lipofectamine 2000 cd transfection reagent  (Thermo Fisher)


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    Name:
    Lipofectamine 2000 CD Transfection Reagent
    Description:
    Lipofectamine 2000 CD Chemically Defined Transfection Reagent is a proprietary animal origin free formulation for transfecting nucleic acids into eukaryotic cells Using Lipofectamine 2000 CD reagent for transfection provides the following advantages • Highest transfection efficiency in many cell types and formats • The animal origin free formulation ensures that mammalian cell culture and bioproduction processes are free of animal derived materials • DNA Lipofectamine 2000 CD complexes can be added directly to cells in culture medium • It is not necessary to remove complexes or change add medium after transfection but complexes may be removed after 4 6 hours
    Catalog Number:
    12566014
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Bioproduction|Cell Culture|Plasmid Transfection|Transient Protein Production & High-Throughput Screening|Antibody & Cell Culture Production|Transfection
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    Structured Review

    Thermo Fisher lipofectamine 2000 cd transfection reagent
    Innate immune signalling by chronic mtDNA stress requires U-ISGF3 but is not associated with NF-κB or interferon gene activation. a-c, qRT-PCR analysis of ( a ) the indicated ISGs, ( b ) NF-κB target genes and (c) IFN genes in WT and Tfam +/− littermate MEFs. d , Western blot of STAT1, p-STAT1 (Y701), TFAM and GAPDH (loading control) in WT and Tfam +/− littermate MEFs transfected with 2μg Poly I:C or <t>lipofectamine</t> only (Mock) for 12 hours. (n=3 independent experiments) e, qRT-PCR analysis of the indicated ISGs in WT, Tfam +/− , Stat1 −/− and Tfam +/− Stat1 −/− littermate MEFs. f, Western blot of STAT1, p-STAT1 (Y701) and histone H3 (nuclear marker and loading control) in purified nuclear extracts from WT and Tfam +/− littermate MEFs. (n=3 independent experiments). g, qRT-PCR analysis of the indicated ISGs in Tfam +/− MEFs transfected with siRNA against Irf9 or Stat2 . All data were analysed with two-tailed unpaired student’s t tests. Asterisks indicate significance as follows: **p
    Lipofectamine 2000 CD Chemically Defined Transfection Reagent is a proprietary animal origin free formulation for transfecting nucleic acids into eukaryotic cells Using Lipofectamine 2000 CD reagent for transfection provides the following advantages • Highest transfection efficiency in many cell types and formats • The animal origin free formulation ensures that mammalian cell culture and bioproduction processes are free of animal derived materials • DNA Lipofectamine 2000 CD complexes can be added directly to cells in culture medium • It is not necessary to remove complexes or change add medium after transfection but complexes may be removed after 4 6 hours
    https://www.bioz.com/result/lipofectamine 2000 cd transfection reagent/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 2000 cd transfection reagent - by Bioz Stars, 2021-06
    99/100 stars

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    1) Product Images from "Mitochondrial DNA Stress Signalling Protects the Nuclear Genome"

    Article Title: Mitochondrial DNA Stress Signalling Protects the Nuclear Genome

    Journal: Nature metabolism

    doi: 10.1038/s42255-019-0150-8

    Innate immune signalling by chronic mtDNA stress requires U-ISGF3 but is not associated with NF-κB or interferon gene activation. a-c, qRT-PCR analysis of ( a ) the indicated ISGs, ( b ) NF-κB target genes and (c) IFN genes in WT and Tfam +/− littermate MEFs. d , Western blot of STAT1, p-STAT1 (Y701), TFAM and GAPDH (loading control) in WT and Tfam +/− littermate MEFs transfected with 2μg Poly I:C or lipofectamine only (Mock) for 12 hours. (n=3 independent experiments) e, qRT-PCR analysis of the indicated ISGs in WT, Tfam +/− , Stat1 −/− and Tfam +/− Stat1 −/− littermate MEFs. f, Western blot of STAT1, p-STAT1 (Y701) and histone H3 (nuclear marker and loading control) in purified nuclear extracts from WT and Tfam +/− littermate MEFs. (n=3 independent experiments). g, qRT-PCR analysis of the indicated ISGs in Tfam +/− MEFs transfected with siRNA against Irf9 or Stat2 . All data were analysed with two-tailed unpaired student’s t tests. Asterisks indicate significance as follows: **p
    Figure Legend Snippet: Innate immune signalling by chronic mtDNA stress requires U-ISGF3 but is not associated with NF-κB or interferon gene activation. a-c, qRT-PCR analysis of ( a ) the indicated ISGs, ( b ) NF-κB target genes and (c) IFN genes in WT and Tfam +/− littermate MEFs. d , Western blot of STAT1, p-STAT1 (Y701), TFAM and GAPDH (loading control) in WT and Tfam +/− littermate MEFs transfected with 2μg Poly I:C or lipofectamine only (Mock) for 12 hours. (n=3 independent experiments) e, qRT-PCR analysis of the indicated ISGs in WT, Tfam +/− , Stat1 −/− and Tfam +/− Stat1 −/− littermate MEFs. f, Western blot of STAT1, p-STAT1 (Y701) and histone H3 (nuclear marker and loading control) in purified nuclear extracts from WT and Tfam +/− littermate MEFs. (n=3 independent experiments). g, qRT-PCR analysis of the indicated ISGs in Tfam +/− MEFs transfected with siRNA against Irf9 or Stat2 . All data were analysed with two-tailed unpaired student’s t tests. Asterisks indicate significance as follows: **p

