lipofectamine 2000 transfection reagents Search Results


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  • 99
    Thermo Fisher lipofectamine 2000
    Effect of TRPC1 silencing on Ca 2+  influx and cell migration in stable WT-RhoA transfected cells.  A : representative immunoblots of TRPC1 and RhoA. Cells were transfected with control siRNA (C-siRNA) or siTRPC1 by LipofectAMINE 2000, and whole cell lysates
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 485598 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 485598 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 2000 - by Bioz Stars, 2020-09
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    99
    Thermo Fisher lipofectamine rnaimax
    Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using <t>lipofectamine</t> <t>RNAiMAX</t> (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.
    Lipofectamine Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 56803 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 56803 article reviews
    Price from $9.99 to $1999.99
    lipofectamine rnaimax - by Bioz Stars, 2020-09
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    99
    Thermo Fisher lipofectamine 3000
    Spred2 overexpression reduced cytokine responses in macrophages. Spred2 was overexpressed by transfecting macrophages with mixtures of <t>Lipofectamine</t> 3000 and Spred2 overexpression plasmid and control plasmid for 24 h, after which the cells were stimulated with PA (500 μM) for 24 h. Cytokine levels in the culture supernatants were measured by ELISA. ** p
    Lipofectamine 3000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 43082 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine 3000/product/Thermo Fisher
    Average 99 stars, based on 43082 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 3000 - by Bioz Stars, 2020-09
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    99
    Thermo Fisher lipofectamine reagent
    Interactions of ATF-2 with JunD in vitro as measured by glutathionine S -transferase (GST)-ATF-2 pull-down assays. A : human ATF-2 amino acid sequence. The basic region and leucine-zipper area were indicated by green and red colors, respectively. B : GST-ATF-2 fusion proteins: schematic diagram depicting various GST-ATF-2 constructs( a ); and GST-ATF-2 fusion proteins as measured by Coomassie blue staining assays( b ). Constructs were transformed into Eschericia coli BL21, and their expression was induced by treatment with isopropyl-b-d-thiogalactopyranoside (IPTG) at the concentration of 0.5 mM. Expressed GST (without ATF-2) or GST-ATF-2 fusion proteins were harvested and purified by equilibrated MagneGST particles. These fusion proteins were monitored by SDS-PAGE analysis and shown by Coomassie blue staining. C : ATF-2 association with JunD in cells overexpressing JunD. Cells were transfected by using the expression vector containing human junD cDNA by <t>LipofectAMINE</t> technique; whole cell lysates were harvested 48 h after the transfection. The magnetic particles bound to GST or GST-ATF-2 fusion proteins were incubated with cell lysate for 30 min, dissolved in 1× SDS loading buffer, and then subjected to SDS-PAGE. Levels of JunD in the complexes pull-down by using GST or GST-ATF-2 fusion proteins were measured by Western blot analysis with the antibody against JunD ( top ), whereas input GST or GST-ATF-2 fusion proteins were examined by using anti-GST antibody ( bottom ). Three experiments were performed that showed similar results. D : levels of JunD protein in the complexes pull-down by GST-ATF-2 fusion proteins GST-505 ( a ) and GST-176 ( b ) from control cells and cells treated with DFMO alone or DFMO plus Put for 6 days.
    Lipofectamine Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 22388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine reagent/product/Thermo Fisher
    Average 99 stars, based on 22388 article reviews
    Price from $9.99 to $1999.99
    lipofectamine reagent - by Bioz Stars, 2020-09
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    Image Search Results


    Effect of TRPC1 silencing on Ca 2+  influx and cell migration in stable WT-RhoA transfected cells.  A : representative immunoblots of TRPC1 and RhoA. Cells were transfected with control siRNA (C-siRNA) or siTRPC1 by LipofectAMINE 2000, and whole cell lysates

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding

    doi: 10.1152/ajpgi.00185.2015

    Figure Lengend Snippet: Effect of TRPC1 silencing on Ca 2+ influx and cell migration in stable WT-RhoA transfected cells. A : representative immunoblots of TRPC1 and RhoA. Cells were transfected with control siRNA (C-siRNA) or siTRPC1 by LipofectAMINE 2000, and whole cell lysates

    Article Snippet: Tissue culture media, LipofectAMINE 2000, and dialyzed fetal bovine serum (dFBS) were obtained from Invitrogen (Carlsbad, CA), and biochemicals were obtained from Sigma (St. Louis, MO).

    Techniques: Migration, Transfection, Western Blot

    CBX2 is a negative regulator of neuronal differentiation. (A) M17 cells were co-transfected with each construct as indicated in the image and the MCS-EF1-copGFP-T2A-puro vector (System Biosciences) by using Lipofectamine 2000 reagent. Puromycin was added to the culture medium 24 h later at a concentration of 1 μg/ml to eliminate non-transfected cells. Immunoblotting analysis was performed 72 h later. Immunoblotting for CBX2 expression showed that shCBX2-1 and shCBX2-2 reduced the amount of endogenous CBX2 in M17 cells. (B) The morphology of the transfected M17 cells was monitored on the 3rd day after differentiation by observing the GFP fluorescence. (C) Knocked-down of CBX2 increased the neurite length in differentiated M17 cells. Scale bar: 100 μm. The data are presented as mean ± SD, n = 30 cells, ∗∗∗ P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: CBX2 Inhibits Neurite Development by Regulating Neuron-Specific Genes Expression

    doi: 10.3389/fnmol.2018.00046

    Figure Lengend Snippet: CBX2 is a negative regulator of neuronal differentiation. (A) M17 cells were co-transfected with each construct as indicated in the image and the MCS-EF1-copGFP-T2A-puro vector (System Biosciences) by using Lipofectamine 2000 reagent. Puromycin was added to the culture medium 24 h later at a concentration of 1 μg/ml to eliminate non-transfected cells. Immunoblotting analysis was performed 72 h later. Immunoblotting for CBX2 expression showed that shCBX2-1 and shCBX2-2 reduced the amount of endogenous CBX2 in M17 cells. (B) The morphology of the transfected M17 cells was monitored on the 3rd day after differentiation by observing the GFP fluorescence. (C) Knocked-down of CBX2 increased the neurite length in differentiated M17 cells. Scale bar: 100 μm. The data are presented as mean ± SD, n = 30 cells, ∗∗∗ P

    Article Snippet: Twenty-four hours after plating, the neurons were transfected with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Transfection, Construct, Plasmid Preparation, Concentration Assay, Expressing, Fluorescence

    Tetracycline inducibility of BAC E11-IGR-β-catenin-ERα. As a gene of interest, the coding sequence of β-catenin-ERα fusion protein is engineered into BAC. (A) The activity of firefly luciferase, a surrogate marker is assayed to reflect the activity of P tet bi. HTB56 cells cultured in 6-well plates (300,000 per well) were cotransfected with 1 µg supercoiled BAC E11-IGR-β-catenin-ERα and 0.1 µg of renilla luciferase-encoding pRLSV40 DNA by lipofectamine reagent. Cells were left untreated or exposed to indicated concentrations of Tet or Dox 24 hours after transfection and luciferase activity was measured in cell extracts 24 hours later. Data from 3 independent experiments are represented as means plus standard deviation (SD). The SD values are too small to be visible in the first five bars. The relative luciferase activity of Tet and Dox untreated cells were set as 1 arbitrarily (* P

    Journal: PLoS ONE

    Article Title: Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

    doi: 10.1371/journal.pone.0006445

    Figure Lengend Snippet: Tetracycline inducibility of BAC E11-IGR-β-catenin-ERα. As a gene of interest, the coding sequence of β-catenin-ERα fusion protein is engineered into BAC. (A) The activity of firefly luciferase, a surrogate marker is assayed to reflect the activity of P tet bi. HTB56 cells cultured in 6-well plates (300,000 per well) were cotransfected with 1 µg supercoiled BAC E11-IGR-β-catenin-ERα and 0.1 µg of renilla luciferase-encoding pRLSV40 DNA by lipofectamine reagent. Cells were left untreated or exposed to indicated concentrations of Tet or Dox 24 hours after transfection and luciferase activity was measured in cell extracts 24 hours later. Data from 3 independent experiments are represented as means plus standard deviation (SD). The SD values are too small to be visible in the first five bars. The relative luciferase activity of Tet and Dox untreated cells were set as 1 arbitrarily (* P

    Article Snippet: Transfection was performed with Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA, USA) by using 1 µg of super-coiled BAC DNA per well.

