gaussia luciferase assay kit  (New England Biolabs)


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    BioLux Gaussia Luciferase Assay Kit
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    BioLux Gaussia Luciferase Assay Kit 1 000 assays
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    e3300l
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    Cellular Biology
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    New England Biolabs gaussia luciferase assay kit
    BioLux Gaussia Luciferase Assay Kit
    BioLux Gaussia Luciferase Assay Kit 1 000 assays
    https://www.bioz.com/result/gaussia luciferase assay kit/product/New England Biolabs
    Average 99 stars, based on 321 article reviews
    Price from $9.99 to $1999.99
    gaussia luciferase assay kit - by Bioz Stars, 2021-02
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    1) Product Images from "Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies"

    Article Title: Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00793

    Gaussia princeps luciferase (GpL)-tumor necrosis factor (TNF) binding to cells with endogenous coexpression of tumor necrosis factor receptor-2 (TNFR2) and progranulin (PGRN). (A) Human embryonal kidney cells 293 (HEK293) cells were transfected (first transfection) with empty vector (EV) or an expression vector encoding TNFR2, where the TNF receptor associated factor 2 (TRAF2) binding site has been replaced by yellow fluorescence protein (YFP) (TNFR2). The following day, transfecfed cells were split into four aliquots which were transfected a second time (second transfection) with expression plasmids encoding PGRN, membrane TNF (memTNF), soluble Flag-tagged TNF (F-TNF), or EV. After an additional day, aliquots of 30,000 cells (P) and 15 µl SN (S) were analyzed by Western blotting with anti-PGRN, anti-TNF, and anti-Flag along with 100 ng PGRN Adi and 100 ng purified untagged soluble TNF (sTNF). (B) Equilibrium binding studies were performed with the indicated concentrations of GpL-TNF. Specific binding of GpL-TNF in the presence of PGRN (second transfection PGRN), membrane TNF (second transfection memTNF), and Flag-TNF (second transfection F-TNF) or the absence of an potential modulator (second transfection EV) was obtained by subtracting unspecific binding values (first transfection EV) from the corresponding total binding values (first transfection TNFR2). Specific binding values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.
    Figure Legend Snippet: Gaussia princeps luciferase (GpL)-tumor necrosis factor (TNF) binding to cells with endogenous coexpression of tumor necrosis factor receptor-2 (TNFR2) and progranulin (PGRN). (A) Human embryonal kidney cells 293 (HEK293) cells were transfected (first transfection) with empty vector (EV) or an expression vector encoding TNFR2, where the TNF receptor associated factor 2 (TRAF2) binding site has been replaced by yellow fluorescence protein (YFP) (TNFR2). The following day, transfecfed cells were split into four aliquots which were transfected a second time (second transfection) with expression plasmids encoding PGRN, membrane TNF (memTNF), soluble Flag-tagged TNF (F-TNF), or EV. After an additional day, aliquots of 30,000 cells (P) and 15 µl SN (S) were analyzed by Western blotting with anti-PGRN, anti-TNF, and anti-Flag along with 100 ng PGRN Adi and 100 ng purified untagged soluble TNF (sTNF). (B) Equilibrium binding studies were performed with the indicated concentrations of GpL-TNF. Specific binding of GpL-TNF in the presence of PGRN (second transfection PGRN), membrane TNF (second transfection memTNF), and Flag-TNF (second transfection F-TNF) or the absence of an potential modulator (second transfection EV) was obtained by subtracting unspecific binding values (first transfection EV) from the corresponding total binding values (first transfection TNFR2). Specific binding values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

    Techniques Used: Luciferase, Binding Assay, Transfection, Plasmid Preparation, Expressing, Fluorescence, Western Blot, Purification, Software

    Gaussia princeps luciferase (GpL) fusion proteins of progranulin (PGRN) show no relevant binding to tumor necrosis factor receptor-1 (TNFR1) or TNFR2. (A) Human embryonal kidney cells 293 (HEK293) cells were transiently transfected with expression plasmids encoding GpL-PGRN (GpL-PGRN), PGRN-GpL (PGRN-GpL), or empty vector (EV). GpL-PGRN concentrations in supernatants (SNs) and cell lysates were determined by help of a GpL fusion protein of known concentration. SNs and cell lysates, containing approximately 100 ng PGRN-GpL or GpL-PGRN along with 100 ng PGRN Adi , were subjected to Western blotting with a PGRN-specific antibody to verify the integrity of the PGRN GpL fusion proteins. (B) TNFR1-Fc, TNFR2-Fc or, as a control for unspecific binding, hIgG1 were immobilized to black enzyme-linked immunosorbent assay plates. Lysates and SN of the GpL-PGRN (GpL-PGRN lys and GpL-PGRN SN ) and PGRN-GpL (PGRN-GpL lys and PGRN-GpL SN ) transfected cells and GpL-tumor necrosis factor (TNF) were added for 1 h and binding was determined in triplicates. (C) TNFR1 and TNFR2 expressing transfectants (total binding) and EV-transfected HEK293 cells (non-specific binding) were subjected to equilibrium binding studies with the indicated GpL fusion proteins. Specific binding (= total − non-specific binding) values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.
    Figure Legend Snippet: Gaussia princeps luciferase (GpL) fusion proteins of progranulin (PGRN) show no relevant binding to tumor necrosis factor receptor-1 (TNFR1) or TNFR2. (A) Human embryonal kidney cells 293 (HEK293) cells were transiently transfected with expression plasmids encoding GpL-PGRN (GpL-PGRN), PGRN-GpL (PGRN-GpL), or empty vector (EV). GpL-PGRN concentrations in supernatants (SNs) and cell lysates were determined by help of a GpL fusion protein of known concentration. SNs and cell lysates, containing approximately 100 ng PGRN-GpL or GpL-PGRN along with 100 ng PGRN Adi , were subjected to Western blotting with a PGRN-specific antibody to verify the integrity of the PGRN GpL fusion proteins. (B) TNFR1-Fc, TNFR2-Fc or, as a control for unspecific binding, hIgG1 were immobilized to black enzyme-linked immunosorbent assay plates. Lysates and SN of the GpL-PGRN (GpL-PGRN lys and GpL-PGRN SN ) and PGRN-GpL (PGRN-GpL lys and PGRN-GpL SN ) transfected cells and GpL-tumor necrosis factor (TNF) were added for 1 h and binding was determined in triplicates. (C) TNFR1 and TNFR2 expressing transfectants (total binding) and EV-transfected HEK293 cells (non-specific binding) were subjected to equilibrium binding studies with the indicated GpL fusion proteins. Specific binding (= total − non-specific binding) values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

    Techniques Used: Luciferase, Binding Assay, Transfection, Expressing, Plasmid Preparation, Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Software

    2) Product Images from "Antibiotic monensin synergizes with EGFR inhibitors and oxaliplatin to suppress the proliferation of human ovarian cancer cells"

    Article Title: Antibiotic monensin synergizes with EGFR inhibitors and oxaliplatin to suppress the proliferation of human ovarian cancer cells

    Journal: Scientific Reports

    doi: 10.1038/srep17523

    Monensin inhibits multiple cancer-associated signaling pathways, including downstream mediators of EGFR signaling. ( A ) Effect of monensin on intracellular β-catenin level. Subconfluent SKOV3 cells were first transduced with Ad-Wnt3A or AdGFP for 16 h, and treated with varied concentrations of monensin for additional 36 h. Cells were fixed and subjected immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counterstained with DAPI. Control IgG was used as a negative control. Representative results are shown. ( B ) Monensin inhibits Tcf/Lef reporter activity. Subconfluent SKOV3 cells were transfected with TOP-Luc reporter plasmid and infected with Ad-Wnt3A or AdGFP for 16 h, followed by a treatment with varied concentrations of monensin for 48 h. Cells were lysed and subjected to luciferase activity assays using Promega’s firefly Luciferase Assay System. Each assay condition was done in triplicate. ( C ) Effect of monensin on the 11 cancer-associated pathway reporter activities. Subconfluent SKOV3 cells were transfected with the homemade Gaussia luciferase reporters for the 11 cancer-associated pathways and a constitutively active reporter pG2Luc. At 16 h post transfection the cells were treated with varied concentrations of monensin for additional 48 h. the culture medium was collected for Gaussia luciferase activity assay using BioLux Gaussia Luciferase Assay Kit (New England Biolabs). Each assay condition was done in triplicate. ( D ) Monensin inhibits four pathways in dose- and time-dependent manners. The selected four pathway reporters were transfected into SKOV3 cells as described in ( C ), except that Gaussia luciferase activities were measured at 24 h, 48 h and 72 h post treatment. (E) Monensin inhibits the expression of genes involved in cell proliferation. Subconfluent SKOV3 cells were treated with the indicated concentrations of monensin for 48 h. Total RNA was isolated and subjected to qPCR analysis of the expression of the indicated genes. Human GAPDH was used as the reference gene.
    Figure Legend Snippet: Monensin inhibits multiple cancer-associated signaling pathways, including downstream mediators of EGFR signaling. ( A ) Effect of monensin on intracellular β-catenin level. Subconfluent SKOV3 cells were first transduced with Ad-Wnt3A or AdGFP for 16 h, and treated with varied concentrations of monensin for additional 36 h. Cells were fixed and subjected immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counterstained with DAPI. Control IgG was used as a negative control. Representative results are shown. ( B ) Monensin inhibits Tcf/Lef reporter activity. Subconfluent SKOV3 cells were transfected with TOP-Luc reporter plasmid and infected with Ad-Wnt3A or AdGFP for 16 h, followed by a treatment with varied concentrations of monensin for 48 h. Cells were lysed and subjected to luciferase activity assays using Promega’s firefly Luciferase Assay System. Each assay condition was done in triplicate. ( C ) Effect of monensin on the 11 cancer-associated pathway reporter activities. Subconfluent SKOV3 cells were transfected with the homemade Gaussia luciferase reporters for the 11 cancer-associated pathways and a constitutively active reporter pG2Luc. At 16 h post transfection the cells were treated with varied concentrations of monensin for additional 48 h. the culture medium was collected for Gaussia luciferase activity assay using BioLux Gaussia Luciferase Assay Kit (New England Biolabs). Each assay condition was done in triplicate. ( D ) Monensin inhibits four pathways in dose- and time-dependent manners. The selected four pathway reporters were transfected into SKOV3 cells as described in ( C ), except that Gaussia luciferase activities were measured at 24 h, 48 h and 72 h post treatment. (E) Monensin inhibits the expression of genes involved in cell proliferation. Subconfluent SKOV3 cells were treated with the indicated concentrations of monensin for 48 h. Total RNA was isolated and subjected to qPCR analysis of the expression of the indicated genes. Human GAPDH was used as the reference gene.

    Techniques Used: Transduction, Immunofluorescence, Staining, Negative Control, Activity Assay, Transfection, Plasmid Preparation, Infection, Luciferase, Expressing, Isolation, Real-time Polymerase Chain Reaction

    3) Product Images from "EVA-1 Functions as an UNC-40 Co-receptor to Enhance Attraction to the MADD-4 Guidance Cue in Caenorhabditis elegans"

    Article Title: EVA-1 Functions as an UNC-40 Co-receptor to Enhance Attraction to the MADD-4 Guidance Cue in Caenorhabditis elegans

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004521

    EVA-1 functions cell-autonomously in muscles and interacts with MADD-4. A . Muscle-expressed EVA-1::CFP rescues the muscle extension defects of eva-1 mutants. B . A summary of EVA-1 domain function that is fully detailed in Figure S1 . C D . FLAG-tagged receptors were expressed from HEK293 cells, bathed in conditioned media from other HEK293 cells that express HA- and Gaussia luciferase-tagged MADD-4 or SLT-1 ligands, and immunoprecipitated to determine the relative amounts of ligand that co-immunoprecipitates with the receptor (see the materials and methods section for more details). C . The western blot on the left shows the five immunoprecipitated FLAG-tagged receptors. The western blot on the right shows the two HA- and Gaussia luciferase-tagged ligands that were collected from cell culture. D . The normalized relative levels of luciferase signal that immunoprecipitated with each potential ligand-receptor complex. E–I . Shown are animals harbouring one of three different transgenes that drive the expression of either neuronally-expressed MADD-4::YFP (from the trIs66 transgenic array) ( E ), muscle-expressed MADD-4::YFP (from the trIs78 transgenic array) ( F ), or muscle-expressed EVA-1::CFP (from the trIs89 transgenic array) ( G ), or animals harbouring two of the transgenes; trIs66 and trIs89 ( H ) and trIs78 and trIs89 ( I ). The relative levels of MADD-4::YFP expression from trIs66 and trIs78 is shown in Figure S2a . Images show either the CFP channel (top), YFP channel (middle) or a merged view (bottom). Arrows in ‘H’ indicate the localization of MADD-4::YFP to EVA-1::CFP expressing muscles; arrows in ‘I’ indicate the vesicularization of MADD-4::YFP and EVA-1::CFP in the muscle cells. J . The quantification of neuronally-secreted MADD-4::YFP localization to muscles over-expressing the indicated receptor. K . The quantification of CFP vesicles in animals that over-express the indicated CFP-tagged receptors (x-axis) in muscles in either the presence of MADD-4::YFP expressed from dorsal muscles (mMADD-4) or pan-neuronally (nMADD-4). The colocalization of MADD-4 and EVA-1 with the RAB-11 and RAB-5 endosomal markers are shown in Figure S2b and S2c . L M . MADD-4::YFP fails to induce obvious vesicularization of UNC-40::CFP in a wild type background (L), but YFP-CFP vesicles are obvious in animals that lack UNC-6 (M). In A, J, and K, statistical significance ( p
    Figure Legend Snippet: EVA-1 functions cell-autonomously in muscles and interacts with MADD-4. A . Muscle-expressed EVA-1::CFP rescues the muscle extension defects of eva-1 mutants. B . A summary of EVA-1 domain function that is fully detailed in Figure S1 . C D . FLAG-tagged receptors were expressed from HEK293 cells, bathed in conditioned media from other HEK293 cells that express HA- and Gaussia luciferase-tagged MADD-4 or SLT-1 ligands, and immunoprecipitated to determine the relative amounts of ligand that co-immunoprecipitates with the receptor (see the materials and methods section for more details). C . The western blot on the left shows the five immunoprecipitated FLAG-tagged receptors. The western blot on the right shows the two HA- and Gaussia luciferase-tagged ligands that were collected from cell culture. D . The normalized relative levels of luciferase signal that immunoprecipitated with each potential ligand-receptor complex. E–I . Shown are animals harbouring one of three different transgenes that drive the expression of either neuronally-expressed MADD-4::YFP (from the trIs66 transgenic array) ( E ), muscle-expressed MADD-4::YFP (from the trIs78 transgenic array) ( F ), or muscle-expressed EVA-1::CFP (from the trIs89 transgenic array) ( G ), or animals harbouring two of the transgenes; trIs66 and trIs89 ( H ) and trIs78 and trIs89 ( I ). The relative levels of MADD-4::YFP expression from trIs66 and trIs78 is shown in Figure S2a . Images show either the CFP channel (top), YFP channel (middle) or a merged view (bottom). Arrows in ‘H’ indicate the localization of MADD-4::YFP to EVA-1::CFP expressing muscles; arrows in ‘I’ indicate the vesicularization of MADD-4::YFP and EVA-1::CFP in the muscle cells. J . The quantification of neuronally-secreted MADD-4::YFP localization to muscles over-expressing the indicated receptor. K . The quantification of CFP vesicles in animals that over-express the indicated CFP-tagged receptors (x-axis) in muscles in either the presence of MADD-4::YFP expressed from dorsal muscles (mMADD-4) or pan-neuronally (nMADD-4). The colocalization of MADD-4 and EVA-1 with the RAB-11 and RAB-5 endosomal markers are shown in Figure S2b and S2c . L M . MADD-4::YFP fails to induce obvious vesicularization of UNC-40::CFP in a wild type background (L), but YFP-CFP vesicles are obvious in animals that lack UNC-6 (M). In A, J, and K, statistical significance ( p

    Techniques Used: Luciferase, Immunoprecipitation, Western Blot, Cell Culture, Expressing, Transgenic Assay

    4) Product Images from "Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation"

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.19108

    Susceptibility of directed differentiated cells from Hdo cells to HCV, HBV, and JEV replication A . Control, non-induced cells (white circles), hepatic induction cells (black squares), and cholangiocytic induction cells (black triangles) were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 1b [Con1, SGR-Con1/GLuc (solid lines) and GND (dashed lines)] in upper graphs and 2a [JFH-1, SGR-JFH1/GLuc (solid lines) and GND (dashed lines)] in lower graphs. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . Expression of miR-200a in HuH-7, Hdo-17, Hdo-23, and their respective hepatic induction cells and cholangiocytic induction cells were examined by qRT-PCR using the TaqMan microRNA Reverse Transcription Kit with miR-200a specific RT primer from the TaqMan microRNA Assay. Expression of miR-200a was normalized to that of Z30 snoRNA (SNORD7), and relative expression levels to HuH-7 cells with no induction treatment are shown. C . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pUC19, pUC-HBV-Bj (GT-B), or -Ce (GT-C). At 3 days or 5 days after transfection, particle-associated HBV DNAs in culture supernatants of transfected cells were measured by qRT-PCR (upper panel). Cell lysates were subjected to immunoblotting of HBs antigens and GAPDH (lower panel). D . HuH-7, Hdo-17, and Hdo-23 cells were infected with JEV (MOI=0.01). Cells were fixed and stained with dsRNA antibody (green) and DAPI (blue) at 3 days post-infection. Bar indicates 200 μm. All assays were performed in triplicate. (A)-(C) Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with control cells with no induction treatment (B) or HuH-7 cells (C) are shown. * p
    Figure Legend Snippet: Susceptibility of directed differentiated cells from Hdo cells to HCV, HBV, and JEV replication A . Control, non-induced cells (white circles), hepatic induction cells (black squares), and cholangiocytic induction cells (black triangles) were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 1b [Con1, SGR-Con1/GLuc (solid lines) and GND (dashed lines)] in upper graphs and 2a [JFH-1, SGR-JFH1/GLuc (solid lines) and GND (dashed lines)] in lower graphs. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . Expression of miR-200a in HuH-7, Hdo-17, Hdo-23, and their respective hepatic induction cells and cholangiocytic induction cells were examined by qRT-PCR using the TaqMan microRNA Reverse Transcription Kit with miR-200a specific RT primer from the TaqMan microRNA Assay. Expression of miR-200a was normalized to that of Z30 snoRNA (SNORD7), and relative expression levels to HuH-7 cells with no induction treatment are shown. C . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pUC19, pUC-HBV-Bj (GT-B), or -Ce (GT-C). At 3 days or 5 days after transfection, particle-associated HBV DNAs in culture supernatants of transfected cells were measured by qRT-PCR (upper panel). Cell lysates were subjected to immunoblotting of HBs antigens and GAPDH (lower panel). D . HuH-7, Hdo-17, and Hdo-23 cells were infected with JEV (MOI=0.01). Cells were fixed and stained with dsRNA antibody (green) and DAPI (blue) at 3 days post-infection. Bar indicates 200 μm. All assays were performed in triplicate. (A)-(C) Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with control cells with no induction treatment (B) or HuH-7 cells (C) are shown. * p

    Techniques Used: Transfection, Luciferase, Derivative Assay, Expressing, Quantitative RT-PCR, TaqMan microRNA Assay, Infection, Staining

    Susceptibility of Hdo cells to HCV RNA replication and involvement of miR-200a in replication A . HuH-7 (white circles), Hdo-17 (black squares), and Hdo-23 (black triangles) cells were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 2a [JFH-1, SGR-JFH1/GLuc wild (solid lines) and GND (dashed lines)] in left panel and 1b [Con1, SGR-Con1/GLuc wild (solid lines) and GND (dashed lines)] in right panel. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pRLucHCVLuc (white bars) or pRLucEMCVLuc (black bars). At 60 h after transfection, luciferase activity in cell lysates was measured by the dual-luciferase reporter assay system. The vertical axis indicates activity of firefly luciferase to that of Renilla luciferase. C . HuH-7, Hdo-17, Hdo-23, and Hec1B cells were infected with a lentivirus encoding miR-122 (white circles) or AcGFP as control (black triangles). Cells were transfected with pHH/SGR-JFH1/GLuc (solid lines) or pHH/SGR-JFH1/GLuc/GND (dashed lines) and pSV40-CLuc 48 h post-infection. Luciferase activity in the supernatant was measured by the BioLux Dual Luciferase Starter Kit at the indicated time points. Relative expression levels of secreted GLuc to CLuc in the supernatants are shown. D . HuH 5-15 cells, which are HCV genotype 1b-replicon cells, were transfected with miRNAs, miRNA inhibitors, and non-target miRNA control (miR cont) by ScreenFect A. At 3 days after transfection, HCV copies in cells were measured by qRT-PCR (grey bars). Cell viability was determined by the ATP concentration in cell lysates with the CellTiter-Glo Luminescent Cell Viability Assay (white bars). E . Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVcc (J6/JFH1, MOI=0.5). HCV copies in cells were measured by qRT-PCR at 3 days post-infection (upper graph). Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVpp (grey bars) or VSVpp (white bars). NanoLuc activity was measured at 1 day post-infection (lower graph). All assays were performed in triplicate. Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with HuH-7 cells (B) or miR cont (D) and (E) are shown. * p
    Figure Legend Snippet: Susceptibility of Hdo cells to HCV RNA replication and involvement of miR-200a in replication A . HuH-7 (white circles), Hdo-17 (black squares), and Hdo-23 (black triangles) cells were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 2a [JFH-1, SGR-JFH1/GLuc wild (solid lines) and GND (dashed lines)] in left panel and 1b [Con1, SGR-Con1/GLuc wild (solid lines) and GND (dashed lines)] in right panel. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pRLucHCVLuc (white bars) or pRLucEMCVLuc (black bars). At 60 h after transfection, luciferase activity in cell lysates was measured by the dual-luciferase reporter assay system. The vertical axis indicates activity of firefly luciferase to that of Renilla luciferase. C . HuH-7, Hdo-17, Hdo-23, and Hec1B cells were infected with a lentivirus encoding miR-122 (white circles) or AcGFP as control (black triangles). Cells were transfected with pHH/SGR-JFH1/GLuc (solid lines) or pHH/SGR-JFH1/GLuc/GND (dashed lines) and pSV40-CLuc 48 h post-infection. Luciferase activity in the supernatant was measured by the BioLux Dual Luciferase Starter Kit at the indicated time points. Relative expression levels of secreted GLuc to CLuc in the supernatants are shown. D . HuH 5-15 cells, which are HCV genotype 1b-replicon cells, were transfected with miRNAs, miRNA inhibitors, and non-target miRNA control (miR cont) by ScreenFect A. At 3 days after transfection, HCV copies in cells were measured by qRT-PCR (grey bars). Cell viability was determined by the ATP concentration in cell lysates with the CellTiter-Glo Luminescent Cell Viability Assay (white bars). E . Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVcc (J6/JFH1, MOI=0.5). HCV copies in cells were measured by qRT-PCR at 3 days post-infection (upper graph). Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVpp (grey bars) or VSVpp (white bars). NanoLuc activity was measured at 1 day post-infection (lower graph). All assays were performed in triplicate. Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with HuH-7 cells (B) or miR cont (D) and (E) are shown. * p