    Techniques Used: Activation Assay, Quantitative RT-PCR, Western Blot, Transfection, Marker, Purification, Two Tailed Test

    Additional analysis of the STAT1 null (Stat1 −/− ) condition in WT (Tfam +/+ Stat1 +/+ ) and Tfam +/− MEFs. a, Western blot of STAT1, TFAM and GAPDH (loading control) in littermate MEFs of the indicated Tfam and Stat1 genotypes (bottom) that were transfected with 2 μg of Poly(I:C) or lipofectamine only (Mock) for 12 hours. (n=3 independent experiments). MEFs described in a were analysed (all normalized to WT) for b, mtDNA abundance (relative mtDNA copy number) by qPCR with D-loop primers; c, mitochondrial mass using MitoTracker Green (MTG) and flow cytometry (mean fluorescence intensity, MFI, in arbitrary units, A.U., is plotted), and d, mitochondrial membrane potential using MitoTracker Deep Red (MTDR) and flow cytometry (MFI in A.U. is plotted). e, MEFs of the indicated genotypes were analysed by immunofluorescence for mitochondrial and mtDNA nucleoid morphology using antibodies against HSP60 (Mito., magenta) and DNA (DNA, cyan), respectively (n=3 independent experiments). Images are Z-stack projections and scale bar represents 10 μm. f, qRT-PCR analysis of the ISGs Cxcl10 and Ifit1 in MEFs of the indicated genotypes. g, qRT-PCR analysis of the indicated ISGs in Tfam +/− MEFs transfected with control (Ctrl) or Irf3 siRNAs for 72 hours. h, qRT-PCR analysis of the indicated ISGs in Tfam +/− Stat1 −/− MEFs transfected with control (Ctrl) or Irf3 siRNAs for 72 hours. i, Western blot showing siRNA knock-down of IRF9 or STAT2 in Tfam +/− MEFs compared to control (Ctrl) siRNA treated cells. GAPDH was probed as the loading control. (n=3 independent experiments). j, qRT-PCR analysis of the indicated ISGs in Tfam +/− Stat1 −/− MEFs transfected with control (Ctrl) or Irf9 siRNAs for 72 hours. k, qRT-PCR analysis of the indicated ISGs in Tfam +/− Stat1 −/− MEFs transfected with control (Ctrl) or Stat2 siRNAs for 72 hours. c-d, error bars indicate mean ± SD of n=3 biological replicates. For b the data shown are from one of two biological replicates with the error bars indicating the mean ± SD of three technical replicates. For g, h, j and k, . Asterisks indicate significance as follows: ** P
    Figure Legend Snippet: Additional analysis of the STAT1 null (Stat1 −/− ) condition in WT (Tfam +/+ Stat1 +/+ ) and Tfam +/− MEFs. a, Western blot of STAT1, TFAM and GAPDH (loading control) in littermate MEFs of the indicated Tfam and Stat1 genotypes (bottom) that were transfected with 2 μg of Poly(I:C) or lipofectamine only (Mock) for 12 hours. (n=3 independent experiments). MEFs described in a were analysed (all normalized to WT) for b, mtDNA abundance (relative mtDNA copy number) by qPCR with D-loop primers; c, mitochondrial mass using MitoTracker Green (MTG) and flow cytometry (mean fluorescence intensity, MFI, in arbitrary units, A.U., is plotted), and d, mitochondrial membrane potential using MitoTracker Deep Red (MTDR) and flow cytometry (MFI in A.U. is plotted). e, MEFs of the indicated genotypes were analysed by immunofluorescence for mitochondrial and mtDNA nucleoid morphology using antibodies against HSP60 (Mito., magenta) and DNA (DNA, cyan), respectively (n=3 independent experiments). Images are Z-stack projections and scale bar represents 10 μm. f, qRT-PCR analysis of the ISGs Cxcl10 and Ifit1 in MEFs of the indicated genotypes. g, qRT-PCR analysis of the indicated ISGs in Tfam +/− MEFs transfected with control (Ctrl) or Irf3 siRNAs for 72 hours. h, qRT-PCR analysis of the indicated ISGs in Tfam +/− Stat1 −/− MEFs transfected with control (Ctrl) or Irf3 siRNAs for 72 hours. i, Western blot showing siRNA knock-down of IRF9 or STAT2 in Tfam +/− MEFs compared to control (Ctrl) siRNA treated cells. GAPDH was probed as the loading control. (n=3 independent experiments). j, qRT-PCR analysis of the indicated ISGs in Tfam +/− Stat1 −/− MEFs transfected with control (Ctrl) or Irf9 siRNAs for 72 hours. k, qRT-PCR analysis of the indicated ISGs in Tfam +/− Stat1 −/− MEFs transfected with control (Ctrl) or Stat2 siRNAs for 72 hours. c-d, error bars indicate mean ± SD of n=3 biological replicates. For b the data shown are from one of two biological replicates with the error bars indicating the mean ± SD of three technical replicates. For g, h, j and k, . Asterisks indicate significance as follows: ** P

