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  • 99
    Thermo Fisher lipofectamine 2000
    Development of YFP-Aβ fusion proteins in HEK-293T cell lines for measuring Aβ aggregates in brains. The indicated cell lines were developed to measure prion-like activity of preparations consisting of synthetic Aβ peptides and mouse brain-derived extracts based on their abilities to induce fluorescent aggregates which we refer to as puncta. ( A ) Diagram illustrating the Aβ constructs used in this study (left). Stably transfected HEK293T cells expressing an Aβ-YFP fusion construct underwent <t>lipofectamine-based</t> transduction with synthetic Aβ fibrils (right). ( B ) Representative confocal images of HEK293T cells expressing Aβ42 fused to YFP at the N-terminus (clone #1), which were treated with PBS (left; control) or exposed to synthetic Aβ40 fibrils (initial monomeric concentration, 1 μM) (right, exposed). The aggregates of Aβ-YFP appear as fluorescent yellow puncta. To measure prion-like activity, we counted the number of puncta-positive cells and expressed this as a percent of the total number of cells in the field of view (% positive cells). Lower panels are higher magnification images of white boxed areas in upper panels. Scale bars: 20 μm (upper panels) or 5 μm (lower panels). ( C ) HEK293T cells transfected with YFP-Aβ42 were treated with two different types of Aβ ranging from 0.03–100 nM (initial monomeric concentration): synthetic Aβ40 (left) or Aβ purified from TgAPP23 mouse brains (right). Puncta-inducing activity in the HEK293T cells was quantified 2 days after the initial exposure to various Aβ preparations. Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 3 independent experiments. ( D ) Cell lines stably expressing four different wildtype (WT) Aβ constructs (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, , and are representative of 2 to 3 independent experiments. ( E ) Cell lines stably expressing four different Aβ40 constructs fused to YFP at the N-terminus (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 2 independent experiments.
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 492053 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 492053 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
    IRF3 is phosphorylated, translocates to the nucleus, and binds the CXCL10 promoter in HT29 cells in response to addition of poly(I:C). A , Western blots of HT29 cells were stimulated with poly(I:C) alone ( Poly(I:C) , 2.5 μg/ml) or transfected with poly(I:C) using <t>Lipofectamine</t> <t>RNAimax</t> ( LF + Poly(I:C) , 0–1200 min) and stained with antibodies against phospho-IRF3 Ser-396 , total IRF3, phosphor-p65 Ser-536 , total p65, or GAPDH. The results are representative of two independent experiments. MW , molecular weight. B and C , nuclear accumulation of IRF3 ( B ) and IRF1 ( C ) in HT29 cells left untreated ( 0 ), stimulated with poly(I:C) (5–2 μg/ml), or transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml) for 3 h or overnight ( o/n ). Stimulated cells were fixed and immunostained for IRF3 or IRF1, and cell nuclei were stained with Hoechst 3342. Cells were visualized by automated imaging, and analysis was done using ScanR. The results show the percentage of cells with positive staining of IRF3 and IRF1 in the nucleus. The results show mean ± S.D. of triplicate samples with a minimum of 1300 cells assayed and are representative of three independent experiments. D , CXCL10 promotor occupancy by IRF3 in HT29 cells after poly(I:C) (2 μg/ml) stimulation for 3 h. IRF3 binding to the CXCL10 promoter was investigated by ChIP followed by qPCR of the CXCL10 promoter region. RNA polymerase II occupancy was measured as a control. E and F , CXCL10 production ( left panels ) and IRF mRNA expression ( right panels ) in HT29 cells left untreated ( No add ), treated with siRNA against IRF3 ( E ) or IRF7 ( F ) (10 n m ), NS RNA (10 n m ), or transfection reagent alone ( LF ) for 24 h. Cells were subsequently stimulated with poly(I:C) (2.5 μg/ml) for 6 h. CXCL10 release was assessed by ELISA, whereas silencing of IRF3 and 7 was confirmed by assessing mRNA expression by qPCR using GAPDH as a reference control. The results show mean ± S.D. of triplicate samples.
    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
    Generation of GalNAc-T-knockout P19 cells. (A) Target sequence derived from the genomic sequence of B4galnt1 exon 1 was inserted into the GeneArt CRISPR nuclease vector. P19 cells were transfected with the vector using the <t>Lipofectamine</t> 3000 reagent. (B) Sequencing of the neighboring target region in exon 1 of the B4galnt1 gene of CRIPR/Cas9 vector-transfected P19 cells. Parts of the B4galnt1 gene from 3 different subcloned cells were amplified by PCR and subcloned into a TOPO cloning vector. Eight TOPO vectors derived from 3 subcloned cells (subclones 1 to 3) were sequenced. All target sites in the vectors contained the 14-nucleotide deletion, and neither other deletions nor original sequence were detected. Gray squares indicate the target sequences. (C) P19 neurons were fixed and stained with an Alexa Fluor 647-labeled CTB (magenta) or anti-GT1b mAb and Alexa Fluor 488-labeled anti-mouse IgG antibody (green). DAPI was used to stain the nuclei (blue). Differential interference contrast (DIC) and fluorescence images were collected using confocal microscopy. Neither CTB, which binds GM1, nor GT1b (using an anti-GT1b mAb) were detected on GalNAc-T-knockout P19 cells. WT and KO indicate wild-type and GalNAc-T-knockout cells, respectively. Scale bars indicate 10 μm.
    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