    Techniques Used: Transfection, Luciferase, Derivative Assay, Activity Assay, Reporter Assay, Infection, Expressing, Quantitative RT-PCR, Concentration Assay, Cell Viability Assay

    5) Product Images from "A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1"

    Article Title: A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1

    Journal: Journal of Virology

    doi: 10.1128/JVI.00856-19

    Functional analysis of GBF1/BIG2 N-terminal chimeras in replication and secretion. (A) Scheme of the BIG2-derived substitutions in the GBF1 sequence. All GBF1 expression constructs are GFP tagged and contain the A795E BFA resistance mutation in the Sec7 domain. (B) Performance of the indicated mutants in the poliovirus replicon replication and cellular secretion assays. For the replication assay, cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. Expression of the 2-17/BIG2 and 47-62/BIG2 constructs, the most compromised in the replication assay, is additionally verified by Western blotting in the samples from the corresponding experiments. For the secretion assay, the cells were cotransfected with plasmids coding for a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control) and a plasmid coding for a secreted Gaussia luciferase. The next day, they were washed and incubated in the medium with the indicated amount of BFA, and the amount of secreted luciferase was determined after 4 h. Secretion data are normalized to the signal obtained without BFA for each construct. The statistical significance of the difference between the signal in the positive control and that in the sample expressing a mutant GBF1 for corresponding concentrations of BFA is indicated.
    Figure Legend Snippet: Functional analysis of GBF1/BIG2 N-terminal chimeras in replication and secretion. (A) Scheme of the BIG2-derived substitutions in the GBF1 sequence. All GBF1 expression constructs are GFP tagged and contain the A795E BFA resistance mutation in the Sec7 domain. (B) Performance of the indicated mutants in the poliovirus replicon replication and cellular secretion assays. For the replication assay, cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. Expression of the 2-17/BIG2 and 47-62/BIG2 constructs, the most compromised in the replication assay, is additionally verified by Western blotting in the samples from the corresponding experiments. For the secretion assay, the cells were cotransfected with plasmids coding for a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control) and a plasmid coding for a secreted Gaussia luciferase. The next day, they were washed and incubated in the medium with the indicated amount of BFA, and the amount of secreted luciferase was determined after 4 h. Secretion data are normalized to the signal obtained without BFA for each construct. The statistical significance of the difference between the signal in the positive control and that in the sample expressing a mutant GBF1 for corresponding concentrations of BFA is indicated.

    Techniques Used: Functional Assay, Derivative Assay, Sequencing, Expressing, Construct, Mutagenesis, Transfection, Positive Control, Plasmid Preparation, Negative Control, Luciferase, Incubation, Western Blot

    GBF1 mutants unable to activate Arf are defective in replication. (A) Schematic of GBF1 constructs containing the inactivating 7A mutation. All GBF1 expression constructs are GFP tagged. (B) GST-Arf1-GBF1 pulldown assay. GST or GST-Δ17 ARF1 was immobilized on glutathione beads and incubated with lysate from cells (designated SM [starting material]) expressing GFP-tagged GBF1/A795E, GBF1/A795E/A794K, or GBF1/A795E/7A. The bound material was analyzed by SDS-PAGE and the gel either stained with Coomassie blue (top) or transferred to nitrocellulose (NC) and immunoblotted with anti-GFP antibodies (bottom). GBF1/795/7A does not bind the ARF substrate, whereas GBF1/795 and GBF1/795/794 bind ARF. (C) Performance of the corresponding mutants in a poliovirus replication assay. Cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. For the secretion assay, the cells were cotransfected with plasmids coding for a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control) and a plasmid coding for a secreted Gaussia luciferase. The next day, they were washed and incubated in the medium with the indicated amount of BFA, and the amount of secreted luciferase was determined after 4 h. Expression of the 7A constructs in the samples from the corresponding replication experiments is additionally verified by Western blotting. Representative positive-control (A795E) and negative-control (vector) samples are shown.
    Figure Legend Snippet: GBF1 mutants unable to activate Arf are defective in replication. (A) Schematic of GBF1 constructs containing the inactivating 7A mutation. All GBF1 expression constructs are GFP tagged. (B) GST-Arf1-GBF1 pulldown assay. GST or GST-Δ17 ARF1 was immobilized on glutathione beads and incubated with lysate from cells (designated SM [starting material]) expressing GFP-tagged GBF1/A795E, GBF1/A795E/A794K, or GBF1/A795E/7A. The bound material was analyzed by SDS-PAGE and the gel either stained with Coomassie blue (top) or transferred to nitrocellulose (NC) and immunoblotted with anti-GFP antibodies (bottom). GBF1/795/7A does not bind the ARF substrate, whereas GBF1/795 and GBF1/795/794 bind ARF. (C) Performance of the corresponding mutants in a poliovirus replication assay. Cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. For the secretion assay, the cells were cotransfected with plasmids coding for a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control) and a plasmid coding for a secreted Gaussia luciferase. The next day, they were washed and incubated in the medium with the indicated amount of BFA, and the amount of secreted luciferase was determined after 4 h. Expression of the 7A constructs in the samples from the corresponding replication experiments is additionally verified by Western blotting. Representative positive-control (A795E) and negative-control (vector) samples are shown.

    Techniques Used: Construct, Mutagenesis, Expressing, Incubation, SDS Page, Staining, Transfection, Positive Control, Plasmid Preparation, Negative Control, Luciferase, Western Blot

    Functional analysis of alanine scanning mutants of the N terminus of GBF1 in replication and secretion. (A) Schematic representation of the alanine substitutions in the GBF1 sequence. All GBF1 expression constructs are GFP tagged and contain the A795E BFA resistance mutation in the Sec7 domain. (B) Performance of the indicated mutants in the poliovirus replicon replication and cellular secretion assays. For the replication assay, cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. For the secretion assay, the cells were cotransfected with plasmids coding for a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control) and a plasmid coding for a secreted Gaussia luciferase. The next day, they were washed and incubated in the medium with the indicated amount of BFA, and the amount of secreted luciferase was determined after 4 h. Secretion data are normalized to the signal obtained without BFA for each construct. Statistical significance of the difference between the signal in the positive control and in the sample expressing a mutant GBF1 for corresponding concentrations of BFA is indicated. RLU, relative light units.
    Figure Legend Snippet: Functional analysis of alanine scanning mutants of the N terminus of GBF1 in replication and secretion. (A) Schematic representation of the alanine substitutions in the GBF1 sequence. All GBF1 expression constructs are GFP tagged and contain the A795E BFA resistance mutation in the Sec7 domain. (B) Performance of the indicated mutants in the poliovirus replicon replication and cellular secretion assays. For the replication assay, cells were transfected with the plasmids expressing a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control). The next day, the cells were transfected with a poliovirus replicon RNA expressing Renilla luciferase and incubated in the presence or absence of 1 μg/ml BFA. For the secretion assay, the cells were cotransfected with plasmids coding for a corresponding GBF1 mutant, a full-length GBF1 A795E (positive control), or an empty vector (negative control) and a plasmid coding for a secreted Gaussia luciferase. The next day, they were washed and incubated in the medium with the indicated amount of BFA, and the amount of secreted luciferase was determined after 4 h. Secretion data are normalized to the signal obtained without BFA for each construct. Statistical significance of the difference between the signal in the positive control and in the sample expressing a mutant GBF1 for corresponding concentrations of BFA is indicated. RLU, relative light units.

    Techniques Used: Functional Assay, Sequencing, Expressing, Construct, Mutagenesis, Transfection, Positive Control, Plasmid Preparation, Negative Control, Luciferase, Incubation

    6) Product Images from "Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors"

    Article Title: Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00357

    Bloodstream and peripheral isolates produce equivalent activation of NF-kB pathway in human monocytes. THP-1 Lucia® NF-kB cells were treated with the cell-free supernatant of 7 pairs of peripheral and bloodstream isolates. Cells were collected at baseline, 2, 4, and 8 h for quantification of luciferase activity by of Biolux® Gaussia luciferase Assay kit. Values obtained by luminescence were normalized to the protein absorbance of the cell lysate as measured by BioRad® DC protein assay. ns, not significant. Negative control = LB broth, positive control = Pam3CSK4 1μg/ml. Data presented represent mean and SEM of 3 independent experiments.
    Figure Legend Snippet: Bloodstream and peripheral isolates produce equivalent activation of NF-kB pathway in human monocytes. THP-1 Lucia® NF-kB cells were treated with the cell-free supernatant of 7 pairs of peripheral and bloodstream isolates. Cells were collected at baseline, 2, 4, and 8 h for quantification of luciferase activity by of Biolux® Gaussia luciferase Assay kit. Values obtained by luminescence were normalized to the protein absorbance of the cell lysate as measured by BioRad® DC protein assay. ns, not significant. Negative control = LB broth, positive control = Pam3CSK4 1μg/ml. Data presented represent mean and SEM of 3 independent experiments.

    Techniques Used: Activation Assay, Luciferase, Activity Assay, DC Protein Assay, Negative Control, Positive Control

    7) Product Images from "TGF?/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells *"

    Article Title: TGF?/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.130518

    Interaction of BMP9 with ALK1 and ALK2 determined by PCA. A ), GLuc can be split into two functionally complemented fragments, GLuc1-(17–93) and GLuc2-(94–185). The extracellular domains of ALK1 and ALK2 were fused to GLuc1, while the full-length BMP9 was fused with GLuc2. FL , full-length; SP , signal peptide. B , subconfluent 293 cells were transfected with ALK1-GLuc1, ALK2-GLuc1, and/or BMP9-Gluc2. Relative Gaussia luciferase activity was determined at 36 h after transfection using the Gaussia luciferase assay kit from New England Biolabs. Each assay condition was done in triplicate.
    Figure Legend Snippet: Interaction of BMP9 with ALK1 and ALK2 determined by PCA. A ), GLuc can be split into two functionally complemented fragments, GLuc1-(17–93) and GLuc2-(94–185). The extracellular domains of ALK1 and ALK2 were fused to GLuc1, while the full-length BMP9 was fused with GLuc2. FL , full-length; SP , signal peptide. B , subconfluent 293 cells were transfected with ALK1-GLuc1, ALK2-GLuc1, and/or BMP9-Gluc2. Relative Gaussia luciferase activity was determined at 36 h after transfection using the Gaussia luciferase assay kit from New England Biolabs. Each assay condition was done in triplicate.

    Techniques Used: Transfection, Luciferase, Activity Assay

    8) Product Images from "Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist"

    Article Title: Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist

    Journal: Immune Network

    doi: 10.4110/in.2019.19.e34

    NGP blocks the activity of LPS. (A) THP-1 cells were treated by vehicle (black bars) or 100 ng/ml of LPS (blank bars) for 16 h in the mixture of NGP conditional medium and empty vector conditional medium in a ratio of 0, 1/4, 1/8, and 1/16. IL-8 was detected from the culture medium (quadruplet mean±SD). (B) THP-1 cells were treated by vehicle or 10 ng/ml of LPS in an empty vector or NGP conditional medium for 30 min. Phospho-NF-κB p65, NF-κB p65, and IκBα were detected by Western blotting. (C) THP-1 cells were treated by vehicle or 10 ng/ml of LPS in an empty vector or NGP conditional medium for 30 min. Phospho-p38 MAPK and p38 MAPK were detected by Western blotting. (D) The conditional medium of empty vector (black) or NGP (blank) was treated overnight. LPS was mixed in different concentrations and then treated to 293 cells transfected with Gaussia luciferase NF-κB construct. The luminescence of cells with NGP medium showed inhibited activity of the reporter (quadruplet mean±SD). * p
    Figure Legend Snippet: NGP blocks the activity of LPS. (A) THP-1 cells were treated by vehicle (black bars) or 100 ng/ml of LPS (blank bars) for 16 h in the mixture of NGP conditional medium and empty vector conditional medium in a ratio of 0, 1/4, 1/8, and 1/16. IL-8 was detected from the culture medium (quadruplet mean±SD). (B) THP-1 cells were treated by vehicle or 10 ng/ml of LPS in an empty vector or NGP conditional medium for 30 min. Phospho-NF-κB p65, NF-κB p65, and IκBα were detected by Western blotting. (C) THP-1 cells were treated by vehicle or 10 ng/ml of LPS in an empty vector or NGP conditional medium for 30 min. Phospho-p38 MAPK and p38 MAPK were detected by Western blotting. (D) The conditional medium of empty vector (black) or NGP (blank) was treated overnight. LPS was mixed in different concentrations and then treated to 293 cells transfected with Gaussia luciferase NF-κB construct. The luminescence of cells with NGP medium showed inhibited activity of the reporter (quadruplet mean±SD). * p

    Techniques Used: Activity Assay, Plasmid Preparation, Western Blot, Transfection, Luciferase, Construct

    9) Product Images from "Trichodysplasia spinulosa-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids"

    Article Title: Trichodysplasia spinulosa-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005112

    Cell surface glycans featuring terminal sialic acids promote TSPyV attachment and pseudovirus infection. (A) Binding of Alexa Fluor 488-conjugated TSPyV VP1 pentamers to mock (PBS) or with 1 U/ml Clostridium perfringens type V neuraminidase pre-treated cells was analysed by flow cytometry. 30,000 gated events were measured for each sample. The average from three independent experiments is shown, and error bars indicate the standard deviation. Data was standardized to signals of mock treated cells alone. S1 Fig shows non-standardized data of individual experimental replicates. (B) Attachment of Alexa Fluor 488-conjugated TSPsV to mock (PBS) or with 1 U/ml Clostridium perfringens type V neuraminidase pre-treated HEK293 and SVGA cells. The analysis was carried out by flow cytometry with 10,000 events measured for each sample. The average from three replicates is shown, and error bars indicate the standard deviation. (C-D) TSPyV pseudovirus (TSPsV) infections of HEK293 and SVGA cells (mock-treated cells and cells incubated with Clostridium perfringens neuraminidase for 30 min prior to PsV infection). BK Polyomavirus pseudovirus (BKPsV) was used as positive control for a neuraminidase-sensitive infection. The efficiency of the PsV infection was measured by transduction of the reporter plasmid phGluc coding for a secreted form of Gaussia luciferase 72 h post infection by detection of the secreted luciferase (measured in relative light units, RLUs using the BioLux Gaussia Luciferase Assay Kit). PsV experiments were done in quadruplicate and repeated three times and the average relative transduction is shown. Error bars indicate standard deviations and statistic analysis was performed using the two-tailed unpaired t test. The data was standardized to mock PsV transfections. The mock PsV control was generated by harvesting HEK293TT cells transfected with control plasmid instead of the capsid expression plasmids and then purifying according to the PsV purification protocol to measure background signal and non-specific transfer of luciferase to infected cells.
    Figure Legend Snippet: Cell surface glycans featuring terminal sialic acids promote TSPyV attachment and pseudovirus infection. (A) Binding of Alexa Fluor 488-conjugated TSPyV VP1 pentamers to mock (PBS) or with 1 U/ml Clostridium perfringens type V neuraminidase pre-treated cells was analysed by flow cytometry. 30,000 gated events were measured for each sample. The average from three independent experiments is shown, and error bars indicate the standard deviation. Data was standardized to signals of mock treated cells alone. S1 Fig shows non-standardized data of individual experimental replicates. (B) Attachment of Alexa Fluor 488-conjugated TSPsV to mock (PBS) or with 1 U/ml Clostridium perfringens type V neuraminidase pre-treated HEK293 and SVGA cells. The analysis was carried out by flow cytometry with 10,000 events measured for each sample. The average from three replicates is shown, and error bars indicate the standard deviation. (C-D) TSPyV pseudovirus (TSPsV) infections of HEK293 and SVGA cells (mock-treated cells and cells incubated with Clostridium perfringens neuraminidase for 30 min prior to PsV infection). BK Polyomavirus pseudovirus (BKPsV) was used as positive control for a neuraminidase-sensitive infection. The efficiency of the PsV infection was measured by transduction of the reporter plasmid phGluc coding for a secreted form of Gaussia luciferase 72 h post infection by detection of the secreted luciferase (measured in relative light units, RLUs using the BioLux Gaussia Luciferase Assay Kit). PsV experiments were done in quadruplicate and repeated three times and the average relative transduction is shown. Error bars indicate standard deviations and statistic analysis was performed using the two-tailed unpaired t test. The data was standardized to mock PsV transfections. The mock PsV control was generated by harvesting HEK293TT cells transfected with control plasmid instead of the capsid expression plasmids and then purifying according to the PsV purification protocol to measure background signal and non-specific transfer of luciferase to infected cells.

    Techniques Used: Infection, Binding Assay, Flow Cytometry, Cytometry, Standard Deviation, Incubation, Positive Control, Transduction, Plasmid Preparation, Luciferase, Two Tailed Test, Transfection, Generated, Expressing, Purification

    Cell binding and PsV infection of TSPyV mutants. (A) Cell binding analysis of TSPyV wild type (WT) and mutant VP1 pentamers to HEK293 and SVGA cells by flow cytometry. VP1 pentamers were covalently thio-labelled using Alexa Fluor 488 C5 maleimide. The histogram depicts the relative fluorescence signal of mutants compared to the binding signal of wild type pentamers. Data was measured in three independent experiments and was standardized for the background signal from cells alone. Error bars indicate standard deviations. (B) WT and mutant TSPsV binding (Alexa Fluor 488 amine-conjugated) to HEK293 and SVGA cells (mock-treated cells and cells incubated with Clostridium perfringens neuraminidase for 30 min prior to PsV infection) examined by flow cytometry. Relative fluorescence signals of mutants compared to the binding signal of WT PsV were plotted and error bars indicate standard deviations for three experimental replicates. Labeled PsV content was normalized by western blotting detection with purified PAB597, a hybridoma supernatant that produces a monoclonal antibody against JCPyV VP1, which cross-reacts with TSPyV VP1. The two-tailed unpaired t test was performed for binding of WT and mutant TSPsVs to mock-treated cells. (C) TSPsV infection of HEK293 and SVGA cells assayed 72 h post infection by detection of the secreted luciferase due to transduction of the reporter plasmid phGluc. The luciferase was quantified using a BioLux Gaussia Luciferase Assay Kit. The secreted luciferase catalyzes a photo-oxidation that emits light, which is measured in relative light units, RLUs. RLUs are given in logarithmic scale. Mock PsV infections were done with a control sample obtained according the PsV purification protocol from cells only transfected with phGluc and control plasmid measure background and nonspecific transfer of luciferase. PsV experiments were done in quadruplicate and repeated two times. Statistic analysis was performed using the two-tailed unpaired t test.
    Figure Legend Snippet: Cell binding and PsV infection of TSPyV mutants. (A) Cell binding analysis of TSPyV wild type (WT) and mutant VP1 pentamers to HEK293 and SVGA cells by flow cytometry. VP1 pentamers were covalently thio-labelled using Alexa Fluor 488 C5 maleimide. The histogram depicts the relative fluorescence signal of mutants compared to the binding signal of wild type pentamers. Data was measured in three independent experiments and was standardized for the background signal from cells alone. Error bars indicate standard deviations. (B) WT and mutant TSPsV binding (Alexa Fluor 488 amine-conjugated) to HEK293 and SVGA cells (mock-treated cells and cells incubated with Clostridium perfringens neuraminidase for 30 min prior to PsV infection) examined by flow cytometry. Relative fluorescence signals of mutants compared to the binding signal of WT PsV were plotted and error bars indicate standard deviations for three experimental replicates. Labeled PsV content was normalized by western blotting detection with purified PAB597, a hybridoma supernatant that produces a monoclonal antibody against JCPyV VP1, which cross-reacts with TSPyV VP1. The two-tailed unpaired t test was performed for binding of WT and mutant TSPsVs to mock-treated cells. (C) TSPsV infection of HEK293 and SVGA cells assayed 72 h post infection by detection of the secreted luciferase due to transduction of the reporter plasmid phGluc. The luciferase was quantified using a BioLux Gaussia Luciferase Assay Kit. The secreted luciferase catalyzes a photo-oxidation that emits light, which is measured in relative light units, RLUs. RLUs are given in logarithmic scale. Mock PsV infections were done with a control sample obtained according the PsV purification protocol from cells only transfected with phGluc and control plasmid measure background and nonspecific transfer of luciferase. PsV experiments were done in quadruplicate and repeated two times. Statistic analysis was performed using the two-tailed unpaired t test.