    Techniques Used: Western Blot, Transfection, Real-time Polymerase Chain Reaction, Flow Cytometry, Fluorescence, Immunofluorescence, Quantitative RT-PCR

    Additional analysis of innate immune signalling in MEFs. a, Heat map of normalized expression values of the indicated ISGs (red font), interferon genes (black font), and NF-κB target genes (blue font) from our previously published microarray analysis4 of WT and Tfam+/− MEFs (two of each). b-d, qRT-PCR analyses of the indicated ISGs (b) , NF-κB target genes (c) and interferon genes (d) in WT MEFs transfected with control (Ctrl) or Tfam siRNA for 72 hours. e, Western blot probing the indicated NF-κB pathway proteins, TFAM and VDAC (loading control) in WT and Tfam+/− littermate MEFs. (n=3 independent experiments.) f, qRT-PCR analysis of interferon β (Ifnb) gene expression in WT MEFs transfected with 2 μg poly(I:C) or lipofectamine only (Mock) for 9 hours. g, qRT-PCR analysis of the indicated ISGs in WT MEFs cultured overnight in the presence of control media (plain), media conditioned by WT or Tfam+/− MEFs, or media conditioned by WT MEFs stimulated with poly(I:C) for 9 hours (PIC-stimulated). The data shown are from one of two ( f and g ) or three ( b-d . All data were analysed with two-tailed unpaired student’s t tests. Asterisks indicate significance as follows: ** P
    Figure Legend Snippet: Additional analysis of innate immune signalling in MEFs. a, Heat map of normalized expression values of the indicated ISGs (red font), interferon genes (black font), and NF-κB target genes (blue font) from our previously published microarray analysis4 of WT and Tfam+/− MEFs (two of each). b-d, qRT-PCR analyses of the indicated ISGs (b) , NF-κB target genes (c) and interferon genes (d) in WT MEFs transfected with control (Ctrl) or Tfam siRNA for 72 hours. e, Western blot probing the indicated NF-κB pathway proteins, TFAM and VDAC (loading control) in WT and Tfam+/− littermate MEFs. (n=3 independent experiments.) f, qRT-PCR analysis of interferon β (Ifnb) gene expression in WT MEFs transfected with 2 μg poly(I:C) or lipofectamine only (Mock) for 9 hours. g, qRT-PCR analysis of the indicated ISGs in WT MEFs cultured overnight in the presence of control media (plain), media conditioned by WT or Tfam+/− MEFs, or media conditioned by WT MEFs stimulated with poly(I:C) for 9 hours (PIC-stimulated). The data shown are from one of two ( f and g ) or three ( b-d . All data were analysed with two-tailed unpaired student’s t tests. Asterisks indicate significance as follows: ** P

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR, Transfection, Western Blot, Cell Culture, Two Tailed Test