    Techniques: BAC Assay, Sequencing, Activity Assay, Luciferase, Marker, Cell Culture, Transfection, Standard Deviation

    The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.

    Journal: PLoS ONE

    Article Title: Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

    doi: 10.1371/journal.pone.0006445

    Figure Lengend Snippet: The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.

    Article Snippet: Transfection was performed with Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA, USA) by using 1 µg of super-coiled BAC DNA per well.

    Techniques: Selection, Marker, FACS, Transfection, BAC Assay, Electroporation, Incubation, Fluorescence, Flow Cytometry, Cytometry

    Axin1 expression in the intestinal epithelial cells changes inflammatory responses. Transient transfections were performed with Lipofectamine2000 (LF2000). (A) IL-8 mRNA in Axin1-overexpressing HCT116 cells after Salmonella colonization. (B) IL-8 protein secretion in cell culture media in Axin1-overexpressing HCT116 cells after Salmonella colonization. (C) IL-8 mRNA in cells with Axin1 blockage by siRNA of Axin1 overexpression and IL-8 protein. (D) Salmonella -induced IL-8 protein levels in cells with Axin1 blockage by using siRNA against Axin1. (E) Salmonella -induced IL-8 protein levels in cells with Axin1 mutations. Axin1 ΔDIX lost the effect to inhibit IL-8 secretion. Data are expressed as mean ± SD. * P

    Journal: PLoS ONE

    Article Title: Axin1 Prevents Salmonella Invasiveness and Inflammatory Response in Intestinal Epithelial Cells

    doi: 10.1371/journal.pone.0034942

    Figure Lengend Snippet: Axin1 expression in the intestinal epithelial cells changes inflammatory responses. Transient transfections were performed with Lipofectamine2000 (LF2000). (A) IL-8 mRNA in Axin1-overexpressing HCT116 cells after Salmonella colonization. (B) IL-8 protein secretion in cell culture media in Axin1-overexpressing HCT116 cells after Salmonella colonization. (C) IL-8 mRNA in cells with Axin1 blockage by siRNA of Axin1 overexpression and IL-8 protein. (D) Salmonella -induced IL-8 protein levels in cells with Axin1 blockage by using siRNA against Axin1. (E) Salmonella -induced IL-8 protein levels in cells with Axin1 mutations. Axin1 ΔDIX lost the effect to inhibit IL-8 secretion. Data are expressed as mean ± SD. * P

    Article Snippet: Transient transfections were performed with Lipofectamine2000 (Invitrogen, San Diego, CA) in accordance with the manufacturer's instructions.

    Techniques: Expressing, Transfection, Cell Culture, Over Expression

    The effects of Tg737 over expression on cell adhesion, invasion, and migration in hypoxia-treated HCC cells.  HepG2 and MHCC97-H cells were treated as detailed in the legend to Figure   4 . ( A ) An adhesion assay was used to evaluate the effects of Tg737 on adhesion. The values of the cells incubated with medium supplemented with 10% FBS under normoxia were set at 1. ( B, C ) The stained membrane after cell invasion demonstrated that Tg737 over expression in HepG2 and MHCC97-H cells led to significantly attenuated cell invasion under hypoxic conditions compared to cells without plasmid transfection under hypoxic conditions. The data are presented as the number of invading cells for each group. ( D, E ) The effects of Tg737 over expression on the migration capacity of hypoxia-treated HCC cells were investigated using a transwell migration assay. The data are presented as the number of migrated cells for each group. I: cells without plasmid transfection; II: cells transfected with pcDNA3.1 (−); III: cells incubated with LipofectamineTM 2000; IV: cells transfected with pcDNA3.1-Tg737.  * ,  P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Tg737 signaling is required for hypoxia-enhanced invasion and migration of hepatoma cells

    doi: 10.1186/1756-9966-31-75

    Figure Lengend Snippet: The effects of Tg737 over expression on cell adhesion, invasion, and migration in hypoxia-treated HCC cells. HepG2 and MHCC97-H cells were treated as detailed in the legend to Figure  4 . ( A ) An adhesion assay was used to evaluate the effects of Tg737 on adhesion. The values of the cells incubated with medium supplemented with 10% FBS under normoxia were set at 1. ( B, C ) The stained membrane after cell invasion demonstrated that Tg737 over expression in HepG2 and MHCC97-H cells led to significantly attenuated cell invasion under hypoxic conditions compared to cells without plasmid transfection under hypoxic conditions. The data are presented as the number of invading cells for each group. ( D, E ) The effects of Tg737 over expression on the migration capacity of hypoxia-treated HCC cells were investigated using a transwell migration assay. The data are presented as the number of migrated cells for each group. I: cells without plasmid transfection; II: cells transfected with pcDNA3.1 (−); III: cells incubated with LipofectamineTM 2000; IV: cells transfected with pcDNA3.1-Tg737. * , P

    Article Snippet: Transient transfection and cell adhesion, invasion and migration assays The pcDNA3.1-Tg737 plasmid was transiently transfected into HepG2 and MHCC97-H cells using LipofectamineTM 2000 (Invitrogen).

    Techniques: Over Expression, Migration, Cell Adhesion Assay, Incubation, Staining, Plasmid Preparation, Transfection, Transwell Migration Assay

    ( A) The cells were harvested with ice-cold PBS and lysed, and polycystin-1 levels were determined using western blot analysis.  The expression levels of polycystin-1 in HepG2 and MHCC97-H cells were decreased in response to hypoxia. ( B ) The cells were subjected to ELISA for analysis of the secretion of polycystin-1, IL-8 and TGF-β1. I: cells incubated with medium supplemented with 10% FBS under normoxia; II: cells incubated with medium supplemented with 1% FBS under normoxia; III: cells incubated with medium supplemented with 1% FBS under hypoxia. The values of the cells incubated with medium supplemented with 10% FBS under normoxia were set at 100%. ( C ) Western blot assays showed increased polycystin-1 protein expression levels in hypoxia-cultured HepG2 and MHCC97-H cells transfected with pcDNA3.1-Tg737. ( D ) ELISA revealed increased polycystin-1 secretion and decreased IL-8 secretion and decreased active and total TGF-β1 levels in hypoxia-cultured HepG2 and MHCC97-H cells transfected with pcDNA3.1-Tg737. The values of cells without plasmid transfection were set at 100%. I: cells without plasmid transfection; II: cells transfected with pcDNA3.1 (−); III: cells incubated with LipofectamineTM 2000; IV: cells transfected with pcDNA3.1-Tg737.  * ,  P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Tg737 signaling is required for hypoxia-enhanced invasion and migration of hepatoma cells

    doi: 10.1186/1756-9966-31-75

    Figure Lengend Snippet: ( A) The cells were harvested with ice-cold PBS and lysed, and polycystin-1 levels were determined using western blot analysis. The expression levels of polycystin-1 in HepG2 and MHCC97-H cells were decreased in response to hypoxia. ( B ) The cells were subjected to ELISA for analysis of the secretion of polycystin-1, IL-8 and TGF-β1. I: cells incubated with medium supplemented with 10% FBS under normoxia; II: cells incubated with medium supplemented with 1% FBS under normoxia; III: cells incubated with medium supplemented with 1% FBS under hypoxia. The values of the cells incubated with medium supplemented with 10% FBS under normoxia were set at 100%. ( C ) Western blot assays showed increased polycystin-1 protein expression levels in hypoxia-cultured HepG2 and MHCC97-H cells transfected with pcDNA3.1-Tg737. ( D ) ELISA revealed increased polycystin-1 secretion and decreased IL-8 secretion and decreased active and total TGF-β1 levels in hypoxia-cultured HepG2 and MHCC97-H cells transfected with pcDNA3.1-Tg737. The values of cells without plasmid transfection were set at 100%. I: cells without plasmid transfection; II: cells transfected with pcDNA3.1 (−); III: cells incubated with LipofectamineTM 2000; IV: cells transfected with pcDNA3.1-Tg737. * , P