    Techniques Used: Binding Assay, Infection, Mutagenesis, Flow Cytometry, Cytometry, Fluorescence, Incubation, Labeling, Western Blot, Purification, Two Tailed Test, Luciferase, Transduction, Plasmid Preparation, Transfection

    10) Product Images from "Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells"

    Article Title: Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells

    Journal: Differentiation; research in biological diversity

    doi: 10.1016/j.diff.2009.06.001

    RA induction of α-myosin heavy chain (MyHC) promoter-driven Gaussia luciferase reporter activity in C2C12 cells. ( A ) Construction of the MyHC-GLuc reporter. A 5.5kb α-myosin heavy chain promoter-driven Gaussia luciferase reporter (MyHC-GLuc)
    Figure Legend Snippet: RA induction of α-myosin heavy chain (MyHC) promoter-driven Gaussia luciferase reporter activity in C2C12 cells. ( A ) Construction of the MyHC-GLuc reporter. A 5.5kb α-myosin heavy chain promoter-driven Gaussia luciferase reporter (MyHC-GLuc)

    Techniques Used: Luciferase, Activity Assay

    11) Product Images from "A Novel Role of Serotonin Receptor 2B Agonist as an Anti-Melanogenesis Agent"

    Article Title: A Novel Role of Serotonin Receptor 2B Agonist as an Anti-Melanogenesis Agent

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17040546

    Effect of BW723C86 on the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT, p38 MAPK, protein kinase A (PKA), and cAMP response element-binding protein (CREB), and on MITF promoter activity. ( A ) Changes in the protein expression of MITF and expression and phosphorylation of PI3K, AKT, p38 MAPK, PKA, and CREB were measured by Western blotting. β-actin was used as the protein loading control; ( B ) Melan-A cells containing pMITF-GLuc were cultured for 24 h with BW723C86. Gaussia luciferase activity was determined from culture supernatants using the Gaussia Luciferase Assay Kit. Values are means ± SD from three replicates ( n = 3). Statistical significance was determined using the Student t -test (* p
    Figure Legend Snippet: Effect of BW723C86 on the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT, p38 MAPK, protein kinase A (PKA), and cAMP response element-binding protein (CREB), and on MITF promoter activity. ( A ) Changes in the protein expression of MITF and expression and phosphorylation of PI3K, AKT, p38 MAPK, PKA, and CREB were measured by Western blotting. β-actin was used as the protein loading control; ( B ) Melan-A cells containing pMITF-GLuc were cultured for 24 h with BW723C86. Gaussia luciferase activity was determined from culture supernatants using the Gaussia Luciferase Assay Kit. Values are means ± SD from three replicates ( n = 3). Statistical significance was determined using the Student t -test (* p

    Techniques Used: Binding Assay, Activity Assay, Expressing, Western Blot, Cell Culture, Luciferase

    12) Product Images from "GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for Coxsackievirus B3 RNA Replication ▿"

    Article Title: GBF1, a Guanine Nucleotide Exchange Factor for Arf, Is Crucial for Coxsackievirus B3 RNA Replication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01244-09

    BFA-resistant GBF1 mutants M832L and A795E differ in their abilities to rescue protein secretion and cell viability in the presence of BFA. (A) HeLa cells were cotransfected with plasmid pCMV-Gluc expressing secreted Gaussia luciferase and either a control
    Figure Legend Snippet: BFA-resistant GBF1 mutants M832L and A795E differ in their abilities to rescue protein secretion and cell viability in the presence of BFA. (A) HeLa cells were cotransfected with plasmid pCMV-Gluc expressing secreted Gaussia luciferase and either a control

    Techniques Used: Plasmid Preparation, Expressing, Luciferase

    13) Product Images from "Monensin inhibits cell proliferation and tumor growth of chemo-resistant pancreatic cancer cells by targeting the EGFR signaling pathway"

    Article Title: Monensin inhibits cell proliferation and tumor growth of chemo-resistant pancreatic cancer cells by targeting the EGFR signaling pathway

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36214-5

    Monensin inhibits multiple cancer-associated pathways and targets EGFR signaling in human pancreatic cancer cells. ( A ) Effect of monensin on several important cancer-associated pathways. Subconfluent Panc-1 cells were transfected with the indicated GLuc reporter plasmids and treated with monensin at the indicated concentrations. At 24 h ( a ) and 48 h ( b ) post treatment, culture media were collected for Gaussia luciferase activity assay. Each assay condition was done in triplicate. “**” p
    Figure Legend Snippet: Monensin inhibits multiple cancer-associated pathways and targets EGFR signaling in human pancreatic cancer cells. ( A ) Effect of monensin on several important cancer-associated pathways. Subconfluent Panc-1 cells were transfected with the indicated GLuc reporter plasmids and treated with monensin at the indicated concentrations. At 24 h ( a ) and 48 h ( b ) post treatment, culture media were collected for Gaussia luciferase activity assay. Each assay condition was done in triplicate. “**” p

    Techniques Used: Transfection, Luciferase, Activity Assay

    14) Product Images from "Surfactant Protein A Impairs Genital HPV16 Pseudovirus Infection by Innate Immune Cell Activation in A Murine Model"

    Article Title: Surfactant Protein A Impairs Genital HPV16 Pseudovirus Infection by Innate Immune Cell Activation in A Murine Model

    Journal: Pathogens

    doi: 10.3390/pathogens8040288

    SP-A reduces HPV16-PsVs infection in C57BL/6 mice by increasing macrophage recruitment into the basal epithelium. 6–10 weeks old female wildtype C57BL/6 mice were pre-treated as described in Figure 3 A. ( A ) HPV16-PsVs encapsidating the Gaussia luciferase reporter plasmid pGLuc were pre-incubated with a 10-fold (w/w) excess of purified SP-A protein (or BSA control) for 1 h at room temperature, before intravaginal inoculation. Genital tracts were washed with 2 × 50 µL PBS at 24 h, 48 h, and 72 h post infection, and activity of the secreted Gaussia luciferase in the vaginal lavage fluid as a measure for infection was determined. Data of three independent experiments are presented relative to infectivity of the BSA control group at 72 h which was set as 100%. Statistical significance was determined using one-way ANOVA and Bonferroni’s multiple comparison tests for the individual time points. *= p
    Figure Legend Snippet: SP-A reduces HPV16-PsVs infection in C57BL/6 mice by increasing macrophage recruitment into the basal epithelium. 6–10 weeks old female wildtype C57BL/6 mice were pre-treated as described in Figure 3 A. ( A ) HPV16-PsVs encapsidating the Gaussia luciferase reporter plasmid pGLuc were pre-incubated with a 10-fold (w/w) excess of purified SP-A protein (or BSA control) for 1 h at room temperature, before intravaginal inoculation. Genital tracts were washed with 2 × 50 µL PBS at 24 h, 48 h, and 72 h post infection, and activity of the secreted Gaussia luciferase in the vaginal lavage fluid as a measure for infection was determined. Data of three independent experiments are presented relative to infectivity of the BSA control group at 72 h which was set as 100%. Statistical significance was determined using one-way ANOVA and Bonferroni’s multiple comparison tests for the individual time points. *= p

    Techniques Used: Infection, Mouse Assay, Luciferase, Plasmid Preparation, Incubation, Purification, Activity Assay

    Infection of C57BL/6 mice with HPV16-PsVs does not alter SP-A expression. ( A ) Mouse model for HPV16-PsVs infection using C57BL/6 mice, adapted from Roberts et al., 2007 [ 8 , 37 ]. Briefly, 6–10 weeks old female wildtype C57/BL6 mice were injected with 2 mg Depo-Provera (s.c.) for 4 days, and then pre-treated with 25 µL 4% N-9 in 3% CMC i.vag. for 6 h prior to HPV16-PsVs infection. Six mice per group were i.vag. infected with 3 µg or 1 µg viral particles encapsidating the reporter gene firefly luciferase (pGL3-control) or Gaussia luciferase (pGLuc), respectively. After 1–3 days, tissues were harvested for analysis. ( B ) Western blot assessing SP-A expression in lung, bronchoalveolar lavage fluid (BAL), female reproductive tract (FRT) tissue, and vaginal lavage (vag. lavage) fluid of naïve wildtype C57BL/6 mice. 20 µg of each sample was loaded per lane, while 0. 5 µg of purified human SP-A protein was loaded as control. No endogenous SP-A was detected in the FRT of naïve mice. ( C ) Four mice per group were i.vag. infected with 3 µg HPV16-PsVs encapsidating the pGL3-control reporter plasmid. Genital tract tissue was harvested 24 h later and RNA extracted for gene expression analysis to confirm successful infection. ( D ) RNA from ( C ) was assessed for SP-A expression, with lung RNA used as positive control. Gapdh was used as a reference gene. ( E ) Four mice per group were i.vag. infected with 3 µg HPV16-PsVs. Vaginal lavages were performed 24 h and 72 h p.i. and assessed by Western Blot for the presence of SP-A. 20 µg of each sample was loaded per lane, while 0.5 µg purified human SP-A protein was loaded as control. * Loading control corresponds to a 45 kDa cellular protein that cross-reacts with the CamVir primary antibody [ 38 ]. ( F ) Four mice per group were i.vag. infected with 3 µg HPV16-PsVs and genital tract tissue was harvested 24 h or 72 h p.i., homogenised and assessed by Western blot for the presence of SP-A as described in (E).
    Figure Legend Snippet: Infection of C57BL/6 mice with HPV16-PsVs does not alter SP-A expression. ( A ) Mouse model for HPV16-PsVs infection using C57BL/6 mice, adapted from Roberts et al., 2007 [ 8 , 37 ]. Briefly, 6–10 weeks old female wildtype C57/BL6 mice were injected with 2 mg Depo-Provera (s.c.) for 4 days, and then pre-treated with 25 µL 4% N-9 in 3% CMC i.vag. for 6 h prior to HPV16-PsVs infection. Six mice per group were i.vag. infected with 3 µg or 1 µg viral particles encapsidating the reporter gene firefly luciferase (pGL3-control) or Gaussia luciferase (pGLuc), respectively. After 1–3 days, tissues were harvested for analysis. ( B ) Western blot assessing SP-A expression in lung, bronchoalveolar lavage fluid (BAL), female reproductive tract (FRT) tissue, and vaginal lavage (vag. lavage) fluid of naïve wildtype C57BL/6 mice. 20 µg of each sample was loaded per lane, while 0. 5 µg of purified human SP-A protein was loaded as control. No endogenous SP-A was detected in the FRT of naïve mice. ( C ) Four mice per group were i.vag. infected with 3 µg HPV16-PsVs encapsidating the pGL3-control reporter plasmid. Genital tract tissue was harvested 24 h later and RNA extracted for gene expression analysis to confirm successful infection. ( D ) RNA from ( C ) was assessed for SP-A expression, with lung RNA used as positive control. Gapdh was used as a reference gene. ( E ) Four mice per group were i.vag. infected with 3 µg HPV16-PsVs. Vaginal lavages were performed 24 h and 72 h p.i. and assessed by Western Blot for the presence of SP-A. 20 µg of each sample was loaded per lane, while 0.5 µg purified human SP-A protein was loaded as control. * Loading control corresponds to a 45 kDa cellular protein that cross-reacts with the CamVir primary antibody [ 38 ]. ( F ) Four mice per group were i.vag. infected with 3 µg HPV16-PsVs and genital tract tissue was harvested 24 h or 72 h p.i., homogenised and assessed by Western blot for the presence of SP-A as described in (E).

    Techniques Used: Infection, Mouse Assay, Expressing, Injection, Luciferase, Western Blot, Purification, Plasmid Preparation, Positive Control

    15) Product Images from "Differential Methylation of the HPV 16 Upstream Regulatory Region during Epithelial Differentiation and Neoplastic Transformation"

    Article Title: Differential Methylation of the HPV 16 Upstream Regulatory Region during Epithelial Differentiation and Neoplastic Transformation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024451

    Methylation of the E2BS1 increases the p97 promoter activity. A. To measure the impact of methylation of the E2BS1 on the p97 promoter activity site specific modifications within the E2BS1 of LCR HPV16 of pGLuc reporter vector construct were generated using modified oligonucleotides. Three constructs containing either an unmethylated (wt E2BS1), a mutated (mutE2BS1) or a methylated (methE2BS1) E2BS1 were used. B and C. These plasmid constructs were co-transfected into C33A and NHK cells and increasing amount of E2 expression vector. The secreted Gaussia luciferase activities were normalized using the corresponding internal ß-galactosidase activities. Each value represents the mean ±standard deviation of at least three independent transfection experiments, each performed in triplicate. D. To analyze the impact of methylation of E2BS1 on the early promoter activity in the natural context of the HPV 16 genome where E2 expression is under control of the p97 early promoter, NHK were transiently transfected with full length HPV16 genome (wt or methylated E2BS1). E. The effect of selective E2BS1 methylation in full-length HPV16 on E6 gene expression was measured by real-time PCR. Wild type and HPV16 full-length genome and the full length HPV16 genome with 2 selectively methylated CpGs within E2BS1 were transfected into normal human foreskin keratinocytes (NHK.f.). Total RNA from cells 24, 48 and 72 hours after transfection was assayed for the expression of E6 mRNA. Relative luciferase activities were calculated with respect to the values of the wt construct, which was set to 1, for each time point. The data represent the mean of four independent experiments performed with each sample in triplicate with error bars indicating ± S.D. Mean values and SDs were calculated from Sigma Plot 10.0. F. To test whether methylation recruits other binding of transcription factors to the region of E2BS1, we performed EMSA analysis using nuclear cell extracts isolated from NHK. Methylated and unmethylated DNA probes spanning the regions containing the E2BS1 were used. The methylated probes showed three shifted bands by adding nuclear cell extracts (line 4, 6). Unmethylated probes were not shifted by the same nuclear extract (lane 3, 5). Competition experiments were performed with non-labeled probes (lines 7 and 8).
    Figure Legend Snippet: Methylation of the E2BS1 increases the p97 promoter activity. A. To measure the impact of methylation of the E2BS1 on the p97 promoter activity site specific modifications within the E2BS1 of LCR HPV16 of pGLuc reporter vector construct were generated using modified oligonucleotides. Three constructs containing either an unmethylated (wt E2BS1), a mutated (mutE2BS1) or a methylated (methE2BS1) E2BS1 were used. B and C. These plasmid constructs were co-transfected into C33A and NHK cells and increasing amount of E2 expression vector. The secreted Gaussia luciferase activities were normalized using the corresponding internal ß-galactosidase activities. Each value represents the mean ±standard deviation of at least three independent transfection experiments, each performed in triplicate. D. To analyze the impact of methylation of E2BS1 on the early promoter activity in the natural context of the HPV 16 genome where E2 expression is under control of the p97 early promoter, NHK were transiently transfected with full length HPV16 genome (wt or methylated E2BS1). E. The effect of selective E2BS1 methylation in full-length HPV16 on E6 gene expression was measured by real-time PCR. Wild type and HPV16 full-length genome and the full length HPV16 genome with 2 selectively methylated CpGs within E2BS1 were transfected into normal human foreskin keratinocytes (NHK.f.). Total RNA from cells 24, 48 and 72 hours after transfection was assayed for the expression of E6 mRNA. Relative luciferase activities were calculated with respect to the values of the wt construct, which was set to 1, for each time point. The data represent the mean of four independent experiments performed with each sample in triplicate with error bars indicating ± S.D. Mean values and SDs were calculated from Sigma Plot 10.0. F. To test whether methylation recruits other binding of transcription factors to the region of E2BS1, we performed EMSA analysis using nuclear cell extracts isolated from NHK. Methylated and unmethylated DNA probes spanning the regions containing the E2BS1 were used. The methylated probes showed three shifted bands by adding nuclear cell extracts (line 4, 6). Unmethylated probes were not shifted by the same nuclear extract (lane 3, 5). Competition experiments were performed with non-labeled probes (lines 7 and 8).

    Techniques Used: Methylation, Activity Assay, Plasmid Preparation, Construct, Generated, Modification, Transfection, Expressing, Luciferase, Standard Deviation, Real-time Polymerase Chain Reaction, Binding Assay, Isolation, Labeling

    16) Product Images from "Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum"

    Article Title: Surf4 (Erv29p) binds amino-terminal tripeptide motifs of soluble cargo proteins with different affinities, enabling prioritization of their exit from the endoplasmic reticulum

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2005140

    Surf4 is essential for keeping AMELX, DSPP, and GH at low concentrations within ER. (A) CRISPR/Cas9 technology was used to delete SURF4 alleles from HEK293A cells ( Surf4 KO ). Endogenous Surf4 was detected in HEK293A cell lysate (left lane) but not in Surf4 KO cell lysate (center lane). Right lane shows reexpression of Surf4 using plasmid-encoding HA-tagged Surf4 in Surf4 KO . Introduction of HA-tag slightly increased the M r of Surf4. Detection of Surf4 was with affinity-purified rabbit antibody to carboxy-terminal peptide. Lower panel: Detection of β-Actin serves as loading/protease controls. (B) Trafficking of secreted proteins lacking Surf4-binding motifs were unaffected by loss of Surf4. SEAP and LPO-Gluc were equally well secreted from normal and Surf4 KO cells. Conditioned media were harvested 22 hr posttransfection. SEAP secretion was assayed with 5μl of conditioned media using QUANTI-Blue kit. Luciferase activity was determined using 5 μl of conditioned media with BioLux Gaussia Luciferase Assay kit following Assay Protocol II. (Error bars are SEM with a transfection sample size of n = 5 [SEAP] and n = 6 [LPO-Gluc]) (C) AMELX myc starting with MPL (Lane 1) well trafficked out of wild-type cells, but mutant EPL-AMELX (Lane 2) was not. Neither protein was efficiently trafficked out of Surf4 KO cells (Lanes 3 and 4). AMELX was detected using primary antibody to Myc-tag. (D) Trafficking of wild-type AMELX (MPL) in Surf4 KO cells was rescued by coexpression of either HA-Surf4 (Lane 1) or yeast’s Erv29p (Lane 3), but trafficking of EPL-AMELX was not rescued by either cargo receptor (Lanes 2 and 4). Coexpression of Surf4 lacking proposed motif for COPI recycling to ER (HA-Surf4-AAK) also could not rescue trafficking of MPL-AMELX (Lane 5). (E) Trafficking of IPV-DSPP in HEK293A cells (Lane 1) was lost in Surf4 KO cells (Lane 3). There was negligible trafficking of IPD-DSPP in either wild-type (Lane 2) or Surf4 KO cells (Lane 4). (F) Coexpression of HA-Surf4 (Lane 1) or HA-Erv29p (Lane 3) rescued IPV-DSPP trafficking in Surf4 KO cells but not for IPD-DSPP (Lanes 2 and 4). Primary antibody to mDSP domain was used to detect intact DSPP and its DSP fragment. (G) Evidence for aggregate formation by DSPP and AMELX (Myc-tagged) in Surf4 KO cells. Top panel: Surf4 KO cells expressing DSPP were briefly pelleted and then treated for 10 min with buffer containing digitonin (CEB) with (+) or without (-) 10 mM Ca 2+ and pelleted at > 100,000 x g. As observed on western blots, 10 mM Ca 2+ stabilized a portion of DSPP in the pellet fraction. In the bottom panel, AMELX (Myc-tagged) formed stable aggregate in Surf4 KO cells with most remaining in > 100,000 x g pellet after solubilizing cells with an MEB for 10 min. (H) Surf4-trafficked cargo with motifs other than Φ-P-Φ. Trafficking of GH lacking one hydrophobic amino acid (FPT), serine replacing proline at position 2 (ISV), or both lacking the proline, plus replacement of one hydrophobic with a positive-charged amino acid (RSV) were all rescued in Surf4 KO cells upon coexpression of HA-Surf4 protein. Trafficking of di-acidic EET-GH was not rescued by HA-Surf4. (I) LPO-Gluc, noted in Panel B as not using Surf4, acquired lower steady-state levels when wild-type motif QTT was replaced with strong ER-ESCAPE motifs (RSV or IPV). Same proteins expressed in Surf4 KO cells retained their high steady-state levels. Cells were harvested 22 hr posttransfection. The Luciferase activity was normalized to total protein (Luciferase units/mg protein). Error bars are SEM with sample size of n = 6 and P
    Figure Legend Snippet: Surf4 is essential for keeping AMELX, DSPP, and GH at low concentrations within ER. (A) CRISPR/Cas9 technology was used to delete SURF4 alleles from HEK293A cells ( Surf4 KO ). Endogenous Surf4 was detected in HEK293A cell lysate (left lane) but not in Surf4 KO cell lysate (center lane). Right lane shows reexpression of Surf4 using plasmid-encoding HA-tagged Surf4 in Surf4 KO . Introduction of HA-tag slightly increased the M r of Surf4. Detection of Surf4 was with affinity-purified rabbit antibody to carboxy-terminal peptide. Lower panel: Detection of β-Actin serves as loading/protease controls. (B) Trafficking of secreted proteins lacking Surf4-binding motifs were unaffected by loss of Surf4. SEAP and LPO-Gluc were equally well secreted from normal and Surf4 KO cells. Conditioned media were harvested 22 hr posttransfection. SEAP secretion was assayed with 5μl of conditioned media using QUANTI-Blue kit. Luciferase activity was determined using 5 μl of conditioned media with BioLux Gaussia Luciferase Assay kit following Assay Protocol II. (Error bars are SEM with a transfection sample size of n = 5 [SEAP] and n = 6 [LPO-Gluc]) (C) AMELX myc starting with MPL (Lane 1) well trafficked out of wild-type cells, but mutant EPL-AMELX (Lane 2) was not. Neither protein was efficiently trafficked out of Surf4 KO cells (Lanes 3 and 4). AMELX was detected using primary antibody to Myc-tag. (D) Trafficking of wild-type AMELX (MPL) in Surf4 KO cells was rescued by coexpression of either HA-Surf4 (Lane 1) or yeast’s Erv29p (Lane 3), but trafficking of EPL-AMELX was not rescued by either cargo receptor (Lanes 2 and 4). Coexpression of Surf4 lacking proposed motif for COPI recycling to ER (HA-Surf4-AAK) also could not rescue trafficking of MPL-AMELX (Lane 5). (E) Trafficking of IPV-DSPP in HEK293A cells (Lane 1) was lost in Surf4 KO cells (Lane 3). There was negligible trafficking of IPD-DSPP in either wild-type (Lane 2) or Surf4 KO cells (Lane 4). (F) Coexpression of HA-Surf4 (Lane 1) or HA-Erv29p (Lane 3) rescued IPV-DSPP trafficking in Surf4 KO cells but not for IPD-DSPP (Lanes 2 and 4). Primary antibody to mDSP domain was used to detect intact DSPP and its DSP fragment. (G) Evidence for aggregate formation by DSPP and AMELX (Myc-tagged) in Surf4 KO cells. Top panel: Surf4 KO cells expressing DSPP were briefly pelleted and then treated for 10 min with buffer containing digitonin (CEB) with (+) or without (-) 10 mM Ca 2+ and pelleted at > 100,000 x g. As observed on western blots, 10 mM Ca 2+ stabilized a portion of DSPP in the pellet fraction. In the bottom panel, AMELX (Myc-tagged) formed stable aggregate in Surf4 KO cells with most remaining in > 100,000 x g pellet after solubilizing cells with an MEB for 10 min. (H) Surf4-trafficked cargo with motifs other than Φ-P-Φ. Trafficking of GH lacking one hydrophobic amino acid (FPT), serine replacing proline at position 2 (ISV), or both lacking the proline, plus replacement of one hydrophobic with a positive-charged amino acid (RSV) were all rescued in Surf4 KO cells upon coexpression of HA-Surf4 protein. Trafficking of di-acidic EET-GH was not rescued by HA-Surf4. (I) LPO-Gluc, noted in Panel B as not using Surf4, acquired lower steady-state levels when wild-type motif QTT was replaced with strong ER-ESCAPE motifs (RSV or IPV). Same proteins expressed in Surf4 KO cells retained their high steady-state levels. Cells were harvested 22 hr posttransfection. The Luciferase activity was normalized to total protein (Luciferase units/mg protein). Error bars are SEM with sample size of n = 6 and P