    Development of YFP-Aβ fusion proteins in HEK-293T cell lines for measuring Aβ aggregates in brains. The indicated cell lines were developed to measure prion-like activity of preparations consisting of synthetic Aβ peptides and mouse brain-derived extracts based on their abilities to induce fluorescent aggregates which we refer to as puncta. ( A ) Diagram illustrating the Aβ constructs used in this study (left). Stably transfected HEK293T cells expressing an Aβ-YFP fusion construct underwent lipofectamine-based transduction with synthetic Aβ fibrils (right). ( B ) Representative confocal images of HEK293T cells expressing Aβ42 fused to YFP at the N-terminus (clone #1), which were treated with PBS (left; control) or exposed to synthetic Aβ40 fibrils (initial monomeric concentration, 1 μM) (right, exposed). The aggregates of Aβ-YFP appear as fluorescent yellow puncta. To measure prion-like activity, we counted the number of puncta-positive cells and expressed this as a percent of the total number of cells in the field of view (% positive cells). Lower panels are higher magnification images of white boxed areas in upper panels. Scale bars: 20 μm (upper panels) or 5 μm (lower panels). ( C ) HEK293T cells transfected with YFP-Aβ42 were treated with two different types of Aβ ranging from 0.03–100 nM (initial monomeric concentration): synthetic Aβ40 (left) or Aβ purified from TgAPP23 mouse brains (right). Puncta-inducing activity in the HEK293T cells was quantified 2 days after the initial exposure to various Aβ preparations. Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 3 independent experiments. ( D ) Cell lines stably expressing four different wildtype (WT) Aβ constructs (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, , and are representative of 2 to 3 independent experiments. ( E ) Cell lines stably expressing four different Aβ40 constructs fused to YFP at the N-terminus (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 2 independent experiments.

    Journal: Science translational medicine

    Article Title: Aβ and tau prion-like activities decline with longevity in Alzheimer’s disease brains

    doi: 10.1126/scitranslmed.aat8462

    Figure Lengend Snippet: Development of YFP-Aβ fusion proteins in HEK-293T cell lines for measuring Aβ aggregates in brains. The indicated cell lines were developed to measure prion-like activity of preparations consisting of synthetic Aβ peptides and mouse brain-derived extracts based on their abilities to induce fluorescent aggregates which we refer to as puncta. ( A ) Diagram illustrating the Aβ constructs used in this study (left). Stably transfected HEK293T cells expressing an Aβ-YFP fusion construct underwent lipofectamine-based transduction with synthetic Aβ fibrils (right). ( B ) Representative confocal images of HEK293T cells expressing Aβ42 fused to YFP at the N-terminus (clone #1), which were treated with PBS (left; control) or exposed to synthetic Aβ40 fibrils (initial monomeric concentration, 1 μM) (right, exposed). The aggregates of Aβ-YFP appear as fluorescent yellow puncta. To measure prion-like activity, we counted the number of puncta-positive cells and expressed this as a percent of the total number of cells in the field of view (% positive cells). Lower panels are higher magnification images of white boxed areas in upper panels. Scale bars: 20 μm (upper panels) or 5 μm (lower panels). ( C ) HEK293T cells transfected with YFP-Aβ42 were treated with two different types of Aβ ranging from 0.03–100 nM (initial monomeric concentration): synthetic Aβ40 (left) or Aβ purified from TgAPP23 mouse brains (right). Puncta-inducing activity in the HEK293T cells was quantified 2 days after the initial exposure to various Aβ preparations. Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 3 independent experiments. ( D ) Cell lines stably expressing four different wildtype (WT) Aβ constructs (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, , and are representative of 2 to 3 independent experiments. ( E ) Cell lines stably expressing four different Aβ40 constructs fused to YFP at the N-terminus (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 2 independent experiments.

    Article Snippet: A mixture of Lipofectamine 2000 (1.5% final volume; Thermo Fisher), OptiMEM (78.5% final volume; Thermo Fisher) and sample (20% final volume) were incubated at room temperature for 2 hours and plated in four replicate wells (10 μL/well).

    Techniques: Activity Assay, Derivative Assay, Construct, Stable Transfection, Transfection, Expressing, Transduction, Concentration Assay, Purification

    Integrin αvβ3 and fibronectin cooperate to maintain slug expression (A), total cell extracts from HT1080 and 786-0 cells after treatment with β3 integrin, fibronectin or control siRNA compared to Lipofectamine 2000 alone (Mock) were probed for FN, β3 integrin, snail, twist and slug. Ponceau S (PS) staining shows protein loading. (B), total cell extracts from HT1080 and 786-0 cells after treatment with slug or control siRNA were probed for slug, β3 integrin and FN. Ponceau S (PS) staining shows protein loading. (C), 786-0 and HT1080 cells embedded in fibrin for 24 hours were analyzed for invadopodia formation after transfection with siRNA against slug or control siRNA. ***P