    Article Snippet: Transient transfection and cell adhesion, invasion and migration assays The pcDNA3.1-Tg737 plasmid was transiently transfected into HepG2 and MHCC97-H cells using LipofectamineTM 2000 (Invitrogen).

    Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture, Transfection, Plasmid Preparation

    Western blot assay was performed to determine the expression levels of Tg737 in the different cells. The HepG2 and MHCC97-H cells were transiently transfected with the pcDNA3.1-Tg737 plasmid. To exclude liposome/vector-related effects, HepG2 and MHCC97-H cells transfected with pcDNA3.1 (−) or incubated with LipofectamineTM 2000 alone were used as controls. HepG2 and MHCC97-H cells without plasmid transfection also served as blank controls. The cells were incubated with fresh DMEM (1% FBS) for 12 h under hypoxia, then lysed and subjected to immunoblot analysis.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Tg737 signaling is required for hypoxia-enhanced invasion and migration of hepatoma cells

    doi: 10.1186/1756-9966-31-75

    Figure Lengend Snippet: Western blot assay was performed to determine the expression levels of Tg737 in the different cells. The HepG2 and MHCC97-H cells were transiently transfected with the pcDNA3.1-Tg737 plasmid. To exclude liposome/vector-related effects, HepG2 and MHCC97-H cells transfected with pcDNA3.1 (−) or incubated with LipofectamineTM 2000 alone were used as controls. HepG2 and MHCC97-H cells without plasmid transfection also served as blank controls. The cells were incubated with fresh DMEM (1% FBS) for 12 h under hypoxia, then lysed and subjected to immunoblot analysis.

    Article Snippet: Transient transfection and cell adhesion, invasion and migration assays The pcDNA3.1-Tg737 plasmid was transiently transfected into HepG2 and MHCC97-H cells using LipofectamineTM 2000 (Invitrogen).

    Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Incubation

    (A, B) HepG2 and MHCC97-H cells were treated as detailed in the legend to Figure 4 . Annexin V assays revealed that the cell viability of HepG2 and MHCC97-H cells transfected with pcDNA3.1-Tg737 and further incubated with fresh DMEM (1% FBS) for 12 h under hypoxia were not significantly different from cells without plasmid transfection. The data from HepG2 and MHCC97-H cells transfected with pcDNA3.1 (−) or incubated with LipofectamineTM 2000 excluded any liposome/pEGFP-C1-related effects on cell viability.I: cells without plasmid transfection; II: cells transfected with pcDNA3.1 (−); III: cells incubated with LipofectamineTM 2000; IV: cells transfected with pcDNA3.1-Tg737.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Tg737 signaling is required for hypoxia-enhanced invasion and migration of hepatoma cells

    doi: 10.1186/1756-9966-31-75

    Figure Lengend Snippet: (A, B) HepG2 and MHCC97-H cells were treated as detailed in the legend to Figure 4 . Annexin V assays revealed that the cell viability of HepG2 and MHCC97-H cells transfected with pcDNA3.1-Tg737 and further incubated with fresh DMEM (1% FBS) for 12 h under hypoxia were not significantly different from cells without plasmid transfection. The data from HepG2 and MHCC97-H cells transfected with pcDNA3.1 (−) or incubated with LipofectamineTM 2000 excluded any liposome/pEGFP-C1-related effects on cell viability.I: cells without plasmid transfection; II: cells transfected with pcDNA3.1 (−); III: cells incubated with LipofectamineTM 2000; IV: cells transfected with pcDNA3.1-Tg737.

    Article Snippet: Transient transfection and cell adhesion, invasion and migration assays The pcDNA3.1-Tg737 plasmid was transiently transfected into HepG2 and MHCC97-H cells using LipofectamineTM 2000 (Invitrogen).

    Techniques: Transfection, Incubation, Plasmid Preparation

    Endogenous (constitutive) activation of NF-κB in C4-2B cells. a : Measurement of constitutive NF-κB activation in C4-2B and LNCaP cells by luciferase assay following Lipofectamine 2000 transient (24 hr) transfection of cells with either

    Journal: The Prostate

    Article Title: PI3K/Akt-Dependent Transcriptional Regulation and Activation of BMP-2-Smad Signaling by NF-?B in Metastatic Prostate Cancer Cells

    doi: 10.1002/pros.20870

    Figure Lengend Snippet: Endogenous (constitutive) activation of NF-κB in C4-2B cells. a : Measurement of constitutive NF-κB activation in C4-2B and LNCaP cells by luciferase assay following Lipofectamine 2000 transient (24 hr) transfection of cells with either

    Article Snippet: The transfection reagent Lipofectamine 2000 was obtained from Invitrogen.

    Techniques: Activation Assay, Luciferase, Transfection

    In vitro expression studies showed that the p.L339P substitution affects A antigen expression. (A) The median fluorescence index (MFI) of HeLa cells after transfection with wild-type GTA, the p.L339P mutant, or a control construct. (B) The relative percentage of antigen-expressing cells (AEC%) of HeLa cells after transfection with wild-type GTA, the p.L339P mutant, or a control construct. (C–E), The transfected HeLa cells were incubated with anti-A antibody and cell agglutination was observed using a phase contrast microscope. The aggregation of cells transfected with the mutant p.L339P construct (C), a control construct (D), or wild-type GTA construct (E) is shown.

    Journal: Blood Transfusion

    Article Title: Molecular genetic analysis of weak ABO subgroups in the Chinese population reveals ten novel ABO subgroup alleles

    doi: 10.2450/2018.0091-18

    Figure Lengend Snippet: In vitro expression studies showed that the p.L339P substitution affects A antigen expression. (A) The median fluorescence index (MFI) of HeLa cells after transfection with wild-type GTA, the p.L339P mutant, or a control construct. (B) The relative percentage of antigen-expressing cells (AEC%) of HeLa cells after transfection with wild-type GTA, the p.L339P mutant, or a control construct. (C–E), The transfected HeLa cells were incubated with anti-A antibody and cell agglutination was observed using a phase contrast microscope. The aggregation of cells transfected with the mutant p.L339P construct (C), a control construct (D), or wild-type GTA construct (E) is shown.

    Article Snippet: HeLa cells (5×105 ) maintained in Dulbecco’s modified Eagle’s medium containing 10% foetal calf serum were transiently transfected with 1 μg of expression vector using transfection reagent (Lipofectamine 2000, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions.