    Additional data supporting mtDNA stress-mediated ISG induction in MEFs. a, Western blot probing STAT1, p-STAT1 (Y701), γH2A.X (DNA damage marker) and GAPDH (loading control) in WT MEFs treated with the indicated doses of doxorubicin (Dox) for 24 hours. (n=3 independent experiments). b, qRT-PCR analysis of the indicated ISGs in WT ( Stat1 +/+ ) and Stat1 null ( Stat1 −/− ) littermate MEFs challenged with (+Dox) or without 500 nM Dox for 24 hours. c, WT MEFs treated with (Dox) or without (Ctrl) 1μM doxorubicin were analysed by immunofluorescence for mitochondrial and mtDNA nucleoid morphology by immunofluorescence against HSP60 (Mito., magenta) and DNA (DNA, cyan). (n=3 independent experiments). Images are Z-stack projections and scale bar represents 10 μm. d and f , mtDNA abundance (relative mtDNA copy number) by qPCR with D-loop primers in WT MEFs exposed to (d) 100 μM 2’−3’-dideoxycytidine (ddC) or (f) 450 ng/mL ethidium bromide (EtBr) for 10–12 days. e, g, qRT-PCR analysis of the ISGs Ifit1 and Ifit3 in (e) Control (Ctrl) and ddC-treated MEFs (described in d) challenged with (+Dox) or without (-Dox) 500nM for doxorubicin for 16 hours, and (g) Control (Ctrl) and EtBr-treated MEFs (described in f ) transfected with 2 μg dsDNA90 or lipofectamine only (Mock) for 9 hours. h , mtDNA abundance (relative mtDNA copy number) by qPCR with D-loop primers in LMTK- cells with (ρ+) or without (ρ˚) mtDNA. i , qRT-PCR analysis of the ISGs Ifit1 and Ifit3 in LMTK- ρ+ and ρ˚ cells (described in h) challenged with 500 nM Dox for 24 hours. j and k, MEFs were pre-treated with 100 μM chloramphenicol (Chlo) for 24 hours followed by 500 nM Doxorubicin (Dox) for 24 hours. j, Western blot using an OXPHOS complex cocktail (mtDNA-encoded subunit is in red, nucleus-encoded subunits in black), γH2A.X (DNA damage marker) or actin (loading control) (n=3 independent experiments). k, qRT-PCR analysis of the ISGs Ifit1 and Ifit3. For e and g the data shown are from one of two biological replicates with the error bars indicating the mean ± SD of three technical replicates. For b, d, f, h and i, . All data were analysed with two-tailed unpaired student’s t tests. Asterisks indicate significance as follows: ** P
    Figure Legend Snippet: Additional data supporting mtDNA stress-mediated ISG induction in MEFs. a, Western blot probing STAT1, p-STAT1 (Y701), γH2A.X (DNA damage marker) and GAPDH (loading control) in WT MEFs treated with the indicated doses of doxorubicin (Dox) for 24 hours. (n=3 independent experiments). b, qRT-PCR analysis of the indicated ISGs in WT ( Stat1 +/+ ) and Stat1 null ( Stat1 −/− ) littermate MEFs challenged with (+Dox) or without 500 nM Dox for 24 hours. c, WT MEFs treated with (Dox) or without (Ctrl) 1μM doxorubicin were analysed by immunofluorescence for mitochondrial and mtDNA nucleoid morphology by immunofluorescence against HSP60 (Mito., magenta) and DNA (DNA, cyan). (n=3 independent experiments). Images are Z-stack projections and scale bar represents 10 μm. d and f , mtDNA abundance (relative mtDNA copy number) by qPCR with D-loop primers in WT MEFs exposed to (d) 100 μM 2’−3’-dideoxycytidine (ddC) or (f) 450 ng/mL ethidium bromide (EtBr) for 10–12 days. e, g, qRT-PCR analysis of the ISGs Ifit1 and Ifit3 in (e) Control (Ctrl) and ddC-treated MEFs (described in d) challenged with (+Dox) or without (-Dox) 500nM for doxorubicin for 16 hours, and (g) Control (Ctrl) and EtBr-treated MEFs (described in f ) transfected with 2 μg dsDNA90 or lipofectamine only (Mock) for 9 hours. h , mtDNA abundance (relative mtDNA copy number) by qPCR with D-loop primers in LMTK- cells with (ρ+) or without (ρ˚) mtDNA. i , qRT-PCR analysis of the ISGs Ifit1 and Ifit3 in LMTK- ρ+ and ρ˚ cells (described in h) challenged with 500 nM Dox for 24 hours. j and k, MEFs were pre-treated with 100 μM chloramphenicol (Chlo) for 24 hours followed by 500 nM Doxorubicin (Dox) for 24 hours. j, Western blot using an OXPHOS complex cocktail (mtDNA-encoded subunit is in red, nucleus-encoded subunits in black), γH2A.X (DNA damage marker) or actin (loading control) (n=3 independent experiments). k, qRT-PCR analysis of the ISGs Ifit1 and Ifit3. For e and g the data shown are from one of two biological replicates with the error bars indicating the mean ± SD of three technical replicates. For b, d, f, h and i, . All data were analysed with two-tailed unpaired student’s t tests. Asterisks indicate significance as follows: ** P