    Journal: Cancer research

    Article Title: Integrin αvβ3 and fibronectin upregulate Slug in cancer cells to promote clot invasion and metastasis

    doi: 10.1158/0008-5472.CAN-13-0602

    Figure Lengend Snippet: Integrin αvβ3 and fibronectin cooperate to maintain slug expression (A), total cell extracts from HT1080 and 786-0 cells after treatment with β3 integrin, fibronectin or control siRNA compared to Lipofectamine 2000 alone (Mock) were probed for FN, β3 integrin, snail, twist and slug. Ponceau S (PS) staining shows protein loading. (B), total cell extracts from HT1080 and 786-0 cells after treatment with slug or control siRNA were probed for slug, β3 integrin and FN. Ponceau S (PS) staining shows protein loading. (C), 786-0 and HT1080 cells embedded in fibrin for 24 hours were analyzed for invadopodia formation after transfection with siRNA against slug or control siRNA. ***P

    Article Snippet: Cells were transfected in Opti-MEM medium (Invitrogen) using LipofectAMINE 2000 reagent (Invitrogen) according to manufacturer instructions.

    Techniques: Expressing, Staining, Transfection

    ARV-p10 protein expression in B16 melanoma cells using NP-ARV. (a) The expression of the ARV-p10 protein (upper panel) and housekeeping GAPDH (lower panel) transcripts was determined by RT-PCR 48 hours posttransfection. A representative gel containing DNA ladder (L, lane 1), PCR blank control (control, lane 2), nontransfected cells (parental, lane 3), Lipofectamine pIRES-ARV-transfected cells (Lipo-ARV, lane 4), and NP-ARV-transfected cells (lane 5). (b) Expression of ARV-p10 protein determined by immunofluorescence. B16 (upper panel), Lipo-ARV-transfected B16 cells (middle panel), and NP-ARV-transfected B16 cells (lower panel) stained with DAPI (left column) and with an antibody against ARV-p10 protein (middle column). Merge is shown in the right column. (c) Syncytium formation 48 h posttransfection of B16 cells (upper panel), Lipo-ARV-transfected B16 cells (middle panel), and NP-ARV-transfected B16 cells (lower panel) stained with DAPI (left column) and CellMask (middle column). Merge is shown in the right column. White arrows mark fusion points. (d) Quantification of syncytia 48 hours posttransfection by hemacolor stain. (e) Cell viability was evaluated at 24, 48, and 120 hours posttransfection and was normalized against nontransfected cells. Graphs correspond to average ± standard error of three independent experiments. Statistical analyses were performed using the Mann-Whitney test ( ∗ p

    Journal: BioMed Research International

    Article Title: Chitosan-Based Delivery of Avian Reovirus Fusogenic Protein p10 Gene: In Vitro and In Vivo Studies towards a New Vaccine against Melanoma

    doi: 10.1155/2020/4045760

    Figure Lengend Snippet: ARV-p10 protein expression in B16 melanoma cells using NP-ARV. (a) The expression of the ARV-p10 protein (upper panel) and housekeeping GAPDH (lower panel) transcripts was determined by RT-PCR 48 hours posttransfection. A representative gel containing DNA ladder (L, lane 1), PCR blank control (control, lane 2), nontransfected cells (parental, lane 3), Lipofectamine pIRES-ARV-transfected cells (Lipo-ARV, lane 4), and NP-ARV-transfected cells (lane 5). (b) Expression of ARV-p10 protein determined by immunofluorescence. B16 (upper panel), Lipo-ARV-transfected B16 cells (middle panel), and NP-ARV-transfected B16 cells (lower panel) stained with DAPI (left column) and with an antibody against ARV-p10 protein (middle column). Merge is shown in the right column. (c) Syncytium formation 48 h posttransfection of B16 cells (upper panel), Lipo-ARV-transfected B16 cells (middle panel), and NP-ARV-transfected B16 cells (lower panel) stained with DAPI (left column) and CellMask (middle column). Merge is shown in the right column. White arrows mark fusion points. (d) Quantification of syncytia 48 hours posttransfection by hemacolor stain. (e) Cell viability was evaluated at 24, 48, and 120 hours posttransfection and was normalized against nontransfected cells. Graphs correspond to average ± standard error of three independent experiments. Statistical analyses were performed using the Mann-Whitney test ( ∗ p

    Article Snippet: ARV-p10 Protein ExpressionFor ARV-p10 detection, B16 cells at a confluence of 40-60% were transfected with NPs of chitosan and pIRES-ARV (N/P 20) and Lipofectamine (Invitrogen, 11668027).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Transfection, Immunofluorescence, Staining, MANN-WHITNEY

    NO 2 -FAs suppress STING signaling and release of type I IFN. ( A and B ) THP-1 cells and ( C and D ) BMMs (WT mice) were treated with indicated NO 2 -FAs (5–10 µM) or OA/LA (10 µM) 15 min before stimulation with dsDNA (4 µg/mL) or infection with HSV-2 (MOI 1) or left untreated (Ut). After 20 h, supernatants were harvested and analyzed for type I IFN. Data represent one of two independent experiments and are presented as mean ± SEM. ( E – G ) THP-1 cells were treated with NO 2 -FAs (10 µM) or OA/LA (10 µM) 15 min before stimulation with cGAMP (4 µg/mL) or dsDNA (4 µg/mL) using Lipofectamine2000 (Lipo). After 3 h, lysates were separated by SDS/PAGE, and indicated proteins were detected by Western blotting using specific antibodies. STING and IRF3 dimers were detected using nondenaturing and nonreducing conditions. Vinculin was used as loading control.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling

    doi: 10.1073/pnas.1806239115

    Figure Lengend Snippet: NO 2 -FAs suppress STING signaling and release of type I IFN. ( A and B ) THP-1 cells and ( C and D ) BMMs (WT mice) were treated with indicated NO 2 -FAs (5–10 µM) or OA/LA (10 µM) 15 min before stimulation with dsDNA (4 µg/mL) or infection with HSV-2 (MOI 1) or left untreated (Ut). After 20 h, supernatants were harvested and analyzed for type I IFN. Data represent one of two independent experiments and are presented as mean ± SEM. ( E – G ) THP-1 cells were treated with NO 2 -FAs (10 µM) or OA/LA (10 µM) 15 min before stimulation with cGAMP (4 µg/mL) or dsDNA (4 µg/mL) using Lipofectamine2000 (Lipo). After 3 h, lysates were separated by SDS/PAGE, and indicated proteins were detected by Western blotting using specific antibodies. STING and IRF3 dimers were detected using nondenaturing and nonreducing conditions. Vinculin was used as loading control.

    Article Snippet: For transfection setups, 4 µg/mL dsDNA (HSV-60; InvivoGen) and 4 µL/mL Lipofectamine2000 (Invitrogen) were used according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Infection, SDS Page, Western Blot

    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

    Transfection efficiency of PEI-Fe 3 O 4 /siRNA complexes. (A) Fluorescence microscopy image of LX-2 cells transfected with PEI-Fe 3 O 4 /siRNA complexes (original magnification, ×200). Red fluorescence indicates Cy3-labeled siRNA, while blue fluorescence represents DAPI-stained nuclei. The scale bar represents 100 µm. (B) Transfection efficiency (percentage of cells transfected) of LX-2 cells transfected with PEI-Fe 3 O 4 /siRNA complexes and Lipo 2000/siRNA. PEI-Fe 3 O 4 , polyethyleneimine-functionalized magnetic iron oxide; siRNA, small interfering RNA; cy3, Cyanine 3; Lipo 2000, Lipofectamine 2000.

    Journal: Molecular Medicine Reports

    Article Title: Antifibrotic effects of specific siRNA targeting connective tissue growth factor delivered by polyethyleneimine-functionalized magnetic iron oxide nanoparticles on LX-2 cells

    doi: 10.3892/mmr.2019.10834

    Figure Lengend Snippet: Transfection efficiency of PEI-Fe 3 O 4 /siRNA complexes. (A) Fluorescence microscopy image of LX-2 cells transfected with PEI-Fe 3 O 4 /siRNA complexes (original magnification, ×200). Red fluorescence indicates Cy3-labeled siRNA, while blue fluorescence represents DAPI-stained nuclei. The scale bar represents 100 µm. (B) Transfection efficiency (percentage of cells transfected) of LX-2 cells transfected with PEI-Fe 3 O 4 /siRNA complexes and Lipo 2000/siRNA. PEI-Fe 3 O 4 , polyethyleneimine-functionalized magnetic iron oxide; siRNA, small interfering RNA; cy3, Cyanine 3; Lipo 2000, Lipofectamine 2000.

    Article Snippet: The transfection efficiency of PEI-Fe3 O4 NPs was measured and compared with that of the naked siRNA and the standard transfection reagent Lipofectamine® 2000 (Lipo 2000; Invitrogen; Thermo Fisher Scientific, Inc.).

    Techniques: Transfection, Fluorescence, Microscopy, Labeling, Staining, Small Interfering RNA

    Upregulation of Cx43 rescues rMC-1 from HG-induced apoptosis. Western blot analysis shows Cx43 plasmid transfection upregulates Cx43 expression in rMC-1 grown in HG. ( A ) Representative WB image shows Cx43 expression is increased through Cx43p plasmid

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: High Glucose Alters Cx43 Expression and Gap Junction Intercellular Communication in Retinal Müller Cells: Promotes Müller Cell and Pericyte Apoptosis

    doi: 10.1167/iovs.14-14606

    Figure Lengend Snippet: Upregulation of Cx43 rescues rMC-1 from HG-induced apoptosis. Western blot analysis shows Cx43 plasmid transfection upregulates Cx43 expression in rMC-1 grown in HG. ( A ) Representative WB image shows Cx43 expression is increased through Cx43p plasmid

    Article Snippet: To determine whether Cx43 upregulation rescues cells from HG-induced apoptosis, rMC-1 were grown for 7 days in HG medium and transfected with plasmid pEGFPN1 containing full-length Cx43 cDNA or empty vector as control using transfection reagent (Lipofectamine 2000; Invitrogen) at a ratio of 1 μL of transfection reagent (Invitrogen) for every 1 μg plasmid DNA.