    Techniques: In Vitro, Expressing, Fluorescence, Transfection, Mutagenesis, Construct, Incubation, Agglutination, Microscopy

    Transfection efficiency of neuronal cultures is increased when preceded with Hyaluronidase treatment. Neuronal cultures were transfected with Lipofectamine2000™ and two different amounts of DNA (0.8 and 1.6 µg DNA), resulting in similar transfection efficiencies, with both transfections strongly enhanced by hyaluronidase treatment. Pretreating cultures 1 h before transfection was more effective than treatment 24 h before transfection. (N = 4 per group) ***p

    Journal: PLoS ONE

    Article Title: Tackling Obstacles for Gene Therapy Targeting Neurons: Disrupting Perineural Nets with Hyaluronidase Improves Transduction

    doi: 10.1371/journal.pone.0053269

    Figure Lengend Snippet: Transfection efficiency of neuronal cultures is increased when preceded with Hyaluronidase treatment. Neuronal cultures were transfected with Lipofectamine2000™ and two different amounts of DNA (0.8 and 1.6 µg DNA), resulting in similar transfection efficiencies, with both transfections strongly enhanced by hyaluronidase treatment. Pretreating cultures 1 h before transfection was more effective than treatment 24 h before transfection. (N = 4 per group) ***p

    Article Snippet: Two different amounts of DNA were used, with the higher amount being 1.6 µg of pCDH1-MCS1-EF1-copGFP DNA and 4 µl Lipofectamine2000™ reagent in 100 µl OptiMEM® (Invitrogen) each, or the lower amount with 0.8 µg of pCDH1-MCS1-EF1-copGFP DNA and 2 µl Lipofectamine2000™ reagent in 50 µl OptiMEM® each.

    Techniques: Transfection

    CryJ-LAMP DNA construct and expression in vitro . (a) Plasmid construction of LAMP Vaccine. CryJ1-LAMP and CryJ2-LAMP plasmids were generated by inserting synthetic CryJ1 or CryJ2 gene into the N LAMP-C LAMP gene to create N LAMP-CryJ1-C LAMP or N LAMP-CryJ2-C LAMP. (b) 293T cells were transfected with CryJ1-LAMP or CryJ2-LAMP. Transfection reagents Opti-MEM and Lipofectamine 2000 solution only were used as a transfection control. Cell lysates were examined for expression of CryJ1-LAMP or CryJ2-LAMP by using antibodies against CryJ1 (left), CryJ2 (middle), or human-LAMP (right), respectively.

    Journal: Journal of Immunology Research

    Article Title: CryJ-LAMP DNA Vaccines for Japanese Red Cedar Allergy Induce Robust Th1-Type Immune Responses in Murine Model

    doi: 10.1155/2016/4857869

    Figure Lengend Snippet: CryJ-LAMP DNA construct and expression in vitro . (a) Plasmid construction of LAMP Vaccine. CryJ1-LAMP and CryJ2-LAMP plasmids were generated by inserting synthetic CryJ1 or CryJ2 gene into the N LAMP-C LAMP gene to create N LAMP-CryJ1-C LAMP or N LAMP-CryJ2-C LAMP. (b) 293T cells were transfected with CryJ1-LAMP or CryJ2-LAMP. Transfection reagents Opti-MEM and Lipofectamine 2000 solution only were used as a transfection control. Cell lysates were examined for expression of CryJ1-LAMP or CryJ2-LAMP by using antibodies against CryJ1 (left), CryJ2 (middle), or human-LAMP (right), respectively.

    Article Snippet: To verify the expression of CryJ1-LAMP and CryJ2-LAMP plasmids in mammalian cells, 293T cells were transfected with CryJ1-LAMP or CryJ2-LAMP plasmids with a Lipofectamine® 2000 Kit (Life Technologies, Grand Island, NY).

    Techniques: Construct, Expressing, In Vitro, Plasmid Preparation, Generated, Transfection

    Effects of AC on NF-AT activation . Jurkat cells (5 × 10 4 ) were transfected with pGL4.30 (luc2P/NFAT-RE/Hygro) by Lipofectamin™ 2000 (Invitrogen, USA) for 24 hours according to the manufacturer's instructions. Then, the cells were cultured with anti-CD3 (1 μg/ml)/CD28 (3 μg/ml) Ab in the presence or absence of AC (6.25, 12.5 and 25 μM) or CsA (2.5 μM) for four hours. Total cell lysates were extracted with 1× reporter lysis buffer (Promega, USA), then 10 μg of total cell lysates were used to determine luciferase activity by the Luciferase Assay System (Promega, USA). Each bar is the mean ± SD of three independent experiments.  ##  P

    Journal: Chinese Medicine

    Article Title: Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    doi: 10.1186/1749-8546-6-12

    Figure Lengend Snippet: Effects of AC on NF-AT activation . Jurkat cells (5 × 10 4 ) were transfected with pGL4.30 (luc2P/NFAT-RE/Hygro) by Lipofectamin™ 2000 (Invitrogen, USA) for 24 hours according to the manufacturer's instructions. Then, the cells were cultured with anti-CD3 (1 μg/ml)/CD28 (3 μg/ml) Ab in the presence or absence of AC (6.25, 12.5 and 25 μM) or CsA (2.5 μM) for four hours. Total cell lysates were extracted with 1× reporter lysis buffer (Promega, USA), then 10 μg of total cell lysates were used to determine luciferase activity by the Luciferase Assay System (Promega, USA). Each bar is the mean ± SD of three independent experiments. ## P

    Article Snippet: Luciferase assay Jurkat cells (5 × 104 ) were transfected by pGL4.30 (luc2P/NFAT-RE/Hygro) with Lipofectamin™ 2000 (Invitrogen, USA) for 24 hours according to the manufacturer's instructions.

    Techniques: Activation Assay, Transfection, Cell Culture, Lysis, Luciferase, Activity Assay

    Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using lipofectamine RNAiMAX (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.

    Journal: Virology

    Article Title: Normal human cell proteins that interact with the adenovirus type 5 E1B 55 kDa protein

    doi: 10.1016/j.virol.2017.01.013

    Figure Lengend Snippet: Impact of ANP32A knockdown on viral late gene expression. A. A double-stranded siRNA targeting ANP32A mRNA (ANP) or a universal, non-targeting control siRNA (C) were introduced in A549 cells using lipofectamine RNAiMAX (InVitrogen) as described in Materials and Methods, or were exposed only to lipofectamine (LF) or only to medium (−). Whole cell lysates were prepared 3 days thereafter and ANP32A and β-actin examined by immunoblotting. B. Proliferating A549 cells were treated with ANP32A (ANP) or control (C) siRNAs, or exposed to lipofectamine (LF) or medium (−) only for 72 h. They were then infected with 10 pfu/cell AdEasyE1 or AdeasyE1Δ2347 or mock infected for 24 h. Viral protein V and cellular β-actin in whole cell lysates were examined by immunoblotting.

    Article Snippet: All three siRNAs were introduced into HFFs using lipofectamine RNAiMAX (InVitrogen), raising the possibility that this reagent is deleterious to HFFs and/or viral genome replication.

    Techniques: Expressing, Aqueous Normal-phase Chromatography, Infection

    Functional characteristics of miR-548aq-3p as anti-angiogenic miRNAs. ( A ) Healthy ECFCs were transduced with mock lentivirus (M) or lentivirus overexpressing miR-548aq-3p (OE). 48 hours after transduction, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( B ) Representative images from the tube formation assays of non-transduced controls treated with or without 10 nM vinblastine (VB), mock infected (M) and miR-548aq-3p overexpressed (OE) healthy ECFCs (original magnification 10×, scale bar 300 μm). ( C ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( B ). ( D ) CAD ECFCs were transfected with mock (M) or miR-548aq-3p inhibitor (KD) using Lipofectamine RNAiMAX. 48 hours after transfection, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( E ) Representative images from the tube formation assay of CAD ECFCs treated with or without 10 nM vinblastine (VB), mock transfected (M) and miR-548aq-3p knockdown (KD) (original magnification 10×, scale bar 300 μm). ( F ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( E ).