    Techniques Used: Western Blot, Marker, Quantitative RT-PCR, Immunofluorescence, Real-time Polymerase Chain Reaction, Transfection, Two Tailed Test

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    Article Snippet: Cell culture, transfections and cell lines MCF-7, MDA MB 231, HEK 293, Phoenix, A549, MiaPaca2, HT1080, HCT 116, NIH 3T3 and 293T cell lines were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium (Hyclone) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml of penicillin/streptomycin, in a humidified incubator in an atmosphere of 5% CO2 at 37°C. .. For retrovirus production, Phoenix cells were transfected with 4 μg of the pBabe-puro HRAS V12 and HRAS loop mutants plasmids described below using 12 μl of L-lipofectamine 2000 transfection reagent (Thermofisher) according to the manufacturers' instructions. .. 48 hours after transfection the target cells were infected with Phoenix supernatants and selected with 2 μg/ml of puromycin.

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    Construct:

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    Thermo Fisher lipofectamine 2000
    miR-221 targets PTEN leading to activation of the Akt pathway. A , putative miR-221-binding sites (red) in the 3′-UTRs of PTEN ( top ). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B , SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C , SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using <t>Lipofectamine</t> 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D , similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine 2000/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 2000 - by Bioz Stars, 2021-06
    99/100 stars
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    miR-221 targets PTEN leading to activation of the Akt pathway. A , putative miR-221-binding sites (red) in the 3′-UTRs of PTEN ( top ). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B , SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C , SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D , similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p

    Journal: PLoS ONE

    Article Title: MicroRNA-221 Induces Cell Survival and Cisplatin Resistance through PI3K/Akt Pathway in Human Osteosarcoma

    doi: 10.1371/journal.pone.0053906

    Figure Lengend Snippet: miR-221 targets PTEN leading to activation of the Akt pathway. A , putative miR-221-binding sites (red) in the 3′-UTRs of PTEN ( top ). The middle panel shows sequence alignment of the miR-221 sequences (red color) with a region of the PTEN 3′-UTR (red) from indicated species. The bottom shows a mutant of PTEN 3′-UTR for pMIR-REPORT. B , SOSP-9607 cells were transfected with miR-221 mimic, miR-221 inhibitor, scramble oligonucleotide or nothing (blank). And then indicated protein and mRNA were detected. Rows 1 to 7, the expression of PTEN, phospho-Akt (p-Akt), total-Akt (t-Akt), Cyclin D1 (CCND1), p27 and p57 were analyzed by Western blot; row 8, β-actin was used as a loading control; row 9, PTEN mRNA level was measured by RT-PCR; row 10, GAPDH was used as an control. C , SOSP-9607 cells were co-transfected with miR-221 mimic or scramble oligonucleotide, and pMIR-REPORT vector consisting of a luciferase gene containing the wild type (PTEN-3′-UTR) or mutated (PTEN-mut-3′-UTR) miR-221 binding sites in its 3′-UTR region by using Lipofectamine 2000 reagent. The cells were also transfected with 50 ng pRL-TK vector as an internal standard. 24 h after transfection, luciferase activity was measured by using a dual luciferase reporter assay (Promega). D , similar experiments were performed in MG63 cells as described in panel C except that miR-221 mimic was instead by miR-221 inhibitor. The results were presented as relative luciferase activity (firefly Luc/Renilla Luc). All experiments were repeated three times in triplicate. Columns, mean of three independent experiments; bars, SD. **, p

    Article Snippet: For the luciferase assay in SOSP-9607 cells, cells at the density of 1.2×105 per well in 24-well plates were cotransfected with 0.8 ug pMIR-REPORT luciferase reporters with 3′-UTR or mut-3′-UTR of PTEN, 100 nM miR-221 mimic or scramble oligonucleotide using Lipofectamine 2000 reagent.