    Techniques: Western Blot, Plasmid Preparation, Transfection, Expressing

    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

    IRF3 is phosphorylated, translocates to the nucleus, and binds the CXCL10 promoter in HT29 cells in response to addition of poly(I:C). A , Western blots of HT29 cells were stimulated with poly(I:C) alone ( Poly(I:C) , 2.5 μg/ml) or transfected with poly(I:C) using Lipofectamine RNAimax ( LF + Poly(I:C) , 0–1200 min) and stained with antibodies against phospho-IRF3 Ser-396 , total IRF3, phosphor-p65 Ser-536 , total p65, or GAPDH. The results are representative of two independent experiments. MW , molecular weight. B and C , nuclear accumulation of IRF3 ( B ) and IRF1 ( C ) in HT29 cells left untreated ( 0 ), stimulated with poly(I:C) (5–2 μg/ml), or transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml) for 3 h or overnight ( o/n ). Stimulated cells were fixed and immunostained for IRF3 or IRF1, and cell nuclei were stained with Hoechst 3342. Cells were visualized by automated imaging, and analysis was done using ScanR. The results show the percentage of cells with positive staining of IRF3 and IRF1 in the nucleus. The results show mean ± S.D. of triplicate samples with a minimum of 1300 cells assayed and are representative of three independent experiments. D , CXCL10 promotor occupancy by IRF3 in HT29 cells after poly(I:C) (2 μg/ml) stimulation for 3 h. IRF3 binding to the CXCL10 promoter was investigated by ChIP followed by qPCR of the CXCL10 promoter region. RNA polymerase II occupancy was measured as a control. E and F , CXCL10 production ( left panels ) and IRF mRNA expression ( right panels ) in HT29 cells left untreated ( No add ), treated with siRNA against IRF3 ( E ) or IRF7 ( F ) (10 n m ), NS RNA (10 n m ), or transfection reagent alone ( LF ) for 24 h. Cells were subsequently stimulated with poly(I:C) (2.5 μg/ml) for 6 h. CXCL10 release was assessed by ELISA, whereas silencing of IRF3 and 7 was confirmed by assessing mRNA expression by qPCR using GAPDH as a reference control. The results show mean ± S.D. of triplicate samples.

    Journal: The Journal of Biological Chemistry

    Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

    doi: 10.1074/jbc.M117.784090

    Figure Lengend Snippet: IRF3 is phosphorylated, translocates to the nucleus, and binds the CXCL10 promoter in HT29 cells in response to addition of poly(I:C). A , Western blots of HT29 cells were stimulated with poly(I:C) alone ( Poly(I:C) , 2.5 μg/ml) or transfected with poly(I:C) using Lipofectamine RNAimax ( LF + Poly(I:C) , 0–1200 min) and stained with antibodies against phospho-IRF3 Ser-396 , total IRF3, phosphor-p65 Ser-536 , total p65, or GAPDH. The results are representative of two independent experiments. MW , molecular weight. B and C , nuclear accumulation of IRF3 ( B ) and IRF1 ( C ) in HT29 cells left untreated ( 0 ), stimulated with poly(I:C) (5–2 μg/ml), or transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml) for 3 h or overnight ( o/n ). Stimulated cells were fixed and immunostained for IRF3 or IRF1, and cell nuclei were stained with Hoechst 3342. Cells were visualized by automated imaging, and analysis was done using ScanR. The results show the percentage of cells with positive staining of IRF3 and IRF1 in the nucleus. The results show mean ± S.D. of triplicate samples with a minimum of 1300 cells assayed and are representative of three independent experiments. D , CXCL10 promotor occupancy by IRF3 in HT29 cells after poly(I:C) (2 μg/ml) stimulation for 3 h. IRF3 binding to the CXCL10 promoter was investigated by ChIP followed by qPCR of the CXCL10 promoter region. RNA polymerase II occupancy was measured as a control. E and F , CXCL10 production ( left panels ) and IRF mRNA expression ( right panels ) in HT29 cells left untreated ( No add ), treated with siRNA against IRF3 ( E ) or IRF7 ( F ) (10 n m ), NS RNA (10 n m ), or transfection reagent alone ( LF ) for 24 h. Cells were subsequently stimulated with poly(I:C) (2.5 μg/ml) for 6 h. CXCL10 release was assessed by ELISA, whereas silencing of IRF3 and 7 was confirmed by assessing mRNA expression by qPCR using GAPDH as a reference control. The results show mean ± S.D. of triplicate samples.

    Article Snippet: Silencing of TLR3 and TRIF SW620 or HT29 cells were transfected for 24–48 h with 10–20 nm siRNA against TLR3 (TLR3_5, Qiagen, SI02630768; TLR3_8, Qiagen, SI02655156) or non-silencing control siRNA (Qiagen, SI03650325) using Lipofectamine RNAiMAX (Invitrogen) for TLR3 knockdown or with siRNA against TICAM-1/TRIF (Ambion, s45115) for TICAM/TRIF knockdown using Lipofectamine RNAiMAX. siRNA and transfection reagent (ratio, 1:2) were preincubated for 15 min in RPMI medium before being added to newly seeded cells.