    Techniques Used: CRISPR, Plasmid Preparation, Affinity Purification, Binding Assay, Luciferase, Activity Assay, Transfection, Mutagenesis, Expressing, Western Blot

    17) Product Images from "The Kr?ppel-like zinc finger protein Glis3 directly and indirectly activates insulin gene transcription"

    Article Title: The Kr?ppel-like zinc finger protein Glis3 directly and indirectly activates insulin gene transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp122

    Transcriptional activity of Glis3 and Glis3-NDH1. ( A ) A β cell line (832/13) and a non-β cell line (NIH3T3) were co-transfected with RIP-Gluc, the internal control SRα-SEAP, and Glis3 or Glis3-NDH1 expression constructs as indicated. Gaussia luciferase assay was performed at 48 h after transfection. The fold change of RIP promoter activity caused by the c-myc-Glis3 constructs were compared to that of the BOS vector expressing the c-myc tag alone. Results are presented as mean ± SD. ( B ) Dose-dependent induction of RIP-mediated luciferase expression by Glis3. 832/13 cells were co-transfected with RIP-Luc and BOS-c-myc together with increasing amounts of c-myc-Glis3 as indicated. The amount of BOS-c-myc vector was adjusted to maintain the same amount of total DNA in each well. ( C ) Inhibition of Glis3-induced luciferase expression by the Glis3-NDH1 mutant. 832/13 or NIH3T3 cells were co-transfected with RIP-Gluc, c-myc-Glis3 and BOS-c-myc together with increasing amounts of c-myc-Glis3-NDH1 as indicated.
    Figure Legend Snippet: Transcriptional activity of Glis3 and Glis3-NDH1. ( A ) A β cell line (832/13) and a non-β cell line (NIH3T3) were co-transfected with RIP-Gluc, the internal control SRα-SEAP, and Glis3 or Glis3-NDH1 expression constructs as indicated. Gaussia luciferase assay was performed at 48 h after transfection. The fold change of RIP promoter activity caused by the c-myc-Glis3 constructs were compared to that of the BOS vector expressing the c-myc tag alone. Results are presented as mean ± SD. ( B ) Dose-dependent induction of RIP-mediated luciferase expression by Glis3. 832/13 cells were co-transfected with RIP-Luc and BOS-c-myc together with increasing amounts of c-myc-Glis3 as indicated. The amount of BOS-c-myc vector was adjusted to maintain the same amount of total DNA in each well. ( C ) Inhibition of Glis3-induced luciferase expression by the Glis3-NDH1 mutant. 832/13 or NIH3T3 cells were co-transfected with RIP-Gluc, c-myc-Glis3 and BOS-c-myc together with increasing amounts of c-myc-Glis3-NDH1 as indicated.

    Techniques Used: Activity Assay, Transfection, Expressing, Construct, Luciferase, Plasmid Preparation, Inhibition, Mutagenesis

    18) Product Images from "Establishment of a High-Yield Recombinant Adeno-Associated Virus/Human Bocavirus Vector Production System Independent of Bocavirus Nonstructural Proteins"

    Article Title: Establishment of a High-Yield Recombinant Adeno-Associated Virus/Human Bocavirus Vector Production System Independent of Bocavirus Nonstructural Proteins

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2018.173

    Effectiveness of NS-free rAAV2/HBoV1 production system. (a) Comparison of the expression of HBoV1 capsid proteins in transfected HEK293 cells. Expression of VP1, VP2, and VP3 was assessed by Western blot analysis following transfection with codon-optimized HBoV1 cap in helper pcDNAoptVP1(ATG), pcDNAoptVP, or replication-incompetent HBoV1 genome clone pHBoV1KUm630. Cell lysates from the transfections were resolved in same polyacrylamide gel, but several irrelevant lanes were removed. After probing for HBoV1 capsid proteins, the blot was re-probed for β-actin expression. (b and c) Quantitation of vector production. (b) Representative example of a side-by-side comparison of production yields from indicated HBoV1 helper plasmids for forty 150 mm plates of transfected HEK293 cells. (c) Comparison of average yields for NS-free helper pcDNAoptVP and NP1-depedent helper pCMVNS*Cap. Values represent mean ± SEM yield from four side-by-side preparations. (d) Comparison of transduction activities of rAAV2/HBoV1 preparations produced using pcDNAoptVP and pCMVNS*Cap. HAE-ALI cultures apically infected with AV2/HBc-gLuc at an MOI of 10,000 DRP/cell. Values represent mean ± SEM ( n = 4) accumulated gaussia luciferase activity for the previous 24 h period at each time point. (e) Comparison of the content of HBoV1 capsid subunits in virions, as assessed by Western blot analysis. 1.3 × 10 10 DRP of virions were loaded per lane. Lanes 1–3 : virions generated using helper pcDNAoptVP. Lanes 4–6 : virions generated using helper pCMVNS*Cap. VP1, VP2, and VP3 bands were detected using anti-HBoV1 VP3 antibody and visualized by horseradish peroxidase–conjugated anti-rat immunoglobulin G antibody. Note : Anti-HBoV1 VP3 visualized a low-molecular weight band on the blots from the transfection of capsid expression plasmid (a) and also from purified virions (e) . It is unclear if this band is a degradative product or an unknown small VP component incorporated in the virions.
    Figure Legend Snippet: Effectiveness of NS-free rAAV2/HBoV1 production system. (a) Comparison of the expression of HBoV1 capsid proteins in transfected HEK293 cells. Expression of VP1, VP2, and VP3 was assessed by Western blot analysis following transfection with codon-optimized HBoV1 cap in helper pcDNAoptVP1(ATG), pcDNAoptVP, or replication-incompetent HBoV1 genome clone pHBoV1KUm630. Cell lysates from the transfections were resolved in same polyacrylamide gel, but several irrelevant lanes were removed. After probing for HBoV1 capsid proteins, the blot was re-probed for β-actin expression. (b and c) Quantitation of vector production. (b) Representative example of a side-by-side comparison of production yields from indicated HBoV1 helper plasmids for forty 150 mm plates of transfected HEK293 cells. (c) Comparison of average yields for NS-free helper pcDNAoptVP and NP1-depedent helper pCMVNS*Cap. Values represent mean ± SEM yield from four side-by-side preparations. (d) Comparison of transduction activities of rAAV2/HBoV1 preparations produced using pcDNAoptVP and pCMVNS*Cap. HAE-ALI cultures apically infected with AV2/HBc-gLuc at an MOI of 10,000 DRP/cell. Values represent mean ± SEM ( n = 4) accumulated gaussia luciferase activity for the previous 24 h period at each time point. (e) Comparison of the content of HBoV1 capsid subunits in virions, as assessed by Western blot analysis. 1.3 × 10 10 DRP of virions were loaded per lane. Lanes 1–3 : virions generated using helper pcDNAoptVP. Lanes 4–6 : virions generated using helper pCMVNS*Cap. VP1, VP2, and VP3 bands were detected using anti-HBoV1 VP3 antibody and visualized by horseradish peroxidase–conjugated anti-rat immunoglobulin G antibody. Note : Anti-HBoV1 VP3 visualized a low-molecular weight band on the blots from the transfection of capsid expression plasmid (a) and also from purified virions (e) . It is unclear if this band is a degradative product or an unknown small VP component incorporated in the virions.

    Techniques Used: Expressing, Transfection, Western Blot, Quantitation Assay, Plasmid Preparation, Transduction, Produced, Infection, Luciferase, Activity Assay, Generated, Molecular Weight, Purification

    Optimization of NS-free production system. (a) Comparison of production yield and transduction potency. Virions produced using the indicated helper plasmids demonstrated trade-off in production yield versus transduction potency. Values represent mean ± SEM yield (left y -axis) and potency (right y -axis) as percentages relative to those for virion produced using pCMEpoIVNP1Cap-Rep2 (values were set at 100% yield and potency). (b) Schematic illustration of three plasmids used in NS-free production system. HBoV1 capsid proteins VP1, VP2, and VP3 are expressed from the codon-optimized cap genes by using distinct expression plasmids. (c) Comparison of production yield from most efficient NP1-dependent and NS-free production systems. Values represent mean ± SEM yield in three side-by-side preparations using the indicated helper plasmids. (d) Comparison of transduction (luciferase) activities of rAAV2/HBoV1 preparations, as described, for HAE-ALI cultures apically transduced with three side-by-side paired lots of virions, as described in (c) . Transgene expression was assessed on days 2, 4, and 14 post infection. Bars and error bars represent mean ± SEM ( n = 4) gaussia luciferase activity in the culture medium accumulated in 24 h intervals.
    Figure Legend Snippet: Optimization of NS-free production system. (a) Comparison of production yield and transduction potency. Virions produced using the indicated helper plasmids demonstrated trade-off in production yield versus transduction potency. Values represent mean ± SEM yield (left y -axis) and potency (right y -axis) as percentages relative to those for virion produced using pCMEpoIVNP1Cap-Rep2 (values were set at 100% yield and potency). (b) Schematic illustration of three plasmids used in NS-free production system. HBoV1 capsid proteins VP1, VP2, and VP3 are expressed from the codon-optimized cap genes by using distinct expression plasmids. (c) Comparison of production yield from most efficient NP1-dependent and NS-free production systems. Values represent mean ± SEM yield in three side-by-side preparations using the indicated helper plasmids. (d) Comparison of transduction (luciferase) activities of rAAV2/HBoV1 preparations, as described, for HAE-ALI cultures apically transduced with three side-by-side paired lots of virions, as described in (c) . Transgene expression was assessed on days 2, 4, and 14 post infection. Bars and error bars represent mean ± SEM ( n = 4) gaussia luciferase activity in the culture medium accumulated in 24 h intervals.

    Techniques Used: Transduction, Produced, Expressing, Luciferase, Infection, Activity Assay

    19) Product Images from "Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors"

    Article Title: Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00357

    Bloodstream and peripheral isolates produce equivalent activation of NF-kB pathway in human monocytes. THP-1 Lucia® NF-kB cells were treated with the cell-free supernatant of 7 pairs of peripheral and bloodstream isolates. Cells were collected at baseline, 2, 4, and 8 h for quantification of luciferase activity by of Biolux® Gaussia luciferase Assay kit. Values obtained by luminescence were normalized to the protein absorbance of the cell lysate as measured by BioRad® DC protein assay. ns, not significant. Negative control = LB broth, positive control = Pam3CSK4 1μg/ml. Data presented represent mean and SEM of 3 independent experiments.
    Figure Legend Snippet: Bloodstream and peripheral isolates produce equivalent activation of NF-kB pathway in human monocytes. THP-1 Lucia® NF-kB cells were treated with the cell-free supernatant of 7 pairs of peripheral and bloodstream isolates. Cells were collected at baseline, 2, 4, and 8 h for quantification of luciferase activity by of Biolux® Gaussia luciferase Assay kit. Values obtained by luminescence were normalized to the protein absorbance of the cell lysate as measured by BioRad® DC protein assay. ns, not significant. Negative control = LB broth, positive control = Pam3CSK4 1μg/ml. Data presented represent mean and SEM of 3 independent experiments.

    Techniques Used: Activation Assay, Luciferase, Activity Assay, DC Protein Assay, Negative Control, Positive Control

    20) Product Images from "Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression"

    Article Title: Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2008.00619.x

    Induction of cyclin D1 mRNA expression upon Wnt3a stimulation. ( A ) qRT‐PCR was used to quantify cyclin D1 transcripts in mMSC treated with 50–150 ng/ml Wnt3a for 3 days. The cyclin D1 mRNA level of non stimulated cells was set as 100%. ( B ) The supernatant of TCF/LEF‐reporter‐mMSC stimulated with 150 ng/ml Wnt3a for 4 days was analysed for Gaussia luciferase activity by determining the RLU of stimulated cells compared with non stimulated cells. The same cells were lysed and cyclin D1 mRNA expression was monitored by qRT‐PCR. Luciferase activity (RLU) and cyclin D1 expression levels of unstimulated cells were set as 100%. Data are presented as mean ± S.D. of one triplicate experiment that is representative of three independent experiments (* P
    Figure Legend Snippet: Induction of cyclin D1 mRNA expression upon Wnt3a stimulation. ( A ) qRT‐PCR was used to quantify cyclin D1 transcripts in mMSC treated with 50–150 ng/ml Wnt3a for 3 days. The cyclin D1 mRNA level of non stimulated cells was set as 100%. ( B ) The supernatant of TCF/LEF‐reporter‐mMSC stimulated with 150 ng/ml Wnt3a for 4 days was analysed for Gaussia luciferase activity by determining the RLU of stimulated cells compared with non stimulated cells. The same cells were lysed and cyclin D1 mRNA expression was monitored by qRT‐PCR. Luciferase activity (RLU) and cyclin D1 expression levels of unstimulated cells were set as 100%. Data are presented as mean ± S.D. of one triplicate experiment that is representative of three independent experiments (* P

    Techniques Used: Expressing, Quantitative RT-PCR, Luciferase, Activity Assay

    Influence of the Wnt/β‐catenin signal transduction pathway on MT1‐MMP expression and invasion of mMSC. ( A ) mMSC were stimulated with 50–150 ng/ml Wnt3a for 3 days. The mRNA expression level of MT1‐MMP in mMSC stimulated with Wnt3a was determined by qRT‐PCR and compared with that of unstimulated control cells (set as 100%). ( B ) MT1‐MMP mRNA expression was monitored in mMSC transfected with β‐catenin and LRP5 siRNA on days 1 and 4 after transfection. The MT1‐MMP mRNA level of nc‐siRNA‐transfected control cells was set as 100%. ( C ) TCF/LEF‐reporter‐mMSC were stimulated with 150 ng/ml Wnt3a for 4 days. Gaussia luciferase activity measurements were performed using cell culture supernatants. Lysis of the same cells allowed the determination of MT1‐MMP mRNA expression levels of Wnt3a‐stimulated cells in comparison to unstimulated TCF/LEF‐reporter‐mMSC (set as 100%). ( D ) TCF/LEF‐reporter‐mMSC were pre‐treated with different amounts of Wnt3a (0–150 ng/ml) for a duration of 72 hrs and Gaussia luciferase activity was measured in cell culture supernatants (non‐stimulated control cells were set as 100%). ( E ) The same TCF/LEF‐reporter‐mMSC were used to study invasion in the Transwell ® system. mMSC invasion rates through Matrigel were determined after 6 hrs under Wnt3a‐induced stimulatory conditions and compared with those of untreated TCF/LEF‐reporter‐mMSC (set as 100%). ( F ) Three days after transfection with β‐catenin siRNA, mMSC were analysed for their ability to invade Matrigel during a period of 6 hrs. The invasion rate of nc‐siRNA‐transfected mMSC was set as 100%. Data are presented as mean ± S.D. of one triplicate experiment that is representative of three independent experiments (* P
    Figure Legend Snippet: Influence of the Wnt/β‐catenin signal transduction pathway on MT1‐MMP expression and invasion of mMSC. ( A ) mMSC were stimulated with 50–150 ng/ml Wnt3a for 3 days. The mRNA expression level of MT1‐MMP in mMSC stimulated with Wnt3a was determined by qRT‐PCR and compared with that of unstimulated control cells (set as 100%). ( B ) MT1‐MMP mRNA expression was monitored in mMSC transfected with β‐catenin and LRP5 siRNA on days 1 and 4 after transfection. The MT1‐MMP mRNA level of nc‐siRNA‐transfected control cells was set as 100%. ( C ) TCF/LEF‐reporter‐mMSC were stimulated with 150 ng/ml Wnt3a for 4 days. Gaussia luciferase activity measurements were performed using cell culture supernatants. Lysis of the same cells allowed the determination of MT1‐MMP mRNA expression levels of Wnt3a‐stimulated cells in comparison to unstimulated TCF/LEF‐reporter‐mMSC (set as 100%). ( D ) TCF/LEF‐reporter‐mMSC were pre‐treated with different amounts of Wnt3a (0–150 ng/ml) for a duration of 72 hrs and Gaussia luciferase activity was measured in cell culture supernatants (non‐stimulated control cells were set as 100%). ( E ) The same TCF/LEF‐reporter‐mMSC were used to study invasion in the Transwell ® system. mMSC invasion rates through Matrigel were determined after 6 hrs under Wnt3a‐induced stimulatory conditions and compared with those of untreated TCF/LEF‐reporter‐mMSC (set as 100%). ( F ) Three days after transfection with β‐catenin siRNA, mMSC were analysed for their ability to invade Matrigel during a period of 6 hrs. The invasion rate of nc‐siRNA‐transfected mMSC was set as 100%. Data are presented as mean ± S.D. of one triplicate experiment that is representative of three independent experiments (* P

    Techniques Used: Transduction, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Cell Culture, Lysis

    Activation of the Wnt/β‐catenin signal transduction pathway in mMSC. ( A ) mMSC were stimulated with 50 ng/ml recombinant Wnt3a for 2 days, whereas TCF/LEF‐reporter‐mMSC were transiently transfected with pN3‐Wnt3a for endogenous activation of the Wnt pathway and lysed after 4 days. Unstimulated mMSC and pN3‐mock‐transfected TCF/LEF‐reporter‐mMSC served as control. Semiquantitative Western blot analysis was performed to monitor the β‐catenin protein level. β‐actin was used as loading control. ( B ) Gaussia luciferase activity was determined in the supernatants of mMSC stably transfected with the TCF/LEF‐reporter plasmid after stimulation with increasing amounts of Wnt3a for 48 hrs. Luciferase activity of unstimulated mMSC was set as 100%. ( C ) TCF/LEF‐reporter‐mMSC were transiently transfected with pN3‐Wnt3a to analyse reporter activity in the cells after 48 hrs. pN3‐mock‐transfected cells served as a control (set as 100%). Data are presented as mean ± S.D. of one triplicate experiment that is representative of three independent experiments (* P
    Figure Legend Snippet: Activation of the Wnt/β‐catenin signal transduction pathway in mMSC. ( A ) mMSC were stimulated with 50 ng/ml recombinant Wnt3a for 2 days, whereas TCF/LEF‐reporter‐mMSC were transiently transfected with pN3‐Wnt3a for endogenous activation of the Wnt pathway and lysed after 4 days. Unstimulated mMSC and pN3‐mock‐transfected TCF/LEF‐reporter‐mMSC served as control. Semiquantitative Western blot analysis was performed to monitor the β‐catenin protein level. β‐actin was used as loading control. ( B ) Gaussia luciferase activity was determined in the supernatants of mMSC stably transfected with the TCF/LEF‐reporter plasmid after stimulation with increasing amounts of Wnt3a for 48 hrs. Luciferase activity of unstimulated mMSC was set as 100%. ( C ) TCF/LEF‐reporter‐mMSC were transiently transfected with pN3‐Wnt3a to analyse reporter activity in the cells after 48 hrs. pN3‐mock‐transfected cells served as a control (set as 100%). Data are presented as mean ± S.D. of one triplicate experiment that is representative of three independent experiments (* P

    Techniques Used: Activation Assay, Transduction, Recombinant, Transfection, Western Blot, Luciferase, Activity Assay, Stable Transfection, Plasmid Preparation

    21) Product Images from "On-target restoration of a split T cell-engaging antibody for precision immunotherapy"

    Article Title: On-target restoration of a split T cell-engaging antibody for precision immunotherapy

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13196-0

    Biochemical basis for hemibody complementation. a A plasmon resonance chip was coated with V L αCD3-scFvαHLA-A2. V H αCD3-scFvαCD45 was used as an analyte at 100, 200, 400, 800, 1600 (nM) as indicated. Association contact time was 300 s, dissociation contact time 3.600 s at a flow rate of 30 μl/min, data represent one out of three experiments. b FCS autocorrelation functions, normalized to the number of molecules, recorded for 1 nM V L αCD3-scFvαHLA-A2 alone (dark red) and for the same sample in presence of 1 µM V H αCD3-scFvαCD45 (light red) or 100 nM HLA-A2 antigen (blue). Curves are fitted using data derived from three independent experiments c Top: Equilibrium binding study of scFv-GpL ( Gaussia princeps luciferase) fusion proteins specific for CD45, HLA-A2, or CD3 to the respective antigens on 10 5 Jurkat (CD3 + , CD45 + ) and U266 cells (HLA-A2 + , CD45 + ). Specific binding (triangles, solid line) was calculated as the difference of total (circles, dashed line) and non-specific binding (squares, dashed line) determined by using an irrelevant scFv-GpL fusion protein, HLA-A2-negative KMS-12-BM cells or CD3-negative U266 cells as indicated. Bottom: For heterologous competition analysis, cells were incubated with scFvCD45-GpL (2 nM), scFvHLA-A2-GpL (2 nM), or scFvCD3-GpL (4 nM) and the indicated concentrations of the hemibodies or the bispecific BiTE construct. IC 50 values were determined and used to calculate the K i of CD45-, HLA-A2-, and CD3-specific scFv domains by help of the previously determined K D -values of the scFv-GpL fusion proteins. Data show one out of three experiments yielding comparable results. d scFvCD3 or isolated V H CD3 and V L CD3 domains were fused to GpL. No specific binding of individual or combined constructs to CD3-positive Jurkat cells were detected as assessed by cell-bound luciferase activity. Data show one out of three experiments yielding comparable results. e Preservation of T cell triggering activity after incubation of hemibodies and BiTEs in human serum for 72 h at 37 °C. Read out was specific lysis at E:T of 5:1. Data represent the mean (±SD) from three replicate wells from one out of three experiments yielding comparable results. f In vivo serum half-lives after intravenous injection of 8 µg of a hemibody or BiTE construct into Balb/c mice. Values are deduced from > 40 mice as determined by ELISA.
    Figure Legend Snippet: Biochemical basis for hemibody complementation. a A plasmon resonance chip was coated with V L αCD3-scFvαHLA-A2. V H αCD3-scFvαCD45 was used as an analyte at 100, 200, 400, 800, 1600 (nM) as indicated. Association contact time was 300 s, dissociation contact time 3.600 s at a flow rate of 30 μl/min, data represent one out of three experiments. b FCS autocorrelation functions, normalized to the number of molecules, recorded for 1 nM V L αCD3-scFvαHLA-A2 alone (dark red) and for the same sample in presence of 1 µM V H αCD3-scFvαCD45 (light red) or 100 nM HLA-A2 antigen (blue). Curves are fitted using data derived from three independent experiments c Top: Equilibrium binding study of scFv-GpL ( Gaussia princeps luciferase) fusion proteins specific for CD45, HLA-A2, or CD3 to the respective antigens on 10 5 Jurkat (CD3 + , CD45 + ) and U266 cells (HLA-A2 + , CD45 + ). Specific binding (triangles, solid line) was calculated as the difference of total (circles, dashed line) and non-specific binding (squares, dashed line) determined by using an irrelevant scFv-GpL fusion protein, HLA-A2-negative KMS-12-BM cells or CD3-negative U266 cells as indicated. Bottom: For heterologous competition analysis, cells were incubated with scFvCD45-GpL (2 nM), scFvHLA-A2-GpL (2 nM), or scFvCD3-GpL (4 nM) and the indicated concentrations of the hemibodies or the bispecific BiTE construct. IC 50 values were determined and used to calculate the K i of CD45-, HLA-A2-, and CD3-specific scFv domains by help of the previously determined K D -values of the scFv-GpL fusion proteins. Data show one out of three experiments yielding comparable results. d scFvCD3 or isolated V H CD3 and V L CD3 domains were fused to GpL. No specific binding of individual or combined constructs to CD3-positive Jurkat cells were detected as assessed by cell-bound luciferase activity. Data show one out of three experiments yielding comparable results. e Preservation of T cell triggering activity after incubation of hemibodies and BiTEs in human serum for 72 h at 37 °C. Read out was specific lysis at E:T of 5:1. Data represent the mean (±SD) from three replicate wells from one out of three experiments yielding comparable results. f In vivo serum half-lives after intravenous injection of 8 µg of a hemibody or BiTE construct into Balb/c mice. Values are deduced from > 40 mice as determined by ELISA.