    Journal: Scientific Reports

    Article Title: miR-548aq-3p is a novel target of Far infrared radiation which predicts coronary artery disease endothelial colony forming cell responsiveness

    doi: 10.1038/s41598-020-63311-1

    Figure Lengend Snippet: Functional characteristics of miR-548aq-3p as anti-angiogenic miRNAs. ( A ) Healthy ECFCs were transduced with mock lentivirus (M) or lentivirus overexpressing miR-548aq-3p (OE). 48 hours after transduction, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( B ) Representative images from the tube formation assays of non-transduced controls treated with or without 10 nM vinblastine (VB), mock infected (M) and miR-548aq-3p overexpressed (OE) healthy ECFCs (original magnification 10×, scale bar 300 μm). ( C ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( B ). ( D ) CAD ECFCs were transfected with mock (M) or miR-548aq-3p inhibitor (KD) using Lipofectamine RNAiMAX. 48 hours after transfection, total RNA was extracted and the expression levels of miR-548aq-3p were quantified by RT-qPCR. ( E ) Representative images from the tube formation assay of CAD ECFCs treated with or without 10 nM vinblastine (VB), mock transfected (M) and miR-548aq-3p knockdown (KD) (original magnification 10×, scale bar 300 μm). ( F ) Quantitative data of total tube length (left panel), number of tubes ( > 30 μm) (middle panel) and number of branched cells (right panel) in ( E ).

    Article Snippet: To knockdown miR-548aq-3p in ECFCs, a commercial synthetic miRIDIAN microRNA Hairpin Inhibitor (hsa-miR-548aq-3p, IH-302531-01-0005) (Dharmacon, Lafayette, CO, USA) was added to the culture medium at a final concentration of 20 nM at 70~80% cell confluence using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA).

    Techniques: Functional Assay, Transduction, Expressing, Quantitative RT-PCR, Infection, Transfection, Tube Formation Assay

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0187971

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

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

    Techniques: Incubation, Transfection, Negative Control

    Matrigel and spheroid uptake of Opti-MEM-formed siRNA can be improved in the presence of 10% serum. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl Opti-MEM using RNAiMax, but co-incubated in a well containing 1.5 mls DMEM containing 10% serum, at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl DMEM containing 10% serum using RNAiMax, but co-incubated in a well containing 1.5 mls Opti-MEM, at 2, 6 and 24 hours post transfection.

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Matrigel and spheroid uptake of Opti-MEM-formed siRNA can be improved in the presence of 10% serum. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl Opti-MEM using RNAiMax, but co-incubated in a well containing 1.5 mls DMEM containing 10% serum, at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 500 μl DMEM containing 10% serum using RNAiMax, but co-incubated in a well containing 1.5 mls Opti-MEM, at 2, 6 and 24 hours post transfection.

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Incubation, Transfection

    Failure of Opti-MEM prepared siRNA to penetrate matrigel or organoids is not cell line or transfection reagent specific. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection. ( C ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using Lipofectamine 2000 at 2, 6 and 24 hours post transfection.

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Failure of Opti-MEM prepared siRNA to penetrate matrigel or organoids is not cell line or transfection reagent specific. ( A ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. ( B ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection. ( C ) Representative confocal images of LS174T spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using Lipofectamine 2000 at 2, 6 and 24 hours post transfection.

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection

    10 kDa dialyzed serum is also efficient for matrigel and spheroid uptake of siRNA. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed with ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% dialyzed serum using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of dialyzed serum. Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by *** P

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: 10 kDa dialyzed serum is also efficient for matrigel and spheroid uptake of siRNA. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed with ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% dialyzed serum using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of dialyzed serum. Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by *** P

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection, Fluorescence

    Primary organoid uptake of siRNA is more efficient when complexes are formed with serum. Representative confocal images of VilCre ER Apc fl/fl Kras G12D/+ primary mouse organoids showing localization of control-Cy3 siRNA 2, 6 and 24 hours post transfection with siRNA formed in ( A ) Opti-MEM or ( B ) 10% serum using RNAiMax. White scale bars indicate 150 μM. Representative confocal images of primary wild type mouse organoids showing localization of control-Cy3 siRNA 2, 6 and 24 hours post transfection with siRNA formed in ( C ) Opti-MEM or ( D ) 10% serum using RNAiMax. White scale bar indicates 80 μM except for 24 hours where it indicates 130 μM. White/black arrows indicate the location of the matrigel boundary.

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Primary organoid uptake of siRNA is more efficient when complexes are formed with serum. Representative confocal images of VilCre ER Apc fl/fl Kras G12D/+ primary mouse organoids showing localization of control-Cy3 siRNA 2, 6 and 24 hours post transfection with siRNA formed in ( A ) Opti-MEM or ( B ) 10% serum using RNAiMax. White scale bars indicate 150 μM. Representative confocal images of primary wild type mouse organoids showing localization of control-Cy3 siRNA 2, 6 and 24 hours post transfection with siRNA formed in ( C ) Opti-MEM or ( D ) 10% serum using RNAiMax. White scale bar indicates 80 μM except for 24 hours where it indicates 130 μM. White/black arrows indicate the location of the matrigel boundary.

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection

    Optimal siRNA uptake by matrigel and spheroids occurs with a 1–10% serum concentration. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed with ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% serum using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of serum. Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by * P

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Optimal siRNA uptake by matrigel and spheroids occurs with a 1–10% serum concentration. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed with ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% serum using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of serum. Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by * P

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Concentration Assay, Transfection, Fluorescence

    Spheroid uptake of siRNA is more efficient when complexes are formed with serum. ( A ) Workflow of experimental timeframe for spheroid transfection and analysis. ( B ) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. ( C ) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection. White scale bar indicates 250 μM and white/black arrows indicate the location of the matrigel boundary. Summary graphs indicating the level of CTNNB1 mRNA in ( D ) 3D, and ( E ) 2D SW1463 cell cultures, at 48 hours post transfection with control or CTNNB1 siRNA complexes formed in Opti-MEM or 10% serum using RNAiMax. Levels are normalized to the housekeeping gene TATA-binding protein ( TBP ). Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by ** P

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: Spheroid uptake of siRNA is more efficient when complexes are formed with serum. ( A ) Workflow of experimental timeframe for spheroid transfection and analysis. ( B ) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. ( C ) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection. White scale bar indicates 250 μM and white/black arrows indicate the location of the matrigel boundary. Summary graphs indicating the level of CTNNB1 mRNA in ( D ) 3D, and ( E ) 2D SW1463 cell cultures, at 48 hours post transfection with control or CTNNB1 siRNA complexes formed in Opti-MEM or 10% serum using RNAiMax. Levels are normalized to the housekeeping gene TATA-binding protein ( TBP ). Data represents mean ± 1 s.d ( n = 3). Statistical significance is denoted by ** P

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection, Binding Assay

    BSA can facilitate matrigel entry of siRNA, but not spheroid uptake. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed in ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% BSA (no serum present) using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of BSA. Data represents mean ± 1 s.d ( n = 3).

    Journal: Scientific Reports

    Article Title: Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

    doi: 10.1038/s41598-018-26253-3

    Figure Lengend Snippet: BSA can facilitate matrigel entry of siRNA, but not spheroid uptake. Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA at 2, 6 and 24 hours post transfection with siRNA formed in ( A ) 10% ( B ) 1% ( C ) 0.1% or ( D ) 0% BSA (no serum present) using RNAiMax. ( E ) Summary graph showing the mean internal spheroid fluorescence post transfection using siRNA formed with a dose-response of BSA. Data represents mean ± 1 s.d ( n = 3).