    Techniques: Western Blot, Transfection, Staining, Molecular Weight, Imaging, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    IFNβ is induced in IECs in response to transfection with poly(I:C) but not in response to poly(I:C) addition. A , HT29 cells were left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before CXCL10 in the supernatant was assessed by ELISA. B , kinetics of CXCL10 release assessed by ELISA in supernatant from HT29 cells stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 5, 12, 20, and 24 h. The results are presented as mean ± S.D. of triplicates. C , IFNβ mRNA induction in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone ( Poly(I:C) ), transfected with poly(I:C) complexed with Lipofectamine RNAimax ( LF + Poly(I:C) , 2 μg/ml), or treated with only Lipofectamine RNAimax ( LF ) for 20 h. IFNβ mRNA induction was determined by qPCR. The results are presented as relative induction compared with medium-treated Caco-2 cells. GAPDH served as an internal control. Results show mean -fold induction ± S.D. of triplicates. D , IFNβ protein production in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone, transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 20 h. IFNβ in the supernatant was assessed by ELISA, and the results show mean ± S.D. of three samples. E , HT29 cells were stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 6, 12, 20, or 24 h before CXCL10 and IFNβ mRNA induction was determined by qPCR. The results show relative induction with a non-treated sample as reference. GAPDH served as an internal control. The results show mean -fold induction ± S.D. of triplicates. F , CXCL10 mRNA induction in HT29 cells pretreated with cycloheximide (0, 15, or 30 μg/ml) for 30 min prior to stimulation with poly(I:C) (2.5 μg/ml) for 8 h. CXCL10 mRNA was determined by qPCR (normalized to medium control and the endogenous control TBP). G , viability in HT29 cells left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before viability was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. H , viability in IECs left untreated ( 0 ), stimulated with poly(I:C) alone (2 μg/ml), transfected with poly(I:C) using Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 43 h before the viability of the cells was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. The results show mean ± S.D. of five samples. All results are representative of at least two independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Surface Toll-like receptor 3 expression in metastatic intestinal epithelial cells induces inflammatory cytokine production and promotes invasiveness

    doi: 10.1074/jbc.M117.784090

    Figure Lengend Snippet: IFNβ is induced in IECs in response to transfection with poly(I:C) but not in response to poly(I:C) addition. A , HT29 cells were left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before CXCL10 in the supernatant was assessed by ELISA. B , kinetics of CXCL10 release assessed by ELISA in supernatant from HT29 cells stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 5, 12, 20, and 24 h. The results are presented as mean ± S.D. of triplicates. C , IFNβ mRNA induction in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone ( Poly(I:C) ), transfected with poly(I:C) complexed with Lipofectamine RNAimax ( LF + Poly(I:C) , 2 μg/ml), or treated with only Lipofectamine RNAimax ( LF ) for 20 h. IFNβ mRNA induction was determined by qPCR. The results are presented as relative induction compared with medium-treated Caco-2 cells. GAPDH served as an internal control. Results show mean -fold induction ± S.D. of triplicates. D , IFNβ protein production in HT29, HCT116, SW620, SW480, and Caco-2 cells treated with poly(I:C) (2 μg/ml) alone, transfected with poly(I:C) complexed with Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 20 h. IFNβ in the supernatant was assessed by ELISA, and the results show mean ± S.D. of three samples. E , HT29 cells were stimulated with poly(I:C) (2.5 μg/ml) for 0, 3, 6, 12, 20, or 24 h before CXCL10 and IFNβ mRNA induction was determined by qPCR. The results show relative induction with a non-treated sample as reference. GAPDH served as an internal control. The results show mean -fold induction ± S.D. of triplicates. F , CXCL10 mRNA induction in HT29 cells pretreated with cycloheximide (0, 15, or 30 μg/ml) for 30 min prior to stimulation with poly(I:C) (2.5 μg/ml) for 8 h. CXCL10 mRNA was determined by qPCR (normalized to medium control and the endogenous control TBP). G , viability in HT29 cells left untreated ( 0 ) or stimulated with poly(I:C) (50, 25, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.15 μg/ml) for 20 h before viability was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. H , viability in IECs left untreated ( 0 ), stimulated with poly(I:C) alone (2 μg/ml), transfected with poly(I:C) using Lipofectamine RNAimax (2 μg/ml), or treated with only Lipofectamine RNAimax for 43 h before the viability of the cells was assessed using the MTT assay. The MTT assay results were normalized to an untreated sample. The results show mean ± S.D. of five samples. All results are representative of at least two independent experiments.

    Article Snippet: Silencing of TLR3 and TRIF SW620 or HT29 cells were transfected for 24–48 h with 10–20 nm siRNA against TLR3 (TLR3_5, Qiagen, SI02630768; TLR3_8, Qiagen, SI02655156) or non-silencing control siRNA (Qiagen, SI03650325) using Lipofectamine RNAiMAX (Invitrogen) for TLR3 knockdown or with siRNA against TICAM-1/TRIF (Ambion, s45115) for TICAM/TRIF knockdown using Lipofectamine RNAiMAX. siRNA and transfection reagent (ratio, 1:2) were preincubated for 15 min in RPMI medium before being added to newly seeded cells.

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, MTT Assay

    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

    siRNA dose response curve for three active and one inactive polymer. RNAiMax was used as the positive control to benchmark the efficacy. The same polyplexes were prepared as those in and different amount was added to the cells depending on the

    Journal: Polymer

    Article Title: One-pot Synthesis of Functional Poly(amino ester sulfide)s and Utility in Delivering pDNA and siRNA

    doi: 10.1016/j.polymer.2015.02.020

    Figure Lengend Snippet: siRNA dose response curve for three active and one inactive polymer. RNAiMax was used as the positive control to benchmark the efficacy. The same polyplexes were prepared as those in and different amount was added to the cells depending on the

    Article Snippet: Lipofectamine 2000 (LF2000) and RNAiMax was purchased from Invitrogen and used following the supplier’s recommended protocols.