    Techniques Used: Chromatin Immunoprecipitation, Flow Cytometry, Derivative Assay, Binding Assay, Luciferase, Incubation, Construct, Isolation, Activity Assay, Preserving, Lysis, In Vivo, Injection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    22) Product Images from "Establishment and Characterization of the Reversibly Immortalized Mouse Fetal Heart Progenitors"

    Article Title: Establishment and Characterization of the Reversibly Immortalized Mouse Fetal Heart Progenitors

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.6639

    Characterization of the differentiation potential of iCP15 clones. ( A ) Construction of the MyHC-GLuc reporter. A 5.5kb α-myosin heavy chain promoter-driven Gaussia luciferase reporter (MyHC-GLuc) is schematically shown. The promoter region also contains the first three exons (white boxes). ( B ) Retinoic acid induction of MyHC-GLuc reporter activity in iCP15 clones. Cells of the selected eight iCP15 clones were transfected with pMyHC-GLuc in T-25 flasks for 16-20h, replated in 12-well plates and treated with all-trans retinoic acid (1μM) or DMSO. Culture media were collected at the indicated time points for GLuc assay (NEB). Each assay condition was carried out in triplicate. Relative GLuc activity was expressed as mean ± SD. Fold of changes was calculated by dividing the RA-treated group with DMSO control group for each clone/time point. “*” denotes the fold changes over control were statistically significant ( p
    Figure Legend Snippet: Characterization of the differentiation potential of iCP15 clones. ( A ) Construction of the MyHC-GLuc reporter. A 5.5kb α-myosin heavy chain promoter-driven Gaussia luciferase reporter (MyHC-GLuc) is schematically shown. The promoter region also contains the first three exons (white boxes). ( B ) Retinoic acid induction of MyHC-GLuc reporter activity in iCP15 clones. Cells of the selected eight iCP15 clones were transfected with pMyHC-GLuc in T-25 flasks for 16-20h, replated in 12-well plates and treated with all-trans retinoic acid (1μM) or DMSO. Culture media were collected at the indicated time points for GLuc assay (NEB). Each assay condition was carried out in triplicate. Relative GLuc activity was expressed as mean ± SD. Fold of changes was calculated by dividing the RA-treated group with DMSO control group for each clone/time point. “*” denotes the fold changes over control were statistically significant ( p

    Techniques Used: Clone Assay, Luciferase, Activity Assay, Transfection

    23) Product Images from "Vascular CXCR4 limits atherosclerosis by maintaining arterial integrity: evidence from mouse and human studies"

    Article Title: Vascular CXCR4 limits atherosclerosis by maintaining arterial integrity: evidence from mouse and human studies

    Journal: Circulation

    doi: 10.1161/CIRCULATIONAHA.117.027646

    The CXCL12/CXCR4 axis sustains endothelial barrier function through WNT/β-catenin signaling regulating VE-cadherin (A) Experimental scheme. (B) WNT activity in hAoECs as determined in a Gaussia luciferase WNT reporter assay, after stimulation with CXCL12 (100 ng/ml) for indicated periods. Cells were pretreated by the CXCR4 antagonist AMD3100 (1 µg/ml) or CXCR4 knock-down was performed using siRNA (CXCR4-kd). WNT activation was blocked by the WNT inhibitor endo-IWR1 (10 µM) (n=3; 1-way ANOVA with Sidak’s multiple comparison test). (C) β-Catenin protein levels in cytoplasmic and nuclear extracts of hAoECs after stimulation with CXCL12 (100 ng/ml) for 24 h. Cells were pretreated with endo-IWR1 (10 µM). Silencing of CXCR4 was performed using siRNA (CXCR4-kd). β-Catenin levels were normalized to cytoplasmic (tubulin) and nuclear (lamin β1) loading controls, respectively, and presented relative to untreated controls, as quantified by densitometry (n=3; 1-way ANOVA with Tukey’s multiple comparison test). A representative western blot and densitometry quantification is shown (left panel). (D) Permeability of hAoECs to FITC-dextran after stimulation with WNT3a (200 ng/ml) or the WNT activator LiCl (30 mM) for 24 h (n=5-10 wells from 4 independent experiments; Kruskal-Wallis test with Dunn’s post-test). (E) Permeability of hAoECs to FITC-dextran after stimulation with WNT3a (200 ng/ml) or CXCL12 (100 ng/ml) for 24 h and pre-treatment with endo-IWR1 (1 µM) for 1 h, as indicated (n=7-9 wells from 3 independent experiments; 1-way ANOVA with Tukey’s multiple comparison test). (F) VE-cadherin protein levels in cytoplasmic extracts of hAoECs after stimulation with CXCL12 (100 ng/ml) for 24 h and treatment with endo-IWR1 (10 µM), as indicated. Knock-down of CXCR4 was performed using siRNA (CXCR4-kd). VE-cadherin expression was normalized to tubulin and presented relative to untreated, as quantified by densitometry (n=3; 1-way ANOVA with Tukey’s multiple comparison test). A representative Western blot and densitometry quantification is shown. Controls received control siRNA (B,C,F) . (G) Quantification of VE-cadherin expression in carotid artery lysates of BmxCre + vs BmxCre - Cxcr4 fl/fl Apoe −/− mice (n=3; Mann-Whitney test). (H) Permeability of hAoECs to FITC-dextran after stimulation with CXCL12 (100 ng/ml) or WNT3a (200 ng/ml) for 24 h and blockade of SHP2 (5 µM) or PTP1B (25 µM) for 1 h, as indicated (n=8-12 wells from 4 independent experiments; Kruskal-Wallis test with Dunn’s post-test). (I-K) Vascular permeability to Evans blue induced by histamine (10 µg i.v., 10 min) in VE-PTP-FRB + /VE-cadherin-FKBP C57/Bl6 knock-in mice pretreated with the CXCR4 antagonist AMD3465 (125 µg) for 12 h and/or Rapalog (250 µg) for 4 h or vehicle, as indicated (n=8-16; 2-way ANOVA with Bonferroni post-test). (A-K) Data present mean±SD ( A-J ) or mean±SEM ( K ). * P
    Figure Legend Snippet: The CXCL12/CXCR4 axis sustains endothelial barrier function through WNT/β-catenin signaling regulating VE-cadherin (A) Experimental scheme. (B) WNT activity in hAoECs as determined in a Gaussia luciferase WNT reporter assay, after stimulation with CXCL12 (100 ng/ml) for indicated periods. Cells were pretreated by the CXCR4 antagonist AMD3100 (1 µg/ml) or CXCR4 knock-down was performed using siRNA (CXCR4-kd). WNT activation was blocked by the WNT inhibitor endo-IWR1 (10 µM) (n=3; 1-way ANOVA with Sidak’s multiple comparison test). (C) β-Catenin protein levels in cytoplasmic and nuclear extracts of hAoECs after stimulation with CXCL12 (100 ng/ml) for 24 h. Cells were pretreated with endo-IWR1 (10 µM). Silencing of CXCR4 was performed using siRNA (CXCR4-kd). β-Catenin levels were normalized to cytoplasmic (tubulin) and nuclear (lamin β1) loading controls, respectively, and presented relative to untreated controls, as quantified by densitometry (n=3; 1-way ANOVA with Tukey’s multiple comparison test). A representative western blot and densitometry quantification is shown (left panel). (D) Permeability of hAoECs to FITC-dextran after stimulation with WNT3a (200 ng/ml) or the WNT activator LiCl (30 mM) for 24 h (n=5-10 wells from 4 independent experiments; Kruskal-Wallis test with Dunn’s post-test). (E) Permeability of hAoECs to FITC-dextran after stimulation with WNT3a (200 ng/ml) or CXCL12 (100 ng/ml) for 24 h and pre-treatment with endo-IWR1 (1 µM) for 1 h, as indicated (n=7-9 wells from 3 independent experiments; 1-way ANOVA with Tukey’s multiple comparison test). (F) VE-cadherin protein levels in cytoplasmic extracts of hAoECs after stimulation with CXCL12 (100 ng/ml) for 24 h and treatment with endo-IWR1 (10 µM), as indicated. Knock-down of CXCR4 was performed using siRNA (CXCR4-kd). VE-cadherin expression was normalized to tubulin and presented relative to untreated, as quantified by densitometry (n=3; 1-way ANOVA with Tukey’s multiple comparison test). A representative Western blot and densitometry quantification is shown. Controls received control siRNA (B,C,F) . (G) Quantification of VE-cadherin expression in carotid artery lysates of BmxCre + vs BmxCre - Cxcr4 fl/fl Apoe −/− mice (n=3; Mann-Whitney test). (H) Permeability of hAoECs to FITC-dextran after stimulation with CXCL12 (100 ng/ml) or WNT3a (200 ng/ml) for 24 h and blockade of SHP2 (5 µM) or PTP1B (25 µM) for 1 h, as indicated (n=8-12 wells from 4 independent experiments; Kruskal-Wallis test with Dunn’s post-test). (I-K) Vascular permeability to Evans blue induced by histamine (10 µg i.v., 10 min) in VE-PTP-FRB + /VE-cadherin-FKBP C57/Bl6 knock-in mice pretreated with the CXCR4 antagonist AMD3465 (125 µg) for 12 h and/or Rapalog (250 µg) for 4 h or vehicle, as indicated (n=8-16; 2-way ANOVA with Bonferroni post-test). (A-K) Data present mean±SD ( A-J ) or mean±SEM ( K ). * P

    Techniques Used: Activity Assay, Luciferase, Reporter Assay, Activation Assay, Western Blot, Permeability, Expressing, Mouse Assay, MANN-WHITNEY, Knock-In

    24) Product Images from "Anthelmintic mebendazole enhances cisplatin's effect on suppressing cell proliferation and promotes differentiation of head and neck squamous cell carcinoma (HNSCC)"

    Article Title: Anthelmintic mebendazole enhances cisplatin's effect on suppressing cell proliferation and promotes differentiation of head and neck squamous cell carcinoma (HNSCC)

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14673

    MBZ affects multiple cancer-related signaling pathways differently in CAL27 and SCC15 lines ( A ) MBZ activates multiple cancer-related signaling pathways in CAL27 cells. Subconfluent CAL27 cells were transfected with the indicated eight pathway reporters and treated with different concentrations of MBZ for 24 h. The Gaussia luciferase reporter activities were assessed (a). The four activated pathway reporters were further analyzed at 48 h and 72 h (b). ( B ) In SCC15 cells MBZ inhibits the four cancer-related signaling pathways that are activated in CAL27 cells. Subconfluent SCC15 cells were transfected with the indicated four pathway reporters and treated with different concentrations of MBZ. The Gaussia luciferase activities were assessed at 24 h, 48 h and 72 h. * p
    Figure Legend Snippet: MBZ affects multiple cancer-related signaling pathways differently in CAL27 and SCC15 lines ( A ) MBZ activates multiple cancer-related signaling pathways in CAL27 cells. Subconfluent CAL27 cells were transfected with the indicated eight pathway reporters and treated with different concentrations of MBZ for 24 h. The Gaussia luciferase reporter activities were assessed (a). The four activated pathway reporters were further analyzed at 48 h and 72 h (b). ( B ) In SCC15 cells MBZ inhibits the four cancer-related signaling pathways that are activated in CAL27 cells. Subconfluent SCC15 cells were transfected with the indicated four pathway reporters and treated with different concentrations of MBZ. The Gaussia luciferase activities were assessed at 24 h, 48 h and 72 h. * p

    Techniques Used: Transfection, Luciferase

    25) Product Images from "Androgen Receptor Requires JunD as a Co-activator to Switch on an Oxidative Stress Generation Pathway in Prostate Cancer Cells"

    Article Title: Androgen Receptor Requires JunD as a Co-activator to Switch on an Oxidative Stress Generation Pathway in Prostate Cancer Cells

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-09-3596

    Western blot and bioluminescence analysis demonstrating expression of Gluc1-AR and JunD-Gluc2 and reconstitution of Gaussia luciferase activity following androgen stimulation in transfected Hep3B cells
    Figure Legend Snippet: Western blot and bioluminescence analysis demonstrating expression of Gluc1-AR and JunD-Gluc2 and reconstitution of Gaussia luciferase activity following androgen stimulation in transfected Hep3B cells

    Techniques Used: Western Blot, Expressing, Luciferase, Activity Assay, Transfection

    26) Product Images from "Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension"

    Article Title: Hydrogel Design for Supporting Neurite Outgrowth and Promoting Gene Delivery to Maximize Neurite Extension

    Journal: Biotechnology and Bioengineering

    doi: 10.1002/bit.24355

    Expression levels of HT-1080 cells with varying hydrogel PEG content and adhesion. Transfection within hydrogels containing 0, 2.5, or 5 mM, each encapsulating 0.13 μg/μL DNA encoding for Gaussia Luciferase, were monitored once per day up to 5 days for 7.5% (a) and 10% PEG (b). Statistical differences between conditions at each time point is indicated by different letters (A or B) based on ANOVA with p
    Figure Legend Snippet: Expression levels of HT-1080 cells with varying hydrogel PEG content and adhesion. Transfection within hydrogels containing 0, 2.5, or 5 mM, each encapsulating 0.13 μg/μL DNA encoding for Gaussia Luciferase, were monitored once per day up to 5 days for 7.5% (a) and 10% PEG (b). Statistical differences between conditions at each time point is indicated by different letters (A or B) based on ANOVA with p

    Techniques Used: Expressing, Transfection, Luciferase

    27) Product Images from "Rapid, Specific Detection of Alphaviruses from Tissue Cultures Using a Replicon-Defective Reporter Gene Assay"

    Article Title: Rapid, Specific Detection of Alphaviruses from Tissue Cultures Using a Replicon-Defective Reporter Gene Assay

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033007

    EGFP and GLuc expression in replicon-transfected cells. BHK-21 cells were transfected with pVaXJ-EGFP, pVaXJ-EGFPΔnsp4, pVaXJ-GLuc, or pVaXJ-GLucΔnsp4 using Lipofectamine 2000 reagent. (A), (B) EGFP expression was examined 48 h post-transfection using an Olympus IX51 fluorescence microscope. Green color indicates EGFP, and blue color indicates nucleus. (C) GLuc activity was measured at different time points (from 18–170 h) post-transfection using the BioLux™ Gaussia Luciferase Assay Kit and a luminometer. Each data point represents the mean ±SEM of three independent experiments. RLU, relative light units.
    Figure Legend Snippet: EGFP and GLuc expression in replicon-transfected cells. BHK-21 cells were transfected with pVaXJ-EGFP, pVaXJ-EGFPΔnsp4, pVaXJ-GLuc, or pVaXJ-GLucΔnsp4 using Lipofectamine 2000 reagent. (A), (B) EGFP expression was examined 48 h post-transfection using an Olympus IX51 fluorescence microscope. Green color indicates EGFP, and blue color indicates nucleus. (C) GLuc activity was measured at different time points (from 18–170 h) post-transfection using the BioLux™ Gaussia Luciferase Assay Kit and a luminometer. Each data point represents the mean ±SEM of three independent experiments. RLU, relative light units.

    Techniques Used: Expressing, Transfection, Fluorescence, Microscopy, Activity Assay, Luciferase

    28) Product Images from "Balancing Cell Migration with Matrix Degradation Enhances Gene Delivery to Cells Cultured Three-Dimensionally Within Hydrogels"

    Article Title: Balancing Cell Migration with Matrix Degradation Enhances Gene Delivery to Cells Cultured Three-Dimensionally Within Hydrogels

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    doi: 10.1016/j.jconrel.2010.04.032

    Cell migration. HT-1080 (A, B) or NIH/3T3 (C, D) cells were encapsulated within PEG hydrogels containing 0, 2, or 5 mM RGD. Cells were entrapped within a fibrin clot that was encapsulated within 7.5% (A, C) or 10% (B, D) PEG hydrogels. Hydrogels contained 4 μg DNA encoding for Gaussia Luciferase. Significant difference between conditions at each time point is indicated by different letters (A, B, or C) based on ANOVA with p
    Figure Legend Snippet: Cell migration. HT-1080 (A, B) or NIH/3T3 (C, D) cells were encapsulated within PEG hydrogels containing 0, 2, or 5 mM RGD. Cells were entrapped within a fibrin clot that was encapsulated within 7.5% (A, C) or 10% (B, D) PEG hydrogels. Hydrogels contained 4 μg DNA encoding for Gaussia Luciferase. Significant difference between conditions at each time point is indicated by different letters (A, B, or C) based on ANOVA with p

    Techniques Used: Migration, Luciferase

    Gene expression profiles. HT-1080 (A, B) or NIH/3T3 (C, D) cells were encapsulated within PEG hydrogels containing 0, 2, or 5 mM RGD. Cells were entrapped within a fibrin clot that was encapsulated within 7.5% PEG (A, C) or 10% (B, D) PEG hydrogels. Hydrogels contained 4 μg DNA encoding for Gaussia Luciferase. Significant difference between conditions at each time point is indicated by different letters (A, B, or C) based on ANOVA with p
    Figure Legend Snippet: Gene expression profiles. HT-1080 (A, B) or NIH/3T3 (C, D) cells were encapsulated within PEG hydrogels containing 0, 2, or 5 mM RGD. Cells were entrapped within a fibrin clot that was encapsulated within 7.5% PEG (A, C) or 10% (B, D) PEG hydrogels. Hydrogels contained 4 μg DNA encoding for Gaussia Luciferase. Significant difference between conditions at each time point is indicated by different letters (A, B, or C) based on ANOVA with p

    Techniques Used: Expressing, Luciferase

    29) Product Images from "HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations"

    Article Title: HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations

    Journal: eLife

    doi: 10.7554/eLife.34271

    Dually tagged HIV-1 BG505 antibodies are neutralized by trimer specific antibodies. Neutralization of HIV-1 BG505_WT (black) and dually labeled BG505_V1Q3_V4A1 (purple) by the broadly neutralizing antibodies ( A ) PG9 and ( B ) PG16. X-axis depicts increasing concentration of antibodies (μg/ml) and the y-axis shows the relative infectivity compared to control in the absence of ligands. Infectivity was measured using the Gaussia Luciferase assay.
    Figure Legend Snippet: Dually tagged HIV-1 BG505 antibodies are neutralized by trimer specific antibodies. Neutralization of HIV-1 BG505_WT (black) and dually labeled BG505_V1Q3_V4A1 (purple) by the broadly neutralizing antibodies ( A ) PG9 and ( B ) PG16. X-axis depicts increasing concentration of antibodies (μg/ml) and the y-axis shows the relative infectivity compared to control in the absence of ligands. Infectivity was measured using the Gaussia Luciferase assay.

    Techniques Used: Neutralization, Labeling, Concentration Assay, Infection, Luciferase

    Infectivity and Env incorporation of single or dually tagged HIV-1 BG505 viruses. Q3 (GQQQLG) or A1 (GDSLDMLLEWSLM) tags were inserted separately or together into the V1 and V4 loops of full-length Q23_BG505 virus. ( A ) Infectivity was measured from three independent experiments by Gaussia Luciferase assay and normalized to WT (%). ( B ) Env incorporation into virions was detected by Western blotting using an antiserum to HIV-1 gp120 (NIH AIDS Reagent Program) well as an anti-p24 antibody.
    Figure Legend Snippet: Infectivity and Env incorporation of single or dually tagged HIV-1 BG505 viruses. Q3 (GQQQLG) or A1 (GDSLDMLLEWSLM) tags were inserted separately or together into the V1 and V4 loops of full-length Q23_BG505 virus. ( A ) Infectivity was measured from three independent experiments by Gaussia Luciferase assay and normalized to WT (%). ( B ) Env incorporation into virions was detected by Western blotting using an antiserum to HIV-1 gp120 (NIH AIDS Reagent Program) well as an anti-p24 antibody.