    Techniques: Activation Assay, Binding Assay, Sequencing, Mutagenesis, Transfection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay

    DNA-Lipoplex coprecipitation resulted in the highest transfection efficiency compared to all other groups (SNK, n=4, p

    Journal: Biomaterials

    Article Title: Gene Delivery via DNA Incorporation Within a Biomimetic Apatite Coating

    doi: 10.1016/j.biomaterials.2009.09.001

    Figure Lengend Snippet: DNA-Lipoplex coprecipitation resulted in the highest transfection efficiency compared to all other groups (SNK, n=4, p

    Article Snippet: DNA-Lipofectamine 2000® complexes (DNA-Lipoplex) were prepared in polypropylene centrifuge tubes according to the protocol provided by the manufacturer (Molecular Probes, Invitrogen).

    Techniques: Transfection

    High magnification SEM images of the bone-like mineral surface following different DNA incorporation methods: a) Mineralized control b) Plasmid DNA coprecipitation c) DNA-Lipoplex adsorption and d) DNA-Lipoplex coprecipitation. DNA-Lipoplex incorporation via coprecipitation leads to the thickening (white arrows) of the plate-like mineral structures. The “fibrous” coating (black arrows in inset) is most likely due to presence of the cationic lipid because the coprecipitation of DNA alone resulted in minimal changes in apatite morphology.

    Journal: Biomaterials

    Article Title: Gene Delivery via DNA Incorporation Within a Biomimetic Apatite Coating

    doi: 10.1016/j.biomaterials.2009.09.001

    Figure Lengend Snippet: High magnification SEM images of the bone-like mineral surface following different DNA incorporation methods: a) Mineralized control b) Plasmid DNA coprecipitation c) DNA-Lipoplex adsorption and d) DNA-Lipoplex coprecipitation. DNA-Lipoplex incorporation via coprecipitation leads to the thickening (white arrows) of the plate-like mineral structures. The “fibrous” coating (black arrows in inset) is most likely due to presence of the cationic lipid because the coprecipitation of DNA alone resulted in minimal changes in apatite morphology.

    Article Snippet: DNA-Lipofectamine 2000® complexes (DNA-Lipoplex) were prepared in polypropylene centrifuge tubes according to the protocol provided by the manufacturer (Molecular Probes, Invitrogen).

    Techniques: Plasmid Preparation, Adsorption

    Fluorescence images of DNA and lipid agent components from representative samples from each of the following groups: a–b) Mineralized controls, c–d) Plasmid DNA incorporated into PLGA, e–f) Plasmid DNA coprecipitated with mineral, g–h) Plasmid DNA-Lipoplex adsorbed to mineralized films, and i–j) Plasmid DNA-Lipoplex coprecipitated with mineral. Distribution of both the plasmid DNA and the lipid transfection agent on the bone-like mineral was demonstrated by the colocalization of the fluorescent staining (after thorough rinsing) in the adsorption and coprecipitation groups and the absence of staining in the mineralized controls. Scale bars represent 100 μm.

    Journal: Biomaterials

    Article Title: Gene Delivery via DNA Incorporation Within a Biomimetic Apatite Coating

    doi: 10.1016/j.biomaterials.2009.09.001

    Figure Lengend Snippet: Fluorescence images of DNA and lipid agent components from representative samples from each of the following groups: a–b) Mineralized controls, c–d) Plasmid DNA incorporated into PLGA, e–f) Plasmid DNA coprecipitated with mineral, g–h) Plasmid DNA-Lipoplex adsorbed to mineralized films, and i–j) Plasmid DNA-Lipoplex coprecipitated with mineral. Distribution of both the plasmid DNA and the lipid transfection agent on the bone-like mineral was demonstrated by the colocalization of the fluorescent staining (after thorough rinsing) in the adsorption and coprecipitation groups and the absence of staining in the mineralized controls. Scale bars represent 100 μm.

    Article Snippet: DNA-Lipofectamine 2000® complexes (DNA-Lipoplex) were prepared in polypropylene centrifuge tubes according to the protocol provided by the manufacturer (Molecular Probes, Invitrogen).