    Techniques: Positive Control

    Generation of GalNAc-T-knockout P19 cells. (A) Target sequence derived from the genomic sequence of B4galnt1 exon 1 was inserted into the GeneArt CRISPR nuclease vector. P19 cells were transfected with the vector using the Lipofectamine 3000 reagent. (B) Sequencing of the neighboring target region in exon 1 of the B4galnt1 gene of CRIPR/Cas9 vector-transfected P19 cells. Parts of the B4galnt1 gene from 3 different subcloned cells were amplified by PCR and subcloned into a TOPO cloning vector. Eight TOPO vectors derived from 3 subcloned cells (subclones 1 to 3) were sequenced. All target sites in the vectors contained the 14-nucleotide deletion, and neither other deletions nor original sequence were detected. Gray squares indicate the target sequences. (C) P19 neurons were fixed and stained with an Alexa Fluor 647-labeled CTB (magenta) or anti-GT1b mAb and Alexa Fluor 488-labeled anti-mouse IgG antibody (green). DAPI was used to stain the nuclei (blue). Differential interference contrast (DIC) and fluorescence images were collected using confocal microscopy. Neither CTB, which binds GM1, nor GT1b (using an anti-GT1b mAb) were detected on GalNAc-T-knockout P19 cells. WT and KO indicate wild-type and GalNAc-T-knockout cells, respectively. Scale bars indicate 10 μm.

    Journal: PLoS ONE

    Article Title: CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C

    doi: 10.1371/journal.pone.0132363

    Figure Lengend Snippet: Generation of GalNAc-T-knockout P19 cells. (A) Target sequence derived from the genomic sequence of B4galnt1 exon 1 was inserted into the GeneArt CRISPR nuclease vector. P19 cells were transfected with the vector using the Lipofectamine 3000 reagent. (B) Sequencing of the neighboring target region in exon 1 of the B4galnt1 gene of CRIPR/Cas9 vector-transfected P19 cells. Parts of the B4galnt1 gene from 3 different subcloned cells were amplified by PCR and subcloned into a TOPO cloning vector. Eight TOPO vectors derived from 3 subcloned cells (subclones 1 to 3) were sequenced. All target sites in the vectors contained the 14-nucleotide deletion, and neither other deletions nor original sequence were detected. Gray squares indicate the target sequences. (C) P19 neurons were fixed and stained with an Alexa Fluor 647-labeled CTB (magenta) or anti-GT1b mAb and Alexa Fluor 488-labeled anti-mouse IgG antibody (green). DAPI was used to stain the nuclei (blue). Differential interference contrast (DIC) and fluorescence images were collected using confocal microscopy. Neither CTB, which binds GM1, nor GT1b (using an anti-GT1b mAb) were detected on GalNAc-T-knockout P19 cells. WT and KO indicate wild-type and GalNAc-T-knockout cells, respectively. Scale bars indicate 10 μm.

    Article Snippet: Generation of GalNAc-T-depleted P19 cells Undifferentiated P19 cells were transfected with the CRISPR/Cas9 vector and Lipofectamine 3000 (Life Technologies) according to the manufacturer’s instructions.

    Techniques: Knock-Out, Sequencing, Derivative Assay, CRISPR, Plasmid Preparation, Transfection, Amplification, Polymerase Chain Reaction, Clone Assay, Staining, Labeling, CtB Assay, Fluorescence, Confocal Microscopy

    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

    NGB reduces cyclin D1 expression and inhibits cell growth via merlin. (A) Protein and mRNA levels of cyclin D1 are inhibited by NGB. JS1 cells were transfected with increasing amounts of Flag-NGB and immunoblotted with indicated antibodies (panels 1 to 3). Total RNA was isolated from the cells and subjected to semiquantitative reverse transcription-PCR using primers specific for cyclin D1 and β-actin (panels 4 and 5). (B) Knockdown of NGB increases cyclin D1 level. HeLa cells were transfected with NGB-RNAi using Lipofectamine. Following 72 h of incubation, cells were lysed and immunoblotted with anti-NGB (top), -cyclin D1 (middle), and -actin (bottom) antibodies. (C) NGB-reduced cyclin D1 is abrogated by knockdown of merlin. JS1/NGB-59 cells were infected with lentivirus pLKO.1-shRNA/Nf2 and pLKO.1-puro vector and immunoblotted with indicated antibodies. (D and E) Merlin mediates NGB-inhibited cell growth and tumorigenicity. Indicated cells were seeded in 48-well plates at 0.2 × 10 5 /well. The cell number was counted daily for 3 days. The experiment was performed in triplicate (D). The cells were subcutaneously injected into nude mice (3 × 10 6 cells/mouse). Tumor volume was measured every 2 days. Data shown are representative of results from two independent experiments carried out with 16 mice each (8 mice/cell line) (E).