    Techniques Used: Infection, Luciferase, Western Blot

    30) Product Images from "Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1"

    Article Title: Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

    Journal: Cellular microbiology

    doi: 10.1111/j.1462-5822.2010.01482.x

    Truncated GBF1 mutants do not rescue cellular secretion from BFA inhibition A. Schematic map of GBF1 domain organization and of truncated mutants used in this study. Numbers in parenthesis indicate GBF1 amino acids. B. Cells were co-transfected with pCMV-Gluc vector expressing secreted Gaussia luciferase and with vectors expressing full length YFP-GBF1A795E, truncated GBF1 fusions, or an empty vector (control). Cells were incubated for 5 h in the presence of 1 μg/ml BFA or corresponding amount of DMSO. The luciferase activity observed in each sample without BFA was defined as 100%. Western blots show expression of GBF1 species detected with anti-GFP antibodies. Actin immunoblots are shown as a loading control.
    Figure Legend Snippet: Truncated GBF1 mutants do not rescue cellular secretion from BFA inhibition A. Schematic map of GBF1 domain organization and of truncated mutants used in this study. Numbers in parenthesis indicate GBF1 amino acids. B. Cells were co-transfected with pCMV-Gluc vector expressing secreted Gaussia luciferase and with vectors expressing full length YFP-GBF1A795E, truncated GBF1 fusions, or an empty vector (control). Cells were incubated for 5 h in the presence of 1 μg/ml BFA or corresponding amount of DMSO. The luciferase activity observed in each sample without BFA was defined as 100%. Western blots show expression of GBF1 species detected with anti-GFP antibodies. Actin immunoblots are shown as a loading control.

    Techniques Used: Inhibition, Transfection, Plasmid Preparation, Expressing, Luciferase, Incubation, Activity Assay, Western Blot

    31) Product Images from "Dynamics of papillomavirus in vivo disease formation susceptibility to high-level disinfection—Implications for transmission in clinical settings"

    Article Title: Dynamics of papillomavirus in vivo disease formation susceptibility to high-level disinfection—Implications for transmission in clinical settings

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2020.103177

    Quantification of virus infectious titre in vitro (a, b) Measurement of E1^E4 viral gene transcripts or Gaussia luciferase reporter gene activity in HaCaT cells infected with differing doses of HPV18 (red), MmuPV1 (green) and PsV (black). Infectious titre shown with 5-6 log 10 dynamic range. Neutralising assay compares virus titre following incubation with the neutralising antibody or isotypic control. AU, arbitrary unit; NT, not tested; ND, not detected. Data is obtained with biological triplicates and shown as Mean and SD. (c, d) RNAscope® visualisation of E6 and E7 viral gene transcripts in HaCaT cells infected with HPV18 (top) or MmuPV1 (bottom); virus titre shown as VGE/cell; proportion of virus-RNA positive cells (cell boundary in red) given as %.; boxed regions enlarged in (d). Scale bar; 100 μm
    Figure Legend Snippet: Quantification of virus infectious titre in vitro (a, b) Measurement of E1^E4 viral gene transcripts or Gaussia luciferase reporter gene activity in HaCaT cells infected with differing doses of HPV18 (red), MmuPV1 (green) and PsV (black). Infectious titre shown with 5-6 log 10 dynamic range. Neutralising assay compares virus titre following incubation with the neutralising antibody or isotypic control. AU, arbitrary unit; NT, not tested; ND, not detected. Data is obtained with biological triplicates and shown as Mean and SD. (c, d) RNAscope® visualisation of E6 and E7 viral gene transcripts in HaCaT cells infected with HPV18 (top) or MmuPV1 (bottom); virus titre shown as VGE/cell; proportion of virus-RNA positive cells (cell boundary in red) given as %.; boxed regions enlarged in (d). Scale bar; 100 μm

    Techniques Used: In Vitro, Luciferase, Activity Assay, Infection, Incubation

    32) Product Images from "Hypoxia pathway and hypoxia-mediated extensive extramedullary hematopoiesis are involved in ursolic acid's anti-metastatic effect in 4T1 tumor bearing mice"

    Article Title: Hypoxia pathway and hypoxia-mediated extensive extramedullary hematopoiesis are involved in ursolic acid's anti-metastatic effect in 4T1 tumor bearing mice

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12375

    Effect of UA in hypoxia pathway in MDA-MB-231 cells ( A ) Cell was infected with AdRFP, AdRHIF-1α or AdRsiHIF-1α virus for 16 h, followed by UA treatment in 1% FBS DMEM medium for 48 h, cell survival was tested by crystal violet assay. ( B ) Cell stained with crystal violet (show in Figure 6A ) were dissolved with 20% acetic acid and measured at 570 nm. ( C ) MDA-MB-231 -HIF-1α-GLuc cells were infected with AdRFP, AdRHIF-1α or AdRVEGF and treated with UA in serum free DMEM medium for 2 hour. The Gaussia luciferase (GLuc) activity of the cell medium was measured by the Gaussia Luciferase Assay kit. The inhibition was presented as the percentage of the control group.
    Figure Legend Snippet: Effect of UA in hypoxia pathway in MDA-MB-231 cells ( A ) Cell was infected with AdRFP, AdRHIF-1α or AdRsiHIF-1α virus for 16 h, followed by UA treatment in 1% FBS DMEM medium for 48 h, cell survival was tested by crystal violet assay. ( B ) Cell stained with crystal violet (show in Figure 6A ) were dissolved with 20% acetic acid and measured at 570 nm. ( C ) MDA-MB-231 -HIF-1α-GLuc cells were infected with AdRFP, AdRHIF-1α or AdRVEGF and treated with UA in serum free DMEM medium for 2 hour. The Gaussia luciferase (GLuc) activity of the cell medium was measured by the Gaussia Luciferase Assay kit. The inhibition was presented as the percentage of the control group.

    Techniques Used: Multiple Displacement Amplification, Infection, Crystal Violet Assay, Staining, Luciferase, Activity Assay, Inhibition

    33) Product Images from "Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus"

    Article Title: Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004166

    K22 affects replication of diverse coronaviruses including MERS-CoV. ( A-D ) The log reduction of the antiviral activity (bars) and cell toxicity ratio (data points above bars) of K22 during MHV-Gluc ( A ), FCoV-RL ( B ), SARS-CoV ( C ) and IBV ( D ) infection on representative continuous cell lines of murine (L-929 cells; A ), feline (FCWF cells; B ), or primate (Vero cells; C-D ) origin. Data are shown as mean (±SD) of a representative experiment, from two independent experiments performed in triplicate. Toxicity values for Vero cells in panels C and D are derived from the same experiments. ( E-F ). The log reduction of the antiviral activity (bars) and cell toxicity ratio (data points above bars) of K22 in HCoV-229E-ren ( E ) and MERS-CoV ( F ) infected differentiated human airway epithelial (HAE) cultures. Data are shown as mean (±SD) of three independent experiments performed in triplicate (log reduction), or mean (±SD) of a representative experiment, from two independent experiments performed in triplicate (cell viability). ( G-H ) Immunofluorescence analysis of HAE cultures infected with MERS-CoV in presence or absence of K22 in a representative overview ( G , 20x; H , 40x) confocal Z-stack image. Stainings were performed using antibodies directed against ( G ) dsRNA (green), and DAPI (cell nucleus; blue), and ( H ) dsRNA, DAPI, β-tubulin (ciliated cells; white), and ZO1 (tight junctions, red). Scale bars are 50 ( G ) or 20 ( H ) µm.
    Figure Legend Snippet: K22 affects replication of diverse coronaviruses including MERS-CoV. ( A-D ) The log reduction of the antiviral activity (bars) and cell toxicity ratio (data points above bars) of K22 during MHV-Gluc ( A ), FCoV-RL ( B ), SARS-CoV ( C ) and IBV ( D ) infection on representative continuous cell lines of murine (L-929 cells; A ), feline (FCWF cells; B ), or primate (Vero cells; C-D ) origin. Data are shown as mean (±SD) of a representative experiment, from two independent experiments performed in triplicate. Toxicity values for Vero cells in panels C and D are derived from the same experiments. ( E-F ). The log reduction of the antiviral activity (bars) and cell toxicity ratio (data points above bars) of K22 in HCoV-229E-ren ( E ) and MERS-CoV ( F ) infected differentiated human airway epithelial (HAE) cultures. Data are shown as mean (±SD) of three independent experiments performed in triplicate (log reduction), or mean (±SD) of a representative experiment, from two independent experiments performed in triplicate (cell viability). ( G-H ) Immunofluorescence analysis of HAE cultures infected with MERS-CoV in presence or absence of K22 in a representative overview ( G , 20x; H , 40x) confocal Z-stack image. Stainings were performed using antibodies directed against ( G ) dsRNA (green), and DAPI (cell nucleus; blue), and ( H ) dsRNA, DAPI, β-tubulin (ciliated cells; white), and ZO1 (tight junctions, red). Scale bars are 50 ( G ) or 20 ( H ) µm.

    Techniques Used: Activity Assay, Infection, Derivative Assay, Immunofluorescence

    34) Product Images from "Adaptive Evolution of a Tagged Chimeric Gammaretrovirus: Identification of Novel cis-Acting Elements That Modulate Splicing"

    Article Title: Adaptive Evolution of a Tagged Chimeric Gammaretrovirus: Identification of Novel cis-Acting Elements That Modulate Splicing

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2007.04.026

    Comparison of the transcriptional activity of the wild type and mutant LTRs. LNCaP cells were transiently transfected with reporter plasmids containing the wild type or mutant LTRs linked to Gaussia luciferase. Shown for each construct are the average
    Figure Legend Snippet: Comparison of the transcriptional activity of the wild type and mutant LTRs. LNCaP cells were transiently transfected with reporter plasmids containing the wild type or mutant LTRs linked to Gaussia luciferase. Shown for each construct are the average

    Techniques Used: Activity Assay, Mutagenesis, Transfection, Luciferase, Construct

    35) Product Images from "Antibiotic monensin synergizes with EGFR inhibitors and oxaliplatin to suppress the proliferation of human ovarian cancer cells"

    Article Title: Antibiotic monensin synergizes with EGFR inhibitors and oxaliplatin to suppress the proliferation of human ovarian cancer cells

    Journal: Scientific Reports

    doi: 10.1038/srep17523

    Monensin inhibits multiple cancer-associated signaling pathways, including downstream mediators of EGFR signaling. ( A ) Effect of monensin on intracellular β-catenin level. Subconfluent SKOV3 cells were first transduced with Ad-Wnt3A or AdGFP for 16 h, and treated with varied concentrations of monensin for additional 36 h. Cells were fixed and subjected immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counterstained with DAPI. Control IgG was used as a negative control. Representative results are shown. ( B ) Monensin inhibits Tcf/Lef reporter activity. Subconfluent SKOV3 cells were transfected with TOP-Luc reporter plasmid and infected with Ad-Wnt3A or AdGFP for 16 h, followed by a treatment with varied concentrations of monensin for 48 h. Cells were lysed and subjected to luciferase activity assays using Promega’s firefly Luciferase Assay System. Each assay condition was done in triplicate. ( C ) Effect of monensin on the 11 cancer-associated pathway reporter activities. Subconfluent SKOV3 cells were transfected with the homemade Gaussia luciferase reporters for the 11 cancer-associated pathways and a constitutively active reporter pG2Luc. At 16 h post transfection the cells were treated with varied concentrations of monensin for additional 48 h. the culture medium was collected for Gaussia luciferase activity assay using BioLux Gaussia Luciferase Assay Kit (New England Biolabs). Each assay condition was done in triplicate. ( D ) Monensin inhibits four pathways in dose- and time-dependent manners. The selected four pathway reporters were transfected into SKOV3 cells as described in ( C ), except that Gaussia luciferase activities were measured at 24 h, 48 h and 72 h post treatment. (E) Monensin inhibits the expression of genes involved in cell proliferation. Subconfluent SKOV3 cells were treated with the indicated concentrations of monensin for 48 h. Total RNA was isolated and subjected to qPCR analysis of the expression of the indicated genes. Human GAPDH was used as the reference gene.
    Figure Legend Snippet: Monensin inhibits multiple cancer-associated signaling pathways, including downstream mediators of EGFR signaling. ( A ) Effect of monensin on intracellular β-catenin level. Subconfluent SKOV3 cells were first transduced with Ad-Wnt3A or AdGFP for 16 h, and treated with varied concentrations of monensin for additional 36 h. Cells were fixed and subjected immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counterstained with DAPI. Control IgG was used as a negative control. Representative results are shown. ( B ) Monensin inhibits Tcf/Lef reporter activity. Subconfluent SKOV3 cells were transfected with TOP-Luc reporter plasmid and infected with Ad-Wnt3A or AdGFP for 16 h, followed by a treatment with varied concentrations of monensin for 48 h. Cells were lysed and subjected to luciferase activity assays using Promega’s firefly Luciferase Assay System. Each assay condition was done in triplicate. ( C ) Effect of monensin on the 11 cancer-associated pathway reporter activities. Subconfluent SKOV3 cells were transfected with the homemade Gaussia luciferase reporters for the 11 cancer-associated pathways and a constitutively active reporter pG2Luc. At 16 h post transfection the cells were treated with varied concentrations of monensin for additional 48 h. the culture medium was collected for Gaussia luciferase activity assay using BioLux Gaussia Luciferase Assay Kit (New England Biolabs). Each assay condition was done in triplicate. ( D ) Monensin inhibits four pathways in dose- and time-dependent manners. The selected four pathway reporters were transfected into SKOV3 cells as described in ( C ), except that Gaussia luciferase activities were measured at 24 h, 48 h and 72 h post treatment. (E) Monensin inhibits the expression of genes involved in cell proliferation. Subconfluent SKOV3 cells were treated with the indicated concentrations of monensin for 48 h. Total RNA was isolated and subjected to qPCR analysis of the expression of the indicated genes. Human GAPDH was used as the reference gene.

    Techniques Used: Transduction, Immunofluorescence, Staining, Negative Control, Activity Assay, Transfection, Plasmid Preparation, Infection, Luciferase, Expressing, Isolation, Real-time Polymerase Chain Reaction

    36) Product Images from "Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/?-catenin signaling"

    Article Title: Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/?-catenin signaling

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

    doi: 10.1073/pnas.1115083109

    TIMP-1 reduces Wnt/β-catenin activity in a MMP-independent manner. ( A ) Western blot analysis of β-catenin and TIMP-1 in protein extracts obtained from hMSCs transfected with siRNA targeting TIMP-1 (KD) or control siRNA (NC) on days 3 and 7 after transfection. For densitometric quantification, the amount of β-catenin present in cells transfected with control siRNA was set to 100% at each time point. Cellular β-actin was used as a loading control ( n = 3). ( B ) Images of the immunocytochemical staining of β-catenin in hMSCs transfected with siRNA against TIMP-1 (KD) or control siRNA (NC) 3 d after transfection ( n = 3) at magnification 10×. ( C ) TIMP-1 knockdown (KD) and control (NC) hMSCs were transiently transfected with the β-catenin–activated reporter (BAR) and cultivated for up to 7 d in the presence of Wnt3a (150 ng/mL) to stimulate Wnt/β-catenin activity. The activity of secreted Gaussia luciferase, which was transcribed due to β-catenin–dependent TCF/LEF signaling in the cells, was quantified in the conditioned medium at the indicated time points. ( D ) hMSCs transfected with TIMP-1 siRNA or control siRNA were cultured in the presence or absence of Wnt3a (150 ng/mL) and/or recombinant TIMP-1 (20 nM). After 7 d of incubation, the transcription levels of the Wnt/β-catenin target genes cyclin D1 and MT1-MMP were examined using qRT-PCR. The results are given as the percentage of change in mRNA expression relative to that in unstimulated control cells transfected with control siRNA. ( E ) The effects of wild-type TIMP-1 (WT-TIMP-1) and a non-MMP–inhibitory mutant form of TIMP-1 (V-V-TIMP-1) (each 20 nM) on β-catenin reporter activity in BAR-hMSCs without (NC) and with TIMP-1 knockdown (KD) in the absence (basal) and presence of Wnt3a (150 ng/mL) after 24 h of incubation. The data shown in C – E represent the mean ± SD of triplicate measurements ( n = 3). n.s., not significant; * P
    Figure Legend Snippet: TIMP-1 reduces Wnt/β-catenin activity in a MMP-independent manner. ( A ) Western blot analysis of β-catenin and TIMP-1 in protein extracts obtained from hMSCs transfected with siRNA targeting TIMP-1 (KD) or control siRNA (NC) on days 3 and 7 after transfection. For densitometric quantification, the amount of β-catenin present in cells transfected with control siRNA was set to 100% at each time point. Cellular β-actin was used as a loading control ( n = 3). ( B ) Images of the immunocytochemical staining of β-catenin in hMSCs transfected with siRNA against TIMP-1 (KD) or control siRNA (NC) 3 d after transfection ( n = 3) at magnification 10×. ( C ) TIMP-1 knockdown (KD) and control (NC) hMSCs were transiently transfected with the β-catenin–activated reporter (BAR) and cultivated for up to 7 d in the presence of Wnt3a (150 ng/mL) to stimulate Wnt/β-catenin activity. The activity of secreted Gaussia luciferase, which was transcribed due to β-catenin–dependent TCF/LEF signaling in the cells, was quantified in the conditioned medium at the indicated time points. ( D ) hMSCs transfected with TIMP-1 siRNA or control siRNA were cultured in the presence or absence of Wnt3a (150 ng/mL) and/or recombinant TIMP-1 (20 nM). After 7 d of incubation, the transcription levels of the Wnt/β-catenin target genes cyclin D1 and MT1-MMP were examined using qRT-PCR. The results are given as the percentage of change in mRNA expression relative to that in unstimulated control cells transfected with control siRNA. ( E ) The effects of wild-type TIMP-1 (WT-TIMP-1) and a non-MMP–inhibitory mutant form of TIMP-1 (V-V-TIMP-1) (each 20 nM) on β-catenin reporter activity in BAR-hMSCs without (NC) and with TIMP-1 knockdown (KD) in the absence (basal) and presence of Wnt3a (150 ng/mL) after 24 h of incubation. The data shown in C – E represent the mean ± SD of triplicate measurements ( n = 3). n.s., not significant; * P

    Techniques Used: Activity Assay, Western Blot, Transfection, Staining, Luciferase, Cell Culture, Recombinant, Incubation, Quantitative RT-PCR, Expressing, Mutagenesis

    TIMP-1 binding to the cell surface molecule CD63 affects Wnt/β-catenin signaling. ( A ) Confocal microscopic analysis of hMSCs costained with anti-TIMP-1/Alexa 543–conjugated secondary antibody (red staining), anti-CD63/Alexa 488–conjugated secondary antibody (green staining), and Hoechst 33342 (blue staining, for DNA labeling in cell nuclei). The merged image shows the overlay (yellow staining) of the two channels demonstrating TIMP-1 colocalization with CD63. The area marked by a square is shown in higher magnification to display the representative section (white arrow) used for fluorescence intensity profiling analysis. Intensity peaks representing the red (TIMP-1) and green (CD63) emission wavelengths indicate areas of colocalization (black arrows). ( B ) Anti-CD63 immunoprecipitates (IPs) obtained from hMSC lysates 3 d after transfection using a specific siRNA against CD63 (KD) or control siRNA (NC) were subjected to Western blotting, using anti-CD63 and anti-TIMP-1 antibodies. The detection of β-catenin in the cell lysates before immunoprecipitation served as a control. ( n = 3). ( C ) hMSCs were transfected with siRNA against CD63 (KD) or control siRNA (NC). Equal amounts of protein were taken from the cell lysates and conditioned media (CM), and the presence of TIMP-1 was evaluated by Western blotting after 3 d of incubation. For densitometric quantification, the amounts of TIMP-1 present in the control cell lysate and the CM were set as 100%, respectively. TIMP-1 mRNA levels were determined using qRT-PCR after 24 h of incubation ( n = 3). ( D ) BAR-hMSCs were transfected with siRNA targeting CD63 (KD) or a control siRNA (NC) and cultured for 3 d in the absence (basal) or presence of Wnt3a (150 ng/mL) and/or recombinant TIMP-1 (20 nM) (+T). β-Catenin reporter activity was quantified by measuring secreted Gaussia luciferase in the conditioned medium (CM). ( E ) hMSCs transfected with siRNA against CD63 (KD) or control siRNA (NC) were incubated for 3 d in the presence of Wnt3a (150 ng/mL), and the transcription levels of the Wnt/β-catenin target genes cyclin D1 and MT1-MMP were analyzed using qRT-PCR. The data shown in D and E represent the mean ± SD ( n = 3). ** P
    Figure Legend Snippet: TIMP-1 binding to the cell surface molecule CD63 affects Wnt/β-catenin signaling. ( A ) Confocal microscopic analysis of hMSCs costained with anti-TIMP-1/Alexa 543–conjugated secondary antibody (red staining), anti-CD63/Alexa 488–conjugated secondary antibody (green staining), and Hoechst 33342 (blue staining, for DNA labeling in cell nuclei). The merged image shows the overlay (yellow staining) of the two channels demonstrating TIMP-1 colocalization with CD63. The area marked by a square is shown in higher magnification to display the representative section (white arrow) used for fluorescence intensity profiling analysis. Intensity peaks representing the red (TIMP-1) and green (CD63) emission wavelengths indicate areas of colocalization (black arrows). ( B ) Anti-CD63 immunoprecipitates (IPs) obtained from hMSC lysates 3 d after transfection using a specific siRNA against CD63 (KD) or control siRNA (NC) were subjected to Western blotting, using anti-CD63 and anti-TIMP-1 antibodies. The detection of β-catenin in the cell lysates before immunoprecipitation served as a control. ( n = 3). ( C ) hMSCs were transfected with siRNA against CD63 (KD) or control siRNA (NC). Equal amounts of protein were taken from the cell lysates and conditioned media (CM), and the presence of TIMP-1 was evaluated by Western blotting after 3 d of incubation. For densitometric quantification, the amounts of TIMP-1 present in the control cell lysate and the CM were set as 100%, respectively. TIMP-1 mRNA levels were determined using qRT-PCR after 24 h of incubation ( n = 3). ( D ) BAR-hMSCs were transfected with siRNA targeting CD63 (KD) or a control siRNA (NC) and cultured for 3 d in the absence (basal) or presence of Wnt3a (150 ng/mL) and/or recombinant TIMP-1 (20 nM) (+T). β-Catenin reporter activity was quantified by measuring secreted Gaussia luciferase in the conditioned medium (CM). ( E ) hMSCs transfected with siRNA against CD63 (KD) or control siRNA (NC) were incubated for 3 d in the presence of Wnt3a (150 ng/mL), and the transcription levels of the Wnt/β-catenin target genes cyclin D1 and MT1-MMP were analyzed using qRT-PCR. The data shown in D and E represent the mean ± SD ( n = 3). ** P

    Techniques Used: Binding Assay, Staining, DNA Labeling, Fluorescence, Transfection, Western Blot, Immunoprecipitation, Incubation, Quantitative RT-PCR, Cell Culture, Recombinant, Activity Assay, Luciferase

    The miRNA let-7f is implicated in the TIMP-1–mediated inhibition of β-catenin activity and osteogenic differentiation. ( A ) BAR-hMSCs were transfected with control siRNA (NC), siRNA targeting TIMP-1 (KD), and/or siRNA targeting Drosha (KD). After 3 d of culture, β-catenin reporter activity was determined by luminometric analysis of Gaussia luciferase in the culture supernatants. ( B ) hMSCs were transfected with control siRNA (NC) or siRNA targeting TIMP-1 (KD) and incubated for 3 d. RNA was collected and subjected to miRNA analysis, using PCR array technology. The results are shown in a diagram with the crossing point ( Ct ) values in TIMP-1 knockdown cells plotted against the respective Ct s in control cells and fitted well to a straight line, revealing that let-7f was the most up-regulated miRNA tested. ( C ) Quantitation of let-7f transcription in control cells (NC, set as 1) and hMSCs deficient (KD) in TIMP-1 and CD63 using qRT-PCR analysis. The values shown were normalized to GAPDH mRNA. ( D ) BAR-hMSCs were transfected with control siRNA (NC), siRNA against TIMP-1 (KD), a let-7f mimic (MI), and/or a let-7f inhibitor (IN). After 24 h of incubation, β-catenin reporter activity was quantified by luminometric measurement of secreted Gaussia luciferase in the conditioned medium (the control was set as 100%). ( E ) hMSCs were cotransfected with a Renilla luciferase reporter construct bearing the axin 2 3′-UTR and a let-7f mimic (10–40 nM) or control miRNA (40 nM). After 24 h, axin 2 reporter activity was quantified by luminometric measurement of Renilla luciferase in the cell extracts. ( F ) hMSCs were transfected with control siRNA (NC), siRNA against TIMP-1 (KD), a let-7f mimic (MI), and/or a let-7f inhibitor (IN) and cultivated in the presence of Wnt3a (150 ng/mL) to stimulate Wnt/β-catenin signaling. After 24 h, axin 2 protein expression was analyzed using Western blotting, and axin 2 mRNA expression was analyzed using qRT-PCR. β-Actin served as a loading control. For densitometric quantification, the amount of axin 2 protein present in the control cells (NC) was set as 100%. The qRT-PCR values shown were normalized to the GAPDH mRNA levels. ( G–I ) hMSCs transfected with control siRNA (NC), siRNA targeting TIMP-1 (KD), a let-7f mimic (MI), and/or a let-7f inhibitor (IN) were incubated for 14 d in osteogenic differentiation medium. ( G ) Representative images of cellular monolayer mineralization with alizarin red staining. ( H ) The relative absorbance of the alizarin red stain extracts (the NC was set as 100%). ( I ) Relative mRNA levels of the osteogenic markers alkaline phosphatase and osteocalcin were determined using qRT-PCR (the NC was set as 1). The values are normalized to the GAPDH mRNA levels. Data shown in A , C–F , H , and I are given as mean values ± SD of triplicate measurements ( n = 3). n.s., not significant; * P
    Figure Legend Snippet: The miRNA let-7f is implicated in the TIMP-1–mediated inhibition of β-catenin activity and osteogenic differentiation. ( A ) BAR-hMSCs were transfected with control siRNA (NC), siRNA targeting TIMP-1 (KD), and/or siRNA targeting Drosha (KD). After 3 d of culture, β-catenin reporter activity was determined by luminometric analysis of Gaussia luciferase in the culture supernatants. ( B ) hMSCs were transfected with control siRNA (NC) or siRNA targeting TIMP-1 (KD) and incubated for 3 d. RNA was collected and subjected to miRNA analysis, using PCR array technology. The results are shown in a diagram with the crossing point ( Ct ) values in TIMP-1 knockdown cells plotted against the respective Ct s in control cells and fitted well to a straight line, revealing that let-7f was the most up-regulated miRNA tested. ( C ) Quantitation of let-7f transcription in control cells (NC, set as 1) and hMSCs deficient (KD) in TIMP-1 and CD63 using qRT-PCR analysis. The values shown were normalized to GAPDH mRNA. ( D ) BAR-hMSCs were transfected with control siRNA (NC), siRNA against TIMP-1 (KD), a let-7f mimic (MI), and/or a let-7f inhibitor (IN). After 24 h of incubation, β-catenin reporter activity was quantified by luminometric measurement of secreted Gaussia luciferase in the conditioned medium (the control was set as 100%). ( E ) hMSCs were cotransfected with a Renilla luciferase reporter construct bearing the axin 2 3′-UTR and a let-7f mimic (10–40 nM) or control miRNA (40 nM). After 24 h, axin 2 reporter activity was quantified by luminometric measurement of Renilla luciferase in the cell extracts. ( F ) hMSCs were transfected with control siRNA (NC), siRNA against TIMP-1 (KD), a let-7f mimic (MI), and/or a let-7f inhibitor (IN) and cultivated in the presence of Wnt3a (150 ng/mL) to stimulate Wnt/β-catenin signaling. After 24 h, axin 2 protein expression was analyzed using Western blotting, and axin 2 mRNA expression was analyzed using qRT-PCR. β-Actin served as a loading control. For densitometric quantification, the amount of axin 2 protein present in the control cells (NC) was set as 100%. The qRT-PCR values shown were normalized to the GAPDH mRNA levels. ( G–I ) hMSCs transfected with control siRNA (NC), siRNA targeting TIMP-1 (KD), a let-7f mimic (MI), and/or a let-7f inhibitor (IN) were incubated for 14 d in osteogenic differentiation medium. ( G ) Representative images of cellular monolayer mineralization with alizarin red staining. ( H ) The relative absorbance of the alizarin red stain extracts (the NC was set as 100%). ( I ) Relative mRNA levels of the osteogenic markers alkaline phosphatase and osteocalcin were determined using qRT-PCR (the NC was set as 1). The values are normalized to the GAPDH mRNA levels. Data shown in A , C–F , H , and I are given as mean values ± SD of triplicate measurements ( n = 3). n.s., not significant; * P

    Techniques Used: Inhibition, Activity Assay, Transfection, Luciferase, Incubation, Polymerase Chain Reaction, Quantitation Assay, Quantitative RT-PCR, Construct, Expressing, Western Blot, Staining

    37) Product Images from "Ginseng saponin metabolite 20(S)-protopanaxadiol inhibits tumor growth by targeting multiple cancer signaling pathways"

    Article Title: Ginseng saponin metabolite 20(S)-protopanaxadiol inhibits tumor growth by targeting multiple cancer signaling pathways

    Journal: Oncology Reports

    doi: 10.3892/or.2013.2438

    Protopanaxadiol (PPD) targets multiple signaling pathways. (A) The expression of anchor protein 8 (AKAP8L) and phosphatidylinositol transfer protein α (PITPNA) in PPD-treated cancer cells. Subconfluent HCT116 cells were treated with PPD (10 μM). Total RNA was collected at the indicated time points and subjected to semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis with primer pairs for human GAPDH, AKAP8L and PITPNA transcripts. Representative results are shown. (B) The effect of PPD on 8 different signaling pathways in HCT116 cells. Various subconfluent HCT116-GLuc cells lines were seeded in 24-well plates and treated without or with 10 μM PPD. At 24 h, cell culture medium was subjected to Gaussia luciferase assay. Each assay condition was carried out in triplicate.
    Figure Legend Snippet: Protopanaxadiol (PPD) targets multiple signaling pathways. (A) The expression of anchor protein 8 (AKAP8L) and phosphatidylinositol transfer protein α (PITPNA) in PPD-treated cancer cells. Subconfluent HCT116 cells were treated with PPD (10 μM). Total RNA was collected at the indicated time points and subjected to semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis with primer pairs for human GAPDH, AKAP8L and PITPNA transcripts. Representative results are shown. (B) The effect of PPD on 8 different signaling pathways in HCT116 cells. Various subconfluent HCT116-GLuc cells lines were seeded in 24-well plates and treated without or with 10 μM PPD. At 24 h, cell culture medium was subjected to Gaussia luciferase assay. Each assay condition was carried out in triplicate.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Luciferase

    38) Product Images from "A reporter system for replication-competent gammaretroviruses: the inGluc-MLV-DERSE assay"

    Article Title: A reporter system for replication-competent gammaretroviruses: the inGluc-MLV-DERSE assay

    Journal: Gene therapy

    doi: 10.1038/gt.2012.18

    Schematic of inG luc -MLV-DERSE assay The inG luc -MLV-DERSE plasmid consists of a Gaussia Luciferase (G luc ) sequence oriented in a reverse direction with respect to flanking MLV non-coding sequences. Within the non-coding G luc sequence is an intron that is oriented in a forward direction relative to the viral non-coding regions (and can be spliced by the host cell). The plasmid is maintained in 293mCAT1 cells. In the absence of RCR, only minus-strand, spliced G luc sequences are present in the cell. An RCR that infects the DERSE cell can package the RNA containing the minus-strand G luc sequence. In the next round of infection reverse transcription of the encapsidated RNA produces a double-stranded DNA containing an uninterrupted G luc gene. This gene is an intact, coding G luc sequence that is subsequently integrated into the DNA of, and expressed by, the newly infected cell. Expression of G luc is under the control of the cytomegalovirus (CMV) promoter.
    Figure Legend Snippet: Schematic of inG luc -MLV-DERSE assay The inG luc -MLV-DERSE plasmid consists of a Gaussia Luciferase (G luc ) sequence oriented in a reverse direction with respect to flanking MLV non-coding sequences. Within the non-coding G luc sequence is an intron that is oriented in a forward direction relative to the viral non-coding regions (and can be spliced by the host cell). The plasmid is maintained in 293mCAT1 cells. In the absence of RCR, only minus-strand, spliced G luc sequences are present in the cell. An RCR that infects the DERSE cell can package the RNA containing the minus-strand G luc sequence. In the next round of infection reverse transcription of the encapsidated RNA produces a double-stranded DNA containing an uninterrupted G luc gene. This gene is an intact, coding G luc sequence that is subsequently integrated into the DNA of, and expressed by, the newly infected cell. Expression of G luc is under the control of the cytomegalovirus (CMV) promoter.

    Techniques Used: Plasmid Preparation, Luciferase, Sequencing, Infection, Expressing

    39) Product Images from "JunD/AP-1 Antagonizes the Induction of DAPK1 To Promote the Survival of v-Src-Transformed Cells"

    Article Title: JunD/AP-1 Antagonizes the Induction of DAPK1 To Promote the Survival of v-Src-Transformed Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.01925-16

    Identification of regulatory elements of the DAPK1 promoter. (A) Quantification of Gaussia luciferase reporter activity in NY72-4-infected CEFs with 5′ deletion constructs of the DAPK1 upstream region. (B) Schematic representations of the deletion
    Figure Legend Snippet: Identification of regulatory elements of the DAPK1 promoter. (A) Quantification of Gaussia luciferase reporter activity in NY72-4-infected CEFs with 5′ deletion constructs of the DAPK1 upstream region. (B) Schematic representations of the deletion

    Techniques Used: Luciferase, Activity Assay, Infection, Construct

    40) Product Images from "Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies"

    Article Title: Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00793

    Gaussia princeps luciferase (GpL)-tumor necrosis factor (TNF) binding to cells with endogenous coexpression of tumor necrosis factor receptor-2 (TNFR2) and progranulin (PGRN). (A) Human embryonal kidney cells 293 (HEK293) cells were transfected (first transfection) with empty vector (EV) or an expression vector encoding TNFR2, where the TNF receptor associated factor 2 (TRAF2) binding site has been replaced by yellow fluorescence protein (YFP) (TNFR2). The following day, transfecfed cells were split into four aliquots which were transfected a second time (second transfection) with expression plasmids encoding PGRN, membrane TNF (memTNF), soluble Flag-tagged TNF (F-TNF), or EV. After an additional day, aliquots of 30,000 cells (P) and 15 µl SN (S) were analyzed by Western blotting with anti-PGRN, anti-TNF, and anti-Flag along with 100 ng PGRN Adi and 100 ng purified untagged soluble TNF (sTNF). (B) Equilibrium binding studies were performed with the indicated concentrations of GpL-TNF. Specific binding of GpL-TNF in the presence of PGRN (second transfection PGRN), membrane TNF (second transfection memTNF), and Flag-TNF (second transfection F-TNF) or the absence of an potential modulator (second transfection EV) was obtained by subtracting unspecific binding values (first transfection EV) from the corresponding total binding values (first transfection TNFR2). Specific binding values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.
    Figure Legend Snippet: Gaussia princeps luciferase (GpL)-tumor necrosis factor (TNF) binding to cells with endogenous coexpression of tumor necrosis factor receptor-2 (TNFR2) and progranulin (PGRN). (A) Human embryonal kidney cells 293 (HEK293) cells were transfected (first transfection) with empty vector (EV) or an expression vector encoding TNFR2, where the TNF receptor associated factor 2 (TRAF2) binding site has been replaced by yellow fluorescence protein (YFP) (TNFR2). The following day, transfecfed cells were split into four aliquots which were transfected a second time (second transfection) with expression plasmids encoding PGRN, membrane TNF (memTNF), soluble Flag-tagged TNF (F-TNF), or EV. After an additional day, aliquots of 30,000 cells (P) and 15 µl SN (S) were analyzed by Western blotting with anti-PGRN, anti-TNF, and anti-Flag along with 100 ng PGRN Adi and 100 ng purified untagged soluble TNF (sTNF). (B) Equilibrium binding studies were performed with the indicated concentrations of GpL-TNF. Specific binding of GpL-TNF in the presence of PGRN (second transfection PGRN), membrane TNF (second transfection memTNF), and Flag-TNF (second transfection F-TNF) or the absence of an potential modulator (second transfection EV) was obtained by subtracting unspecific binding values (first transfection EV) from the corresponding total binding values (first transfection TNFR2). Specific binding values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

    Techniques Used: Luciferase, Binding Assay, Transfection, Plasmid Preparation, Expressing, Fluorescence, Western Blot, Purification, Software

    Gaussia princeps luciferase (GpL) fusion proteins of progranulin (PGRN) show no relevant binding to tumor necrosis factor receptor-1 (TNFR1) or TNFR2. (A) Human embryonal kidney cells 293 (HEK293) cells were transiently transfected with expression plasmids encoding GpL-PGRN (GpL-PGRN), PGRN-GpL (PGRN-GpL), or empty vector (EV). GpL-PGRN concentrations in supernatants (SNs) and cell lysates were determined by help of a GpL fusion protein of known concentration. SNs and cell lysates, containing approximately 100 ng PGRN-GpL or GpL-PGRN along with 100 ng PGRN Adi , were subjected to Western blotting with a PGRN-specific antibody to verify the integrity of the PGRN GpL fusion proteins. (B) TNFR1-Fc, TNFR2-Fc or, as a control for unspecific binding, hIgG1 were immobilized to black enzyme-linked immunosorbent assay plates. Lysates and SN of the GpL-PGRN (GpL-PGRN lys and GpL-PGRN SN ) and PGRN-GpL (PGRN-GpL lys and PGRN-GpL SN ) transfected cells and GpL-tumor necrosis factor (TNF) were added for 1 h and binding was determined in triplicates. (C) TNFR1 and TNFR2 expressing transfectants (total binding) and EV-transfected HEK293 cells (non-specific binding) were subjected to equilibrium binding studies with the indicated GpL fusion proteins. Specific binding (= total − non-specific binding) values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.
    Figure Legend Snippet: Gaussia princeps luciferase (GpL) fusion proteins of progranulin (PGRN) show no relevant binding to tumor necrosis factor receptor-1 (TNFR1) or TNFR2. (A) Human embryonal kidney cells 293 (HEK293) cells were transiently transfected with expression plasmids encoding GpL-PGRN (GpL-PGRN), PGRN-GpL (PGRN-GpL), or empty vector (EV). GpL-PGRN concentrations in supernatants (SNs) and cell lysates were determined by help of a GpL fusion protein of known concentration. SNs and cell lysates, containing approximately 100 ng PGRN-GpL or GpL-PGRN along with 100 ng PGRN Adi , were subjected to Western blotting with a PGRN-specific antibody to verify the integrity of the PGRN GpL fusion proteins. (B) TNFR1-Fc, TNFR2-Fc or, as a control for unspecific binding, hIgG1 were immobilized to black enzyme-linked immunosorbent assay plates. Lysates and SN of the GpL-PGRN (GpL-PGRN lys and GpL-PGRN SN ) and PGRN-GpL (PGRN-GpL lys and PGRN-GpL SN ) transfected cells and GpL-tumor necrosis factor (TNF) were added for 1 h and binding was determined in triplicates. (C) TNFR1 and TNFR2 expressing transfectants (total binding) and EV-transfected HEK293 cells (non-specific binding) were subjected to equilibrium binding studies with the indicated GpL fusion proteins. Specific binding (= total − non-specific binding) values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

    Techniques Used: Luciferase, Binding Assay, Transfection, Expressing, Plasmid Preparation, Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Software

    Related Articles

    Transfection:

    Article Title: TGF?/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells *
    Article Snippet: .. At 24 and 48 h after transfection, Gaussia luciferase activity was measured by using the Gaussia Luciferase Assay kit (New England Biolabs) according to the manufacturer's instructions. ..

    Luciferase:

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation
    Article Snippet: .. Culture supernatant (20 μl) collected from cells expressing plasmids carrying the GLuc reporter was used to determine GLuc activity by using a BioLux Gaussia Luciferase Assay Kit (New England Biolabs). .. miRNA and DNA transfection miRNAs and non-targeting control miRNA (miR cont) were synthesized by Bonac (Fukuoka, Japan).

    Article Title: EVA-1 Functions as an UNC-40 Co-receptor to Enhance Attraction to the MADD-4 Guidance Cue in Caenorhabditis elegans
    Article Snippet: .. One third of the immunoprecipitate was taken for Gaussia luciferase assays by using the BioLux Kit (NEB) and MicroLumat Plus LB96V luminometer (BERTHOLD). .. Remainder of the immunoprecipitates were analyzed by western blot using rabbit anti-Flag antibodies (NEB).

    Article Title: A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1
    Article Snippet: .. The next day, the cells were washed three times with a serum-free medium and then incubated in a fresh medium supplemented with the indicated amount of BFA for 4 h. At this time point, 20 μl of the medium was used to monitor the amount of secreted Gaussia luciferase using a Gaussia luciferase assay kit (New England BioLabs). .. Secretion data were normalized to the signal obtained from the cells incubated without BFA for each sample.

    Article Title: TGF?/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells *
    Article Snippet: .. At 24 and 48 h after transfection, Gaussia luciferase activity was measured by using the Gaussia Luciferase Assay kit (New England Biolabs) according to the manufacturer's instructions. ..

    Article Title: Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies
    Article Snippet: .. Gaussia princeps luciferase activity was measured with the Gaussia luciferase Assay Kit (New England Biolabs, Frankfurt, Germany) essentially as described by the supplier. .. After starting the reaction by adding substrate-buffer solution, light emission was immediately ( < 10 s) quantified (Lucy 2 or a LUmo Luminometer; both Anthos Labtec Instruments) to minimize errors due to the decay of GpL activity.

    Article Title: Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist
    Article Snippet: .. The culture supernatant was projected to perform luciferase assay with Gaussia Luciferase assay kit and Cypridina Luciferase assay kit (NEB) following their description. ..

    Article Title: Antibiotic monensin synergizes with EGFR inhibitors and oxaliplatin to suppress the proliferation of human ovarian cancer cells
    Article Snippet: .. At 24 h, 48 h or 72 h post treatment, culture media were taken and subjected to Gaussia luciferase assays using the BioLux Gaussia Luciferase Assay Kit (New England Biolabs). ..

    Article Title: Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors
    Article Snippet: .. Luciferase activity of cells, which corresponds to levels of NF-kB activation, collected at 0, 2, 4, and 8 h, was measured by luminescence using Biolux® Gaussia luciferase Assay kit (New England BioLabs Inc.). .. Values obtained by luminescence were normalized to the protein absorbance of the cell lysate as measured by BioRad® DC protein assay.

    Activation Assay:

    Article Title: Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors
    Article Snippet: .. Luciferase activity of cells, which corresponds to levels of NF-kB activation, collected at 0, 2, 4, and 8 h, was measured by luminescence using Biolux® Gaussia luciferase Assay kit (New England BioLabs Inc.). .. Values obtained by luminescence were normalized to the protein absorbance of the cell lysate as measured by BioRad® DC protein assay.

    Incubation:

    Article Title: A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1
    Article Snippet: .. The next day, the cells were washed three times with a serum-free medium and then incubated in a fresh medium supplemented with the indicated amount of BFA for 4 h. At this time point, 20 μl of the medium was used to monitor the amount of secreted Gaussia luciferase using a Gaussia luciferase assay kit (New England BioLabs). .. Secretion data were normalized to the signal obtained from the cells incubated without BFA for each sample.

    Activity Assay:

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation
    Article Snippet: .. Culture supernatant (20 μl) collected from cells expressing plasmids carrying the GLuc reporter was used to determine GLuc activity by using a BioLux Gaussia Luciferase Assay Kit (New England Biolabs). .. miRNA and DNA transfection miRNAs and non-targeting control miRNA (miR cont) were synthesized by Bonac (Fukuoka, Japan).