    Article Snippet: For mRNA assessment, spheroids received either 25 nM CTNNB1 or non-targeting control siRNA (unlabelled) as previously described . siRNA complexes were formed using either Lipofectamine 2000 or RNAiMAX (Invitrogen), in either Opti-MEM (reduced serum) or DMEM (Gibco) containing either 10% normal/dialyzed FBS (Gibco).

    Techniques: Transfection, Fluorescence

    Spred2 overexpression reduced cytokine responses in macrophages. Spred2 was overexpressed by transfecting macrophages with mixtures of Lipofectamine 3000 and Spred2 overexpression plasmid and control plasmid for 24 h, after which the cells were stimulated with PA (500 μM) for 24 h. Cytokine levels in the culture supernatants were measured by ELISA. ** p

    Journal: Frontiers in Immunology

    Article Title: Spred2 Regulates High Fat Diet-Induced Adipose Tissue Inflammation, and Metabolic Abnormalities in Mice

    doi: 10.3389/fimmu.2019.00017

    Figure Lengend Snippet: Spred2 overexpression reduced cytokine responses in macrophages. Spred2 was overexpressed by transfecting macrophages with mixtures of Lipofectamine 3000 and Spred2 overexpression plasmid and control plasmid for 24 h, after which the cells were stimulated with PA (500 μM) for 24 h. Cytokine levels in the culture supernatants were measured by ELISA. ** p

    Article Snippet: For each transfection, 0.75 μL Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) and 0.5 μg Spred2 expression plasmid (kindly provided by Dr. Masakiyo Sakaguchi, Okayama university, Japan) were added to 50 μL Opti-MEM (Life Technologies) and incubated for 10 min at room temperature before the mixtures were added to the cells.

    Techniques: Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    MiR-181d-5p targets KLF6 to ameliorate H/R injury. HK-2 cells were co-transfected with the KLF6 plasmid and miR-181d-5p mimic with Lipofectamine 3000 and, 72 h later, were treated with hypoxia (l% oxygen) for 24 h/reoxygenation for 3 h. (A,B) Quantitative analysis of HIF1-α, KIM-1, and caspase-3 expression in HK-2 cells treated with or without miR-181d-5p and KLF6 ( n = 4 or 5 per group). (C) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 4 per group). The data are presented as the means ± SDs. ∗ P

    Journal: Frontiers in Physiology

    Article Title: MiR-181d-5p Targets KLF6 to Improve Ischemia/Reperfusion-Induced AKI Through Effects on Renal Function, Apoptosis, and Inflammation

    doi: 10.3389/fphys.2020.00510

    Figure Lengend Snippet: MiR-181d-5p targets KLF6 to ameliorate H/R injury. HK-2 cells were co-transfected with the KLF6 plasmid and miR-181d-5p mimic with Lipofectamine 3000 and, 72 h later, were treated with hypoxia (l% oxygen) for 24 h/reoxygenation for 3 h. (A,B) Quantitative analysis of HIF1-α, KIM-1, and caspase-3 expression in HK-2 cells treated with or without miR-181d-5p and KLF6 ( n = 4 or 5 per group). (C) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 4 per group). The data are presented as the means ± SDs. ∗ P

    Article Snippet: After 24 h, 293T cells were co-transfected with the miR-181d mimic or a scrambled miRNA sequence and PGL3-KLF6-wt or PFL3-KLF6-mut using Lipofectamine 3000 transfection reagent (L3000015, Thermo Fisher Scientific, Waltham, MA, United States).

    Techniques: Transfection, Plasmid Preparation, Expressing

    KLF6 overexpression exacerbated the hypoxia-induced decline in renal function, renal tubular cell apoptosis, and inflammatory response. HK-2 cells were transfected with KLF6 plasmid and KLF6 shRNA plasmid or scrambled plasmid with Lipofectamine 3000 and, 72 h later, were incubated in normoxia (control) or treated with hypoxia (1% oxygen) for 24 h/reoxygenation for 3 h. (A) KLF6 protein expression in HK-2 cells treated with or without KLF6 ( n = 3 per group). (B,C) qRT-PCR was used to measure miR-181d-5p, KIM-1 and HIF1-α levels after KLF6 transfection ( n = 5 per group). (D) Annexin V-FITC/PI double staining was utilized to evaluate apoptosis after KLF6 transfection. This experiment was repeated three times. (E) KLF6 increased NF-KB expression. HK-2 cells were transfected with or without the KLF6 plasmid. The results shown are from Western blot analysis of NF-KB and I-KB. β-Actin and Lamin-A were used as internal controls for I-KB and NF-KB, respectively ( n = 3 per group). (F) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 3 per group). The data are presented as the means ± SDs. * P

    Journal: Frontiers in Physiology

    Article Title: MiR-181d-5p Targets KLF6 to Improve Ischemia/Reperfusion-Induced AKI Through Effects on Renal Function, Apoptosis, and Inflammation

    doi: 10.3389/fphys.2020.00510

    Figure Lengend Snippet: KLF6 overexpression exacerbated the hypoxia-induced decline in renal function, renal tubular cell apoptosis, and inflammatory response. HK-2 cells were transfected with KLF6 plasmid and KLF6 shRNA plasmid or scrambled plasmid with Lipofectamine 3000 and, 72 h later, were incubated in normoxia (control) or treated with hypoxia (1% oxygen) for 24 h/reoxygenation for 3 h. (A) KLF6 protein expression in HK-2 cells treated with or without KLF6 ( n = 3 per group). (B,C) qRT-PCR was used to measure miR-181d-5p, KIM-1 and HIF1-α levels after KLF6 transfection ( n = 5 per group). (D) Annexin V-FITC/PI double staining was utilized to evaluate apoptosis after KLF6 transfection. This experiment was repeated three times. (E) KLF6 increased NF-KB expression. HK-2 cells were transfected with or without the KLF6 plasmid. The results shown are from Western blot analysis of NF-KB and I-KB. β-Actin and Lamin-A were used as internal controls for I-KB and NF-KB, respectively ( n = 3 per group). (F) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 3 per group). The data are presented as the means ± SDs. * P

    Article Snippet: After 24 h, 293T cells were co-transfected with the miR-181d mimic or a scrambled miRNA sequence and PGL3-KLF6-wt or PFL3-KLF6-mut using Lipofectamine 3000 transfection reagent (L3000015, Thermo Fisher Scientific, Waltham, MA, United States).

    Techniques: Over Expression, Transfection, Plasmid Preparation, shRNA, Incubation, Expressing, Quantitative RT-PCR, Double Staining, Western Blot

    siRNA-mediated silencing of PCGF isoforms increases  E. chaffeensis  infection. THP-1 cells were transfected with isoform-specific siRNA and then infected with  E. chaffeensis  at 24 h posttransfection. (A) Alexa Fluor 488-conjugated siRNA-transfected cell, showing high efficiency of RNA transfection using Lipofectamine 3000. (B) Western blot analysis of the total cell lysate from control and siRNA-transfected THP-1 cells confirmed the decrease in PCGF2, PCGF3, PCGF4, and PCGF5 48 h posttransfection. GAPDH was used as the loading control. The relative abundance of PCGF isoforms in siRNA-transfected cells was determined after normalization to the loading control and then represented as the percentage remaining after the knockdown. (C) Table representing the percentage increase in ehrlichial morulae and the average number of morulae/cell for each PCGF isoform-specific knockdown. The average morula counts were determined by counting the number of morula present in each field of view and then dividing that by the number of cells counted. The experiment was repeated three times in duplicate, and the values shown are means ± standard deviations (Stdev). (D) The fold change in ehrlichial infection was determined by comparing the ehrlichial  dsb  to the host cell  gapdh  in individual PCGF knockdown using real-time qPCR at 48 hpi ( n  = 3; *,  P  ≤ 0.05).