    Journal: Molecular and Cellular Biology

    Article Title: Identification and Characterization of Putative Tumor Suppressor NGB, a GTP-Binding Protein That Interacts with the Neurofibromatosis 2 Protein ▿

    doi: 10.1128/MCB.00572-06

    Figure Lengend Snippet: NGB reduces cyclin D1 expression and inhibits cell growth via merlin. (A) Protein and mRNA levels of cyclin D1 are inhibited by NGB. JS1 cells were transfected with increasing amounts of Flag-NGB and immunoblotted with indicated antibodies (panels 1 to 3). Total RNA was isolated from the cells and subjected to semiquantitative reverse transcription-PCR using primers specific for cyclin D1 and β-actin (panels 4 and 5). (B) Knockdown of NGB increases cyclin D1 level. HeLa cells were transfected with NGB-RNAi using Lipofectamine. Following 72 h of incubation, cells were lysed and immunoblotted with anti-NGB (top), -cyclin D1 (middle), and -actin (bottom) antibodies. (C) NGB-reduced cyclin D1 is abrogated by knockdown of merlin. JS1/NGB-59 cells were infected with lentivirus pLKO.1-shRNA/Nf2 and pLKO.1-puro vector and immunoblotted with indicated antibodies. (D and E) Merlin mediates NGB-inhibited cell growth and tumorigenicity. Indicated cells were seeded in 48-well plates at 0.2 × 10 5 /well. The cell number was counted daily for 3 days. The experiment was performed in triplicate (D). The cells were subcutaneously injected into nude mice (3 × 10 6 cells/mouse). Tumor volume was measured every 2 days. Data shown are representative of results from two independent experiments carried out with 16 mice each (8 mice/cell line) (E).

    Article Snippet: Cell culture media, protein A/G beads, and Lipofectamine Plus were from Invitrogen (Carlsbad, CA).

    Techniques: Expressing, Transfection, Isolation, Polymerase Chain Reaction, Incubation, Infection, shRNA, Plasmid Preparation, Injection, Mouse Assay

    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

    Pex13 deficiency disrupts protein import into peroxisomes. (A) Peroxisomal protein import in cultured skin fibroblasts from wild-type (+/+) and Pex13-deficient (−/−) mice. PTS1 protein import assessed by indirect immunofluorescence using an anti-SKL antibody (SKL panels), PTS2 protein import assessed by GFP autofluorescence in cells transfected with a plasmid that expresses a PTS2-EGFP fusion protein (GFP panels), and peroxisomal membranes detected by indirect immunofluorescence using an antibody to the peroxisomal integral membrane protein PMP70 (PMP panels) are shown. Note that GFP fluorescence and PMP70 immunofluorescence are from the same cell in each case. Bars, 10 μm. (B) Transfection of Pex13 −/− cells with a plasmid that expresses wild-type, myc-tagged Pex13 rescues import of PTS1 and PTS2-EGFP protein import. myc and SKL immunofluorescence in the same cell (top) and GFP fluorescence and SKL immunofluorescence in the same cell (bottom) are shown. (C) Western blot analysis of liver homogenates to determine the levels of the peroxisomal proteins catalase, peroxisomal bifunctional protein (PBP), and PMP70. Actin levels indicate protein loading. Liver homogenates from wild-type (+/+) and heterozygous (+/−) and homozygous (−/−) knockout animals were used. (D) Electron microscopic analysis of DAB-stained liver for detection of peroxisomes (PO) in wild-type and Pex13 −/− mice. Bars, 1 μm.

    Journal: Molecular and Cellular Biology

    Article Title: Pex13 Inactivation in the Mouse Disrupts Peroxisome Biogenesis and Leads to a Zellweger Syndrome Phenotype

    doi: 10.1128/MCB.23.16.5947-5957.2003

    Figure Lengend Snippet: Pex13 deficiency disrupts protein import into peroxisomes. (A) Peroxisomal protein import in cultured skin fibroblasts from wild-type (+/+) and Pex13-deficient (−/−) mice. PTS1 protein import assessed by indirect immunofluorescence using an anti-SKL antibody (SKL panels), PTS2 protein import assessed by GFP autofluorescence in cells transfected with a plasmid that expresses a PTS2-EGFP fusion protein (GFP panels), and peroxisomal membranes detected by indirect immunofluorescence using an antibody to the peroxisomal integral membrane protein PMP70 (PMP panels) are shown. Note that GFP fluorescence and PMP70 immunofluorescence are from the same cell in each case. Bars, 10 μm. (B) Transfection of Pex13 −/− cells with a plasmid that expresses wild-type, myc-tagged Pex13 rescues import of PTS1 and PTS2-EGFP protein import. myc and SKL immunofluorescence in the same cell (top) and GFP fluorescence and SKL immunofluorescence in the same cell (bottom) are shown. (C) Western blot analysis of liver homogenates to determine the levels of the peroxisomal proteins catalase, peroxisomal bifunctional protein (PBP), and PMP70. Actin levels indicate protein loading. Liver homogenates from wild-type (+/+) and heterozygous (+/−) and homozygous (−/−) knockout animals were used. (D) Electron microscopic analysis of DAB-stained liver for detection of peroxisomes (PO) in wild-type and Pex13 −/− mice. Bars, 1 μm.

    Article Snippet: Cell transfection, using Lipofectamine 2000 (Invitrogen) and 3 μg of relevant plasmid, and indirect immunofluorescence were performed as previously described for human skin fibroblasts ( , ).

    Techniques: Cell Culture, Mouse Assay, Immunofluorescence, Transfection, Plasmid Preparation, Fluorescence, Western Blot, Knock-Out, Staining

    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