    Techniques: Fluorescence, Plasmid Preparation, Transfection, Staining, Adsorption

    Plasmid DNA stability in water, as indicated by a) gel electrophoresis and b) quantification of relative percentages of different forms of DNA. The total intensity of all bands of interest (supercoiled and nicked circular DNA) is equivalent to 100% at each incubation time. Stability of DNA decreases with increasing incubation time as demonstrated by the increasing intensity of the top band (between 23130 and 9416, nicked circular form of DNA) and decreasing intensity of the bottom band, ca. 6557 (supercoiled form of the plasmid DNA). c) DNA-Lipoplex stability in Opti-Mem. Complexation of the DNA and lipid was almost complete due to the absence of bands in these groups. Due to size exclusion, the complexes were not able to leave the wells, resulting in the brightness (white arrow) surrounding the wells. d) Intensity profiles for 2 lanes of the gel: DNA 0 h and DNA-Lipo 48 h demonstrate that the peaks that appear for these wells represent the edges of the wells. Peak intensities for the DNA-Lipoplexes are higher compared to DNA only. Profile intensities for the entire lanes are shown in the inset image.

    Journal: Biomaterials

    Article Title: Gene Delivery via DNA Incorporation Within a Biomimetic Apatite Coating

    doi: 10.1016/j.biomaterials.2009.09.001

    Figure Lengend Snippet: Plasmid DNA stability in water, as indicated by a) gel electrophoresis and b) quantification of relative percentages of different forms of DNA. The total intensity of all bands of interest (supercoiled and nicked circular DNA) is equivalent to 100% at each incubation time. Stability of DNA decreases with increasing incubation time as demonstrated by the increasing intensity of the top band (between 23130 and 9416, nicked circular form of DNA) and decreasing intensity of the bottom band, ca. 6557 (supercoiled form of the plasmid DNA). c) DNA-Lipoplex stability in Opti-Mem. Complexation of the DNA and lipid was almost complete due to the absence of bands in these groups. Due to size exclusion, the complexes were not able to leave the wells, resulting in the brightness (white arrow) surrounding the wells. d) Intensity profiles for 2 lanes of the gel: DNA 0 h and DNA-Lipo 48 h demonstrate that the peaks that appear for these wells represent the edges of the wells. Peak intensities for the DNA-Lipoplexes are higher compared to DNA only. Profile intensities for the entire lanes are shown in the inset image.

    Article Snippet: DNA-Lipofectamine 2000® complexes (DNA-Lipoplex) were prepared in polypropylene centrifuge tubes according to the protocol provided by the manufacturer (Molecular Probes, Invitrogen).

    Techniques: Plasmid Preparation, Nucleic Acid Electrophoresis, Incubation

    Low magnification SEM images of the bone-like mineral surface following different DNA incorporation methods: a) Mineralized control b) Plasmid DNA coprecipitation c) DNA-Lipoplex adsorption and d) DNA-Lipoplex coprecipitation. Neither coprecipitation group demonstrated a difference in the size of the mineral nucleation sites as compared to the mineralized controls.

    Journal: Biomaterials

    Article Title: Gene Delivery via DNA Incorporation Within a Biomimetic Apatite Coating

    doi: 10.1016/j.biomaterials.2009.09.001

    Figure Lengend Snippet: Low magnification SEM images of the bone-like mineral surface following different DNA incorporation methods: a) Mineralized control b) Plasmid DNA coprecipitation c) DNA-Lipoplex adsorption and d) DNA-Lipoplex coprecipitation. Neither coprecipitation group demonstrated a difference in the size of the mineral nucleation sites as compared to the mineralized controls.

    Article Snippet: DNA-Lipofectamine 2000® complexes (DNA-Lipoplex) were prepared in polypropylene centrifuge tubes according to the protocol provided by the manufacturer (Molecular Probes, Invitrogen).

