistry

    Article Title: Antitumor Activity of a Novel Antisense Oligonucleotide Against Akt1

    doi: 10.1002/jcb.22311

    Figure Lengend Snippet: Effect of Akt1 AO on AKT1 protein expression in various human cancer cells. The cells were transfected without (−) or with 0.3 µM of Akt1 AO (+) using Lipofectamine Plus for 3 h and incubated for 24 h with fresh media containing 10% FBS. Cellular protein was isolated and AKT1 protein expression was determined by Western blot analysis. β-Actin was determined as a loading control. Representative data are shown from two independent cell culture experiments.

    Article Snippet: All media and reagents including Lipofectamine Plus and Lipofectamine 2000 used for transfection and M-MLV enzyme kit were obtained from Invitrogen (Carlsbad, CA).

    Techniques: Expressing, Transfection, Incubation, Isolation, Western Blot, Cell Culture

    Combination effect of Akt1 AO with different cytotoxic drugs on the growth of Caki-1 cancer cells. The drugs were used at the following doses: a,b, 2 and 3 nM paclitaxel; c,d, 5 and 10 nM doxorubicin; e,f, 2 and 5 µM fluorouracil; g,h, 0.5 and 1 nM docetaxel, i,j 0.5 and 1.0 µM cisplatin. Data are expressed as percentage growth inhibition in reference to the growth of untreated control cells. Lipofectamine alone (no Akt1 AO) and 0.25% DMSO in media were used as a control for drugs except cisplatin. Lipofectamine alone and water were used as a control for cisplatin. The open portion of the bars represents the percentage growth inhibition values for Akt1 AO. The striped or squared portion of the bars represents the percentage growth inhibition values for the cytotoxic drugs as indicated in the respective legends. The height of the bars on the left represents the sum of the individual agents’ effects and the expected percentage growth inhibition if drugs are additive when used in combination. The total height of the solid bar indicates the actual observed growth inhibition when drugs were used in combination. The data represent means and standard errors of triplicate determination of at least two experiments.

    Journal: Journal of cellular biochemistry

    Article Title: Antitumor Activity of a Novel Antisense Oligonucleotide Against Akt1

    doi: 10.1002/jcb.22311

    Figure Lengend Snippet: Combination effect of Akt1 AO with different cytotoxic drugs on the growth of Caki-1 cancer cells. The drugs were used at the following doses: a,b, 2 and 3 nM paclitaxel; c,d, 5 and 10 nM doxorubicin; e,f, 2 and 5 µM fluorouracil; g,h, 0.5 and 1 nM docetaxel, i,j 0.5 and 1.0 µM cisplatin. Data are expressed as percentage growth inhibition in reference to the growth of untreated control cells. Lipofectamine alone (no Akt1 AO) and 0.25% DMSO in media were used as a control for drugs except cisplatin. Lipofectamine alone and water were used as a control for cisplatin. The open portion of the bars represents the percentage growth inhibition values for Akt1 AO. The striped or squared portion of the bars represents the percentage growth inhibition values for the cytotoxic drugs as indicated in the respective legends. The height of the bars on the left represents the sum of the individual agents’ effects and the expected percentage growth inhibition if drugs are additive when used in combination. The total height of the solid bar indicates the actual observed growth inhibition when drugs were used in combination. The data represent means and standard errors of triplicate determination of at least two experiments.

    Article Snippet: All media and reagents including Lipofectamine Plus and Lipofectamine 2000 used for transfection and M-MLV enzyme kit were obtained from Invitrogen (Carlsbad, CA).

    Techniques: Inhibition

    Knockdown of BiP and caspase-9, caspase-6, and caspase-3 inhibits apoptosis induced by AM2 in human gastric cancer SNU-1 cells. a Cells were plated in 6-well plates and allowed to reach 50 % confluence on the next day. A siRNA against BiP was transfected into SNU-1 cells using lipofectamine. A scrambled siRNA was used as a control. After 30 h of transfection, the cells were exposed to the indicated concentrations of AM2 for 24 h. b Cells were seeded in 60-mm plates and allowed to reach 50 % confluence on the next day. The siRNAs against caspase-3, caspase-6, and caspase-9 were transfected into SNU-1 cells using G-fectin. A scrambled siRNA was used as a control. After 30 h of transfection, the cells were exposed to 50 μg/ml of AM2 for 24 h. Apoptotic cells were quantified with a TdT tagging assay. The results are shown as the mean ± SD of the data from three independent experiments (* P

    Journal: Archives of Pharmacal Research

    Article Title: Ultrafine particles of Ulmus davidiana var. japonica induce apoptosis of gastric cancer cells via activation of caspase and endoplasmic reticulum stress

    doi: 10.1007/s12272-013-0312-2

    Figure Lengend Snippet: Knockdown of BiP and caspase-9, caspase-6, and caspase-3 inhibits apoptosis induced by AM2 in human gastric cancer SNU-1 cells. a Cells were plated in 6-well plates and allowed to reach 50 % confluence on the next day. A siRNA against BiP was transfected into SNU-1 cells using lipofectamine. A scrambled siRNA was used as a control. After 30 h of transfection, the cells were exposed to the indicated concentrations of AM2 for 24 h. b Cells were seeded in 60-mm plates and allowed to reach 50 % confluence on the next day. The siRNAs against caspase-3, caspase-6, and caspase-9 were transfected into SNU-1 cells using G-fectin. A scrambled siRNA was used as a control. After 30 h of transfection, the cells were exposed to 50 μg/ml of AM2 for 24 h. Apoptotic cells were quantified with a TdT tagging assay. The results are shown as the mean ± SD of the data from three independent experiments (* P

    Article Snippet: Cultured cells were transfected with the siRNAs using G-fectin (Genolution., Seoul, Korea) or Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines.

    Techniques: Transfection