    Journal: Infection and Immunity

    Article Title: Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection

    doi: 10.1128/IAI.00845-17

    Figure Lengend Snippet: siRNA-mediated silencing of PCGF isoforms increases E. chaffeensis infection. THP-1 cells were transfected with isoform-specific siRNA and then infected with E. chaffeensis at 24 h posttransfection. (A) Alexa Fluor 488-conjugated siRNA-transfected cell, showing high efficiency of RNA transfection using Lipofectamine 3000. (B) Western blot analysis of the total cell lysate from control and siRNA-transfected THP-1 cells confirmed the decrease in PCGF2, PCGF3, PCGF4, and PCGF5 48 h posttransfection. GAPDH was used as the loading control. The relative abundance of PCGF isoforms in siRNA-transfected cells was determined after normalization to the loading control and then represented as the percentage remaining after the knockdown. (C) Table representing the percentage increase in ehrlichial morulae and the average number of morulae/cell for each PCGF isoform-specific knockdown. The average morula counts were determined by counting the number of morula present in each field of view and then dividing that by the number of cells counted. The experiment was repeated three times in duplicate, and the values shown are means ± standard deviations (Stdev). (D) The fold change in ehrlichial infection was determined by comparing the ehrlichial dsb to the host cell gapdh in individual PCGF knockdown using real-time qPCR at 48 hpi ( n = 3; *, P ≤ 0.05).

    Article Snippet: Briefly, specific siRNA (3 μl) and Lipofectamine 3000 reagent (7.5 μl) were added to Opti-MEM medium (250 μl) (Invitrogen), incubated for 5 min at room temperature, and then added to the cell suspension in a 6-well plate.

    Techniques: Infection, Transfection, Western Blot, Real-time Polymerase Chain Reaction

    Interactions of ATF-2 with JunD in vitro as measured by glutathionine S -transferase (GST)-ATF-2 pull-down assays. A : human ATF-2 amino acid sequence. The basic region and leucine-zipper area were indicated by green and red colors, respectively. B : GST-ATF-2 fusion proteins: schematic diagram depicting various GST-ATF-2 constructs( a ); and GST-ATF-2 fusion proteins as measured by Coomassie blue staining assays( b ). Constructs were transformed into Eschericia coli BL21, and their expression was induced by treatment with isopropyl-b-d-thiogalactopyranoside (IPTG) at the concentration of 0.5 mM. Expressed GST (without ATF-2) or GST-ATF-2 fusion proteins were harvested and purified by equilibrated MagneGST particles. These fusion proteins were monitored by SDS-PAGE analysis and shown by Coomassie blue staining. C : ATF-2 association with JunD in cells overexpressing JunD. Cells were transfected by using the expression vector containing human junD cDNA by LipofectAMINE technique; whole cell lysates were harvested 48 h after the transfection. The magnetic particles bound to GST or GST-ATF-2 fusion proteins were incubated with cell lysate for 30 min, dissolved in 1× SDS loading buffer, and then subjected to SDS-PAGE. Levels of JunD in the complexes pull-down by using GST or GST-ATF-2 fusion proteins were measured by Western blot analysis with the antibody against JunD ( top ), whereas input GST or GST-ATF-2 fusion proteins were examined by using anti-GST antibody ( bottom ). Three experiments were performed that showed similar results. D : levels of JunD protein in the complexes pull-down by GST-ATF-2 fusion proteins GST-505 ( a ) and GST-176 ( b ) from control cells and cells treated with DFMO alone or DFMO plus Put for 6 days.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Induced ATF-2 represses CDK4 transcription through dimerization with JunD inhibiting intestinal epithelial cell growth after polyamine depletion

    doi: 10.1152/ajpcell.00021.2010

    Figure Lengend Snippet: Interactions of ATF-2 with JunD in vitro as measured by glutathionine S -transferase (GST)-ATF-2 pull-down assays. A : human ATF-2 amino acid sequence. The basic region and leucine-zipper area were indicated by green and red colors, respectively. B : GST-ATF-2 fusion proteins: schematic diagram depicting various GST-ATF-2 constructs( a ); and GST-ATF-2 fusion proteins as measured by Coomassie blue staining assays( b ). Constructs were transformed into Eschericia coli BL21, and their expression was induced by treatment with isopropyl-b-d-thiogalactopyranoside (IPTG) at the concentration of 0.5 mM. Expressed GST (without ATF-2) or GST-ATF-2 fusion proteins were harvested and purified by equilibrated MagneGST particles. These fusion proteins were monitored by SDS-PAGE analysis and shown by Coomassie blue staining. C : ATF-2 association with JunD in cells overexpressing JunD. Cells were transfected by using the expression vector containing human junD cDNA by LipofectAMINE technique; whole cell lysates were harvested 48 h after the transfection. The magnetic particles bound to GST or GST-ATF-2 fusion proteins were incubated with cell lysate for 30 min, dissolved in 1× SDS loading buffer, and then subjected to SDS-PAGE. Levels of JunD in the complexes pull-down by using GST or GST-ATF-2 fusion proteins were measured by Western blot analysis with the antibody against JunD ( top ), whereas input GST or GST-ATF-2 fusion proteins were examined by using anti-GST antibody ( bottom ). Three experiments were performed that showed similar results. D : levels of JunD protein in the complexes pull-down by GST-ATF-2 fusion proteins GST-505 ( a ) and GST-176 ( b ) from control cells and cells treated with DFMO alone or DFMO plus Put for 6 days.

    Article Snippet: Transient transfection was performed with Lipofectamine Reagent from Invitrogen (Carlsbad, CA).

    Techniques: In Vitro, Sequencing, Construct, Staining, Transformation Assay, Expressing, Concentration Assay, Purification, SDS Page, Transfection, Plasmid Preparation, Incubation, Western Blot

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

    Journal: Journal of Nanobiotechnology

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

    doi: 10.1186/s12951-015-0125-1

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

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

    Techniques: Flow Cytometry, Expressing, Staining, Transfection

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

    Journal: Journal of Nanobiotechnology

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

    doi: 10.1186/s12951-015-0125-1

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

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

    Techniques: Flow Cytometry, Cytometry, Expressing

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

    Journal: Journal of Nanobiotechnology

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

    doi: 10.1186/s12951-015-0125-1

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

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

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

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

    Journal: Journal of Nanobiotechnology

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

    doi: 10.1186/s12951-015-0125-1

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

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

    Techniques: Staining, Flow Cytometry, Cytometry, Standard Deviation

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

    Journal: Journal of Nanobiotechnology

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

    doi: 10.1186/s12951-015-0125-1

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

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

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

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

    Journal: Journal of Nanobiotechnology

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

    doi: 10.1186/s12951-015-0125-1

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

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

    Techniques: Fluorescence, Transfection

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

    Journal: Journal of Nanobiotechnology

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

    doi: 10.1186/s12951-015-0125-1

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

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

    Techniques: Expressing, Staining

    SOD2 knockdown inhibits both the migration and invasion abilities of TSCC To characterize the role of SOD2 in aiding metastasis, the plasmid containing SOD2 shRNA was transiently transfected into UM1 cells using Lipofectamine Plus reagent. Cells were tested 24 h post-transfection. (A) Significant reduction of SOD2 protein levels and activities were observed in the SOD2 shRNA-transfected UM1 cells compared to the vector control transfected cells. *: P

    Journal: Free radical biology & medicine

    Article Title: Manganese superoxide dismutase induces migration and invasion of tongue squamous cell carcinoma via H2O2-dependent Snail signaling

    doi: 10.1016/j.freeradbiomed.2012.04.031

    Figure Lengend Snippet: SOD2 knockdown inhibits both the migration and invasion abilities of TSCC To characterize the role of SOD2 in aiding metastasis, the plasmid containing SOD2 shRNA was transiently transfected into UM1 cells using Lipofectamine Plus reagent. Cells were tested 24 h post-transfection. (A) Significant reduction of SOD2 protein levels and activities were observed in the SOD2 shRNA-transfected UM1 cells compared to the vector control transfected cells. *: P

    Article Snippet: The constructed plasmids were transiently transfected into UM1 cells using Lipofectamine Plus reagent (Invitrogen, CA, USA), according to the manufacturer's instructions[ ].