    Techniques: Plasmid Preparation, Adsorption

    The S100A10 plasminogen receptor plays a major role in oncogenic RAS-dependent plasminogen activation ( A ) 293T cells stably expressing empty vector (pBabe control) or oncogenic HRAS G12V (HRASG12V) were transfected with 4 μM of pre-designed siRNA (Ambion) specific for S100A10 (S100A10 siRNA) and a non-silencing siRNA control (siRNA control) using Lipofectamine 2000 transfection reagent as per manufacturer's instructions (Invitrogen). Cell lysates were prepared 48 hours after transfection, and total levels of S100A10 and RAS were examined by Western blotting. ( B ) 48 hours after transfection, 25,000 cells were plated in 96 well plates and incubated overnight. Cells were washed 3 times with incubation buffer (Hanks balanced salt solution containing 3 mM CaCl 2 and 1 mM MgCl 2 ) and incubated with 0.5 μM glu-plasminogen for 20–30 minutes before the addition of 500 μM plasmin substrate S2251. The rate of plasmin generation was measured from the A405 nm vs min 2 progress curves ( N = 4). ( C ) 48 hours after transfection, 100,000 cells were plated on the upper chamber of matrigel coated inserts (BD Biosciences) and incubated in serum free medium containing 0.5 μM plasminogen for 48 hours. The lower chamber contained medium with 10% fetal bovine serum (FBS) as a chemoattractant. Invading cells were fixed with methanol and stained with Hematoxylin and Eosin. Cells were quantified by manual counting using a light microscope (20X). Data are expressed as mean number of cells invading per 20× field (5 fields/experimental condition) in duplicates. The plot is representative of three independent experiments. Statistical analysis was performed by two-way ANOVA with Tukey test of significance.

    Journal: Oncotarget

    Article Title: Cell surface protease activation during RAS transformation: Critical role of the plasminogen receptor, S100A10

    doi: 10.18632/oncotarget.10279

    Figure Lengend Snippet: The S100A10 plasminogen receptor plays a major role in oncogenic RAS-dependent plasminogen activation ( A ) 293T cells stably expressing empty vector (pBabe control) or oncogenic HRAS G12V (HRASG12V) were transfected with 4 μM of pre-designed siRNA (Ambion) specific for S100A10 (S100A10 siRNA) and a non-silencing siRNA control (siRNA control) using Lipofectamine 2000 transfection reagent as per manufacturer's instructions (Invitrogen). Cell lysates were prepared 48 hours after transfection, and total levels of S100A10 and RAS were examined by Western blotting. ( B ) 48 hours after transfection, 25,000 cells were plated in 96 well plates and incubated overnight. Cells were washed 3 times with incubation buffer (Hanks balanced salt solution containing 3 mM CaCl 2 and 1 mM MgCl 2 ) and incubated with 0.5 μM glu-plasminogen for 20–30 minutes before the addition of 500 μM plasmin substrate S2251. The rate of plasmin generation was measured from the A405 nm vs min 2 progress curves ( N = 4). ( C ) 48 hours after transfection, 100,000 cells were plated on the upper chamber of matrigel coated inserts (BD Biosciences) and incubated in serum free medium containing 0.5 μM plasminogen for 48 hours. The lower chamber contained medium with 10% fetal bovine serum (FBS) as a chemoattractant. Invading cells were fixed with methanol and stained with Hematoxylin and Eosin. Cells were quantified by manual counting using a light microscope (20X). Data are expressed as mean number of cells invading per 20× field (5 fields/experimental condition) in duplicates. The plot is representative of three independent experiments. Statistical analysis was performed by two-way ANOVA with Tukey test of significance.

    Article Snippet: For retrovirus production, Phoenix cells were transfected with 4 μg of the pBabe-puro HRAS V12 and HRAS loop mutants plasmids described below using 12 μl of L-lipofectamine 2000 transfection reagent (Thermofisher) according to the manufacturers' instructions.

    Techniques: Activation Assay, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Western Blot, Incubation, Staining, Light Microscopy

    A: The normal MCF-7 cells (non-transformed cells), B: transformed MCF-7 cells by pBudCE4.1-azurin-MAM-A recombinant vector using Lipofectamine 2000 reagent, C: treated cells by Lipofectamine compound only, and D: treated MCF-7 s by zeocin antibiotic.

    Journal: Saudi Journal of Biological Sciences

    Article Title: The functions of azurin of Pseudomonas aeruginosa and human mammaglobin-A on proapoptotic and cell cycle regulatory genes expression in the MCF-7 breast cancer cell line

    doi: 10.1016/j.sjbs.2020.04.007

    Figure Lengend Snippet: A: The normal MCF-7 cells (non-transformed cells), B: transformed MCF-7 cells by pBudCE4.1-azurin-MAM-A recombinant vector using Lipofectamine 2000 reagent, C: treated cells by Lipofectamine compound only, and D: treated MCF-7 s by zeocin antibiotic.

    Article Snippet: For this purpose, 7.5 μL of Lipofectamine 2000 reagent was added in 150 μL of RPMI1640 medium without serum and antibiotics and also 2.5 μg/μL of DNA plasmid was prepared in 150 μL of serum and antibiotic-free media.

    Techniques: Transformation Assay, Recombinant, Plasmid Preparation