    Article Title: TGF?/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells *
    Article Snippet: .. At 24 and 48 h after transfection, Gaussia luciferase activity was measured by using the Gaussia Luciferase Assay kit (New England Biolabs) according to the manufacturer's instructions. ..

    Article Title: Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies
    Article Snippet: .. Gaussia princeps luciferase activity was measured with the Gaussia luciferase Assay Kit (New England Biolabs, Frankfurt, Germany) essentially as described by the supplier. .. After starting the reaction by adding substrate-buffer solution, light emission was immediately ( < 10 s) quantified (Lucy 2 or a LUmo Luminometer; both Anthos Labtec Instruments) to minimize errors due to the decay of GpL activity.

    Article Title: Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors
    Article Snippet: .. Luciferase activity of cells, which corresponds to levels of NF-kB activation, collected at 0, 2, 4, and 8 h, was measured by luminescence using Biolux® Gaussia luciferase Assay kit (New England BioLabs Inc.). .. Values obtained by luminescence were normalized to the protein absorbance of the cell lysate as measured by BioRad® DC protein assay.

    Expressing:

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation
    Article Snippet: .. Culture supernatant (20 μl) collected from cells expressing plasmids carrying the GLuc reporter was used to determine GLuc activity by using a BioLux Gaussia Luciferase Assay Kit (New England Biolabs). .. miRNA and DNA transfection miRNAs and non-targeting control miRNA (miR cont) were synthesized by Bonac (Fukuoka, Japan).

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    New England Biolabs gaussia luciferase assay kit
    <t>Gaussia</t> princeps luciferase (GpL)-tumor necrosis factor (TNF) binding to cells with endogenous coexpression of tumor necrosis factor receptor-2 (TNFR2) and progranulin (PGRN). (A) Human embryonal kidney cells 293 (HEK293) cells were transfected (first transfection) with empty vector (EV) or an expression vector encoding TNFR2, where the TNF receptor associated factor 2 (TRAF2) binding site has been replaced by yellow fluorescence protein (YFP) (TNFR2). The following day, transfecfed cells were split into four aliquots which were transfected a second time (second transfection) with expression plasmids encoding PGRN, membrane TNF (memTNF), soluble Flag-tagged TNF (F-TNF), or EV. After an additional day, aliquots of 30,000 cells (P) and 15 µl SN (S) were analyzed by Western blotting with anti-PGRN, anti-TNF, and anti-Flag along with 100 ng PGRN Adi and 100 ng purified untagged soluble TNF (sTNF). (B) Equilibrium binding studies were performed with the indicated concentrations of GpL-TNF. Specific binding of GpL-TNF in the presence of PGRN (second transfection PGRN), membrane TNF (second transfection memTNF), and Flag-TNF (second transfection F-TNF) or the absence of an potential modulator (second transfection EV) was obtained by subtracting unspecific binding values (first transfection EV) from the corresponding total binding values (first transfection TNFR2). Specific binding values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.
    Gaussia Luciferase Assay Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Gaussia princeps luciferase (GpL)-tumor necrosis factor (TNF) binding to cells with endogenous coexpression of tumor necrosis factor receptor-2 (TNFR2) and progranulin (PGRN). (A) Human embryonal kidney cells 293 (HEK293) cells were transfected (first transfection) with empty vector (EV) or an expression vector encoding TNFR2, where the TNF receptor associated factor 2 (TRAF2) binding site has been replaced by yellow fluorescence protein (YFP) (TNFR2). The following day, transfecfed cells were split into four aliquots which were transfected a second time (second transfection) with expression plasmids encoding PGRN, membrane TNF (memTNF), soluble Flag-tagged TNF (F-TNF), or EV. After an additional day, aliquots of 30,000 cells (P) and 15 µl SN (S) were analyzed by Western blotting with anti-PGRN, anti-TNF, and anti-Flag along with 100 ng PGRN Adi and 100 ng purified untagged soluble TNF (sTNF). (B) Equilibrium binding studies were performed with the indicated concentrations of GpL-TNF. Specific binding of GpL-TNF in the presence of PGRN (second transfection PGRN), membrane TNF (second transfection memTNF), and Flag-TNF (second transfection F-TNF) or the absence of an potential modulator (second transfection EV) was obtained by subtracting unspecific binding values (first transfection EV) from the corresponding total binding values (first transfection TNFR2). Specific binding values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

    Journal: Frontiers in Immunology

    Article Title: Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies

    doi: 10.3389/fimmu.2018.00793

    Figure Lengend Snippet: Gaussia princeps luciferase (GpL)-tumor necrosis factor (TNF) binding to cells with endogenous coexpression of tumor necrosis factor receptor-2 (TNFR2) and progranulin (PGRN). (A) Human embryonal kidney cells 293 (HEK293) cells were transfected (first transfection) with empty vector (EV) or an expression vector encoding TNFR2, where the TNF receptor associated factor 2 (TRAF2) binding site has been replaced by yellow fluorescence protein (YFP) (TNFR2). The following day, transfecfed cells were split into four aliquots which were transfected a second time (second transfection) with expression plasmids encoding PGRN, membrane TNF (memTNF), soluble Flag-tagged TNF (F-TNF), or EV. After an additional day, aliquots of 30,000 cells (P) and 15 µl SN (S) were analyzed by Western blotting with anti-PGRN, anti-TNF, and anti-Flag along with 100 ng PGRN Adi and 100 ng purified untagged soluble TNF (sTNF). (B) Equilibrium binding studies were performed with the indicated concentrations of GpL-TNF. Specific binding of GpL-TNF in the presence of PGRN (second transfection PGRN), membrane TNF (second transfection memTNF), and Flag-TNF (second transfection F-TNF) or the absence of an potential modulator (second transfection EV) was obtained by subtracting unspecific binding values (first transfection EV) from the corresponding total binding values (first transfection TNFR2). Specific binding values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

    Article Snippet: Gaussia princeps luciferase activity was measured with the Gaussia luciferase Assay Kit (New England Biolabs, Frankfurt, Germany) essentially as described by the supplier.

    Techniques: Luciferase, Binding Assay, Transfection, Plasmid Preparation, Expressing, Fluorescence, Western Blot, Purification, Software

    Gaussia princeps luciferase (GpL) fusion proteins of progranulin (PGRN) show no relevant binding to tumor necrosis factor receptor-1 (TNFR1) or TNFR2. (A) Human embryonal kidney cells 293 (HEK293) cells were transiently transfected with expression plasmids encoding GpL-PGRN (GpL-PGRN), PGRN-GpL (PGRN-GpL), or empty vector (EV). GpL-PGRN concentrations in supernatants (SNs) and cell lysates were determined by help of a GpL fusion protein of known concentration. SNs and cell lysates, containing approximately 100 ng PGRN-GpL or GpL-PGRN along with 100 ng PGRN Adi , were subjected to Western blotting with a PGRN-specific antibody to verify the integrity of the PGRN GpL fusion proteins. (B) TNFR1-Fc, TNFR2-Fc or, as a control for unspecific binding, hIgG1 were immobilized to black enzyme-linked immunosorbent assay plates. Lysates and SN of the GpL-PGRN (GpL-PGRN lys and GpL-PGRN SN ) and PGRN-GpL (PGRN-GpL lys and PGRN-GpL SN ) transfected cells and GpL-tumor necrosis factor (TNF) were added for 1 h and binding was determined in triplicates. (C) TNFR1 and TNFR2 expressing transfectants (total binding) and EV-transfected HEK293 cells (non-specific binding) were subjected to equilibrium binding studies with the indicated GpL fusion proteins. Specific binding (= total − non-specific binding) values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

    Journal: Frontiers in Immunology

    Article Title: Lack of Evidence for a Direct Interaction of Progranulin and Tumor Necrosis Factor Receptor-1 and Tumor Necrosis Factor Receptor-2 From Cellular Binding Studies

    doi: 10.3389/fimmu.2018.00793

    Figure Lengend Snippet: Gaussia princeps luciferase (GpL) fusion proteins of progranulin (PGRN) show no relevant binding to tumor necrosis factor receptor-1 (TNFR1) or TNFR2. (A) Human embryonal kidney cells 293 (HEK293) cells were transiently transfected with expression plasmids encoding GpL-PGRN (GpL-PGRN), PGRN-GpL (PGRN-GpL), or empty vector (EV). GpL-PGRN concentrations in supernatants (SNs) and cell lysates were determined by help of a GpL fusion protein of known concentration. SNs and cell lysates, containing approximately 100 ng PGRN-GpL or GpL-PGRN along with 100 ng PGRN Adi , were subjected to Western blotting with a PGRN-specific antibody to verify the integrity of the PGRN GpL fusion proteins. (B) TNFR1-Fc, TNFR2-Fc or, as a control for unspecific binding, hIgG1 were immobilized to black enzyme-linked immunosorbent assay plates. Lysates and SN of the GpL-PGRN (GpL-PGRN lys and GpL-PGRN SN ) and PGRN-GpL (PGRN-GpL lys and PGRN-GpL SN ) transfected cells and GpL-tumor necrosis factor (TNF) were added for 1 h and binding was determined in triplicates. (C) TNFR1 and TNFR2 expressing transfectants (total binding) and EV-transfected HEK293 cells (non-specific binding) were subjected to equilibrium binding studies with the indicated GpL fusion proteins. Specific binding (= total − non-specific binding) values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

    Article Snippet: Gaussia princeps luciferase activity was measured with the Gaussia luciferase Assay Kit (New England Biolabs, Frankfurt, Germany) essentially as described by the supplier.

    Techniques: Luciferase, Binding Assay, Transfection, Expressing, Plasmid Preparation, Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Software

    Monensin inhibits multiple cancer-associated signaling pathways, including downstream mediators of EGFR signaling. ( A ) Effect of monensin on intracellular β-catenin level. Subconfluent SKOV3 cells were first transduced with Ad-Wnt3A or AdGFP for 16 h, and treated with varied concentrations of monensin for additional 36 h. Cells were fixed and subjected immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counterstained with DAPI. Control IgG was used as a negative control. Representative results are shown. ( B ) Monensin inhibits Tcf/Lef reporter activity. Subconfluent SKOV3 cells were transfected with TOP-Luc reporter plasmid and infected with Ad-Wnt3A or AdGFP for 16 h, followed by a treatment with varied concentrations of monensin for 48 h. Cells were lysed and subjected to luciferase activity assays using Promega’s firefly Luciferase Assay System. Each assay condition was done in triplicate. ( C ) Effect of monensin on the 11 cancer-associated pathway reporter activities. Subconfluent SKOV3 cells were transfected with the homemade Gaussia luciferase reporters for the 11 cancer-associated pathways and a constitutively active reporter pG2Luc. At 16 h post transfection the cells were treated with varied concentrations of monensin for additional 48 h. the culture medium was collected for Gaussia luciferase activity assay using BioLux Gaussia Luciferase Assay Kit (New England Biolabs). Each assay condition was done in triplicate. ( D ) Monensin inhibits four pathways in dose- and time-dependent manners. The selected four pathway reporters were transfected into SKOV3 cells as described in ( C ), except that Gaussia luciferase activities were measured at 24 h, 48 h and 72 h post treatment. (E) Monensin inhibits the expression of genes involved in cell proliferation. Subconfluent SKOV3 cells were treated with the indicated concentrations of monensin for 48 h. Total RNA was isolated and subjected to qPCR analysis of the expression of the indicated genes. Human GAPDH was used as the reference gene.

    Journal: Scientific Reports

    Article Title: Antibiotic monensin synergizes with EGFR inhibitors and oxaliplatin to suppress the proliferation of human ovarian cancer cells

    doi: 10.1038/srep17523

    Figure Lengend Snippet: Monensin inhibits multiple cancer-associated signaling pathways, including downstream mediators of EGFR signaling. ( A ) Effect of monensin on intracellular β-catenin level. Subconfluent SKOV3 cells were first transduced with Ad-Wnt3A or AdGFP for 16 h, and treated with varied concentrations of monensin for additional 36 h. Cells were fixed and subjected immunofluorescence staining with an anti-β-catenin antibody. The cell nuclei were counterstained with DAPI. Control IgG was used as a negative control. Representative results are shown. ( B ) Monensin inhibits Tcf/Lef reporter activity. Subconfluent SKOV3 cells were transfected with TOP-Luc reporter plasmid and infected with Ad-Wnt3A or AdGFP for 16 h, followed by a treatment with varied concentrations of monensin for 48 h. Cells were lysed and subjected to luciferase activity assays using Promega’s firefly Luciferase Assay System. Each assay condition was done in triplicate. ( C ) Effect of monensin on the 11 cancer-associated pathway reporter activities. Subconfluent SKOV3 cells were transfected with the homemade Gaussia luciferase reporters for the 11 cancer-associated pathways and a constitutively active reporter pG2Luc. At 16 h post transfection the cells were treated with varied concentrations of monensin for additional 48 h. the culture medium was collected for Gaussia luciferase activity assay using BioLux Gaussia Luciferase Assay Kit (New England Biolabs). Each assay condition was done in triplicate. ( D ) Monensin inhibits four pathways in dose- and time-dependent manners. The selected four pathway reporters were transfected into SKOV3 cells as described in ( C ), except that Gaussia luciferase activities were measured at 24 h, 48 h and 72 h post treatment. (E) Monensin inhibits the expression of genes involved in cell proliferation. Subconfluent SKOV3 cells were treated with the indicated concentrations of monensin for 48 h. Total RNA was isolated and subjected to qPCR analysis of the expression of the indicated genes. Human GAPDH was used as the reference gene.

    Article Snippet: At 24 h, 48 h or 72 h post treatment, culture media were taken and subjected to Gaussia luciferase assays using the BioLux Gaussia Luciferase Assay Kit (New England Biolabs).

    Techniques: Transduction, Immunofluorescence, Staining, Negative Control, Activity Assay, Transfection, Plasmid Preparation, Infection, Luciferase, Expressing, Isolation, Real-time Polymerase Chain Reaction

    EVA-1 functions cell-autonomously in muscles and interacts with MADD-4. A . Muscle-expressed EVA-1::CFP rescues the muscle extension defects of eva-1 mutants. B . A summary of EVA-1 domain function that is fully detailed in Figure S1 . C D . FLAG-tagged receptors were expressed from HEK293 cells, bathed in conditioned media from other HEK293 cells that express HA- and Gaussia luciferase-tagged MADD-4 or SLT-1 ligands, and immunoprecipitated to determine the relative amounts of ligand that co-immunoprecipitates with the receptor (see the materials and methods section for more details). C . The western blot on the left shows the five immunoprecipitated FLAG-tagged receptors. The western blot on the right shows the two HA- and Gaussia luciferase-tagged ligands that were collected from cell culture. D . The normalized relative levels of luciferase signal that immunoprecipitated with each potential ligand-receptor complex. E–I . Shown are animals harbouring one of three different transgenes that drive the expression of either neuronally-expressed MADD-4::YFP (from the trIs66 transgenic array) ( E ), muscle-expressed MADD-4::YFP (from the trIs78 transgenic array) ( F ), or muscle-expressed EVA-1::CFP (from the trIs89 transgenic array) ( G ), or animals harbouring two of the transgenes; trIs66 and trIs89 ( H ) and trIs78 and trIs89 ( I ). The relative levels of MADD-4::YFP expression from trIs66 and trIs78 is shown in Figure S2a . Images show either the CFP channel (top), YFP channel (middle) or a merged view (bottom). Arrows in ‘H’ indicate the localization of MADD-4::YFP to EVA-1::CFP expressing muscles; arrows in ‘I’ indicate the vesicularization of MADD-4::YFP and EVA-1::CFP in the muscle cells. J . The quantification of neuronally-secreted MADD-4::YFP localization to muscles over-expressing the indicated receptor. K . The quantification of CFP vesicles in animals that over-express the indicated CFP-tagged receptors (x-axis) in muscles in either the presence of MADD-4::YFP expressed from dorsal muscles (mMADD-4) or pan-neuronally (nMADD-4). The colocalization of MADD-4 and EVA-1 with the RAB-11 and RAB-5 endosomal markers are shown in Figure S2b and S2c . L M . MADD-4::YFP fails to induce obvious vesicularization of UNC-40::CFP in a wild type background (L), but YFP-CFP vesicles are obvious in animals that lack UNC-6 (M). In A, J, and K, statistical significance ( p

    Journal: PLoS Genetics

    Article Title: EVA-1 Functions as an UNC-40 Co-receptor to Enhance Attraction to the MADD-4 Guidance Cue in Caenorhabditis elegans

    doi: 10.1371/journal.pgen.1004521

    Figure Lengend Snippet: EVA-1 functions cell-autonomously in muscles and interacts with MADD-4. A . Muscle-expressed EVA-1::CFP rescues the muscle extension defects of eva-1 mutants. B . A summary of EVA-1 domain function that is fully detailed in Figure S1 . C D . FLAG-tagged receptors were expressed from HEK293 cells, bathed in conditioned media from other HEK293 cells that express HA- and Gaussia luciferase-tagged MADD-4 or SLT-1 ligands, and immunoprecipitated to determine the relative amounts of ligand that co-immunoprecipitates with the receptor (see the materials and methods section for more details). C . The western blot on the left shows the five immunoprecipitated FLAG-tagged receptors. The western blot on the right shows the two HA- and Gaussia luciferase-tagged ligands that were collected from cell culture. D . The normalized relative levels of luciferase signal that immunoprecipitated with each potential ligand-receptor complex. E–I . Shown are animals harbouring one of three different transgenes that drive the expression of either neuronally-expressed MADD-4::YFP (from the trIs66 transgenic array) ( E ), muscle-expressed MADD-4::YFP (from the trIs78 transgenic array) ( F ), or muscle-expressed EVA-1::CFP (from the trIs89 transgenic array) ( G ), or animals harbouring two of the transgenes; trIs66 and trIs89 ( H ) and trIs78 and trIs89 ( I ). The relative levels of MADD-4::YFP expression from trIs66 and trIs78 is shown in Figure S2a . Images show either the CFP channel (top), YFP channel (middle) or a merged view (bottom). Arrows in ‘H’ indicate the localization of MADD-4::YFP to EVA-1::CFP expressing muscles; arrows in ‘I’ indicate the vesicularization of MADD-4::YFP and EVA-1::CFP in the muscle cells. J . The quantification of neuronally-secreted MADD-4::YFP localization to muscles over-expressing the indicated receptor. K . The quantification of CFP vesicles in animals that over-express the indicated CFP-tagged receptors (x-axis) in muscles in either the presence of MADD-4::YFP expressed from dorsal muscles (mMADD-4) or pan-neuronally (nMADD-4). The colocalization of MADD-4 and EVA-1 with the RAB-11 and RAB-5 endosomal markers are shown in Figure S2b and S2c . L M . MADD-4::YFP fails to induce obvious vesicularization of UNC-40::CFP in a wild type background (L), but YFP-CFP vesicles are obvious in animals that lack UNC-6 (M). In A, J, and K, statistical significance ( p

    Article Snippet: One third of the immunoprecipitate was taken for Gaussia luciferase assays by using the BioLux Kit (NEB) and MicroLumat Plus LB96V luminometer (BERTHOLD).

    Techniques: Luciferase, Immunoprecipitation, Western Blot, Cell Culture, Expressing, Transgenic Assay

    Susceptibility of directed differentiated cells from Hdo cells to HCV, HBV, and JEV replication A . Control, non-induced cells (white circles), hepatic induction cells (black squares), and cholangiocytic induction cells (black triangles) were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 1b [Con1, SGR-Con1/GLuc (solid lines) and GND (dashed lines)] in upper graphs and 2a [JFH-1, SGR-JFH1/GLuc (solid lines) and GND (dashed lines)] in lower graphs. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . Expression of miR-200a in HuH-7, Hdo-17, Hdo-23, and their respective hepatic induction cells and cholangiocytic induction cells were examined by qRT-PCR using the TaqMan microRNA Reverse Transcription Kit with miR-200a specific RT primer from the TaqMan microRNA Assay. Expression of miR-200a was normalized to that of Z30 snoRNA (SNORD7), and relative expression levels to HuH-7 cells with no induction treatment are shown. C . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pUC19, pUC-HBV-Bj (GT-B), or -Ce (GT-C). At 3 days or 5 days after transfection, particle-associated HBV DNAs in culture supernatants of transfected cells were measured by qRT-PCR (upper panel). Cell lysates were subjected to immunoblotting of HBs antigens and GAPDH (lower panel). D . HuH-7, Hdo-17, and Hdo-23 cells were infected with JEV (MOI=0.01). Cells were fixed and stained with dsRNA antibody (green) and DAPI (blue) at 3 days post-infection. Bar indicates 200 μm. All assays were performed in triplicate. (A)-(C) Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with control cells with no induction treatment (B) or HuH-7 cells (C) are shown. * p

    Journal: Oncotarget

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation

    doi: 10.18632/oncotarget.19108

    Figure Lengend Snippet: Susceptibility of directed differentiated cells from Hdo cells to HCV, HBV, and JEV replication A . Control, non-induced cells (white circles), hepatic induction cells (black squares), and cholangiocytic induction cells (black triangles) were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 1b [Con1, SGR-Con1/GLuc (solid lines) and GND (dashed lines)] in upper graphs and 2a [JFH-1, SGR-JFH1/GLuc (solid lines) and GND (dashed lines)] in lower graphs. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . Expression of miR-200a in HuH-7, Hdo-17, Hdo-23, and their respective hepatic induction cells and cholangiocytic induction cells were examined by qRT-PCR using the TaqMan microRNA Reverse Transcription Kit with miR-200a specific RT primer from the TaqMan microRNA Assay. Expression of miR-200a was normalized to that of Z30 snoRNA (SNORD7), and relative expression levels to HuH-7 cells with no induction treatment are shown. C . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pUC19, pUC-HBV-Bj (GT-B), or -Ce (GT-C). At 3 days or 5 days after transfection, particle-associated HBV DNAs in culture supernatants of transfected cells were measured by qRT-PCR (upper panel). Cell lysates were subjected to immunoblotting of HBs antigens and GAPDH (lower panel). D . HuH-7, Hdo-17, and Hdo-23 cells were infected with JEV (MOI=0.01). Cells were fixed and stained with dsRNA antibody (green) and DAPI (blue) at 3