    Techniques: Migration, Plasmid Preparation, shRNA, Transfection

    Snail signaling contributes to SOD2-induced migration and invasion of TSCC To characterize the role of Snail signaling in SOD2-induced metastasis of TSCC, western blot analysis was used with actin as the loading control. Plasmids containing SOD2 shRNA were transiently transfected into UM1 cells using Lipofectamine Plus reagent. Cells were tested 24 h post-transfection. The UM2 cells or SOD2 shRNA-transfected UM1 cells were treated with 100μM H 2 O 2 for 24 h. (A) UM1 cells displayed an increase in Snai1, Snai2, MMP-1, ERK1/2 and pERK1/2 protein levels, and decreased protein levels of E-cadtherin compared to UM2 cells. (B) UM1 cells displayed decreased snai1, snai2, MMP-1, ERK1/2 and pERK1/2 protein levels and increased E-cadtherin protein levels upon SOD2 knockdown. (C, D) The addition of H 2 O 2 increased the protein levels of Snai1, Snai2, MMP-1, ERK1/2 and pERK1/2 and decreased protein levels of E-cadtherin in both UM2 cells and the SOD2 shRNA-transfected UMl cells.

    Journal: Free radical biology & medicine

    Article Title: Manganese superoxide dismutase induces migration and invasion of tongue squamous cell carcinoma via H2O2-dependent Snail signaling

    doi: 10.1016/j.freeradbiomed.2012.04.031

    Figure Lengend Snippet: Snail signaling contributes to SOD2-induced migration and invasion of TSCC To characterize the role of Snail signaling in SOD2-induced metastasis of TSCC, western blot analysis was used with actin as the loading control. Plasmids containing SOD2 shRNA were transiently transfected into UM1 cells using Lipofectamine Plus reagent. Cells were tested 24 h post-transfection. The UM2 cells or SOD2 shRNA-transfected UM1 cells were treated with 100μM H 2 O 2 for 24 h. (A) UM1 cells displayed an increase in Snai1, Snai2, MMP-1, ERK1/2 and pERK1/2 protein levels, and decreased protein levels of E-cadtherin compared to UM2 cells. (B) UM1 cells displayed decreased snai1, snai2, MMP-1, ERK1/2 and pERK1/2 protein levels and increased E-cadtherin protein levels upon SOD2 knockdown. (C, D) The addition of H 2 O 2 increased the protein levels of Snai1, Snai2, MMP-1, ERK1/2 and pERK1/2 and decreased protein levels of E-cadtherin in both UM2 cells and the SOD2 shRNA-transfected UMl cells.

    Article Snippet: The constructed plasmids were transiently transfected into UM1 cells using Lipofectamine Plus reagent (Invitrogen, CA, USA), according to the manufacturer's instructions[ ].

    Techniques: Migration, Western Blot, shRNA, Transfection

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-018-06864-0

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

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

    Techniques: Plasmid Preparation, Labeling, shRNA

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

    Journal: Nature Communications

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

    doi: 10.1038/s41467-018-06864-0

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

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

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

    MAGI3 inhibits β-catenin transcriptional activity A. HEK293 cells were co-transfected with plasmids coding for β-catenin (0.2 μg), increasing amounts of GFP-MAGI3 (0, 0.1 or 0.2 μg), Renilla (0.01 μg) and either the TOP or FOP luciferase reporter (0.1 μg each). B. HEK293 cells were co-transfected with increasing amounts of GFP-MAGI3 (0, 0.2 or 0.4 μg) and luciferase reporter plasmids followed by treatment with 30 mM LiCl for 24 h. C. HEK293 cells were co-transfected with Dvl-2 (0.2 μg), increasing amounts of GFP-MAGI3 (0, 0.1 and 0.2 μg) and luciferase reporter plasmids. D. HEK293 cells were co-transfected with increasing amounts of GFP-MAGI3 (0, 0.2 and 0.4 μg) and luciferase reporter plasmids. E. HEK293 cells were co-transfected with luciferase reporter plasmids and either MAGI3 siRNA or scrambled siRNA control (50 nM each) followed by 8 h of stimulation with Wnt3a CM. F. HEK293 cells were co-transfected with GFP-MAGI3 (0.1 μg) and either wild-type β-catenin (WT) or its mutants (T779A, L781A, 0.2 μg each) plus luciferase reporter plasmids. For all transfections, the total amount of DNA was adjusted to 0.5 μg with the empty vector in (A–D) and (F) The reporter activities were determined 48 h after transfection. Values are expressed relative to the ratio of TOP/Renilla versus FOP/Renilla firefly luciferase activity in HEK293 cells transfected with empty vector. Data are expressed as the mean ± SD of triplicate samples. * P

    Journal: Oncotarget

    Article Title: MAGI3 negatively regulates Wnt/β-catenin signaling and suppresses malignant phenotypes of glioma cells

    doi:

    Figure Lengend Snippet: MAGI3 inhibits β-catenin transcriptional activity A. HEK293 cells were co-transfected with plasmids coding for β-catenin (0.2 μg), increasing amounts of GFP-MAGI3 (0, 0.1 or 0.2 μg), Renilla (0.01 μg) and either the TOP or FOP luciferase reporter (0.1 μg each). B. HEK293 cells were co-transfected with increasing amounts of GFP-MAGI3 (0, 0.2 or 0.4 μg) and luciferase reporter plasmids followed by treatment with 30 mM LiCl for 24 h. C. HEK293 cells were co-transfected with Dvl-2 (0.2 μg), increasing amounts of GFP-MAGI3 (0, 0.1 and 0.2 μg) and luciferase reporter plasmids. D. HEK293 cells were co-transfected with increasing amounts of GFP-MAGI3 (0, 0.2 and 0.4 μg) and luciferase reporter plasmids. E. HEK293 cells were co-transfected with luciferase reporter plasmids and either MAGI3 siRNA or scrambled siRNA control (50 nM each) followed by 8 h of stimulation with Wnt3a CM. F. HEK293 cells were co-transfected with GFP-MAGI3 (0.1 μg) and either wild-type β-catenin (WT) or its mutants (T779A, L781A, 0.2 μg each) plus luciferase reporter plasmids. For all transfections, the total amount of DNA was adjusted to 0.5 μg with the empty vector in (A–D) and (F) The reporter activities were determined 48 h after transfection. Values are expressed relative to the ratio of TOP/Renilla versus FOP/Renilla firefly luciferase activity in HEK293 cells transfected with empty vector. Data are expressed as the mean ± SD of triplicate samples. * P

    Article Snippet: Cell transfection was performed using Lipofectamine 2000 (Invitrogen).

    Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation

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

    Journal:

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

    doi: 10.1002/jcb.21561

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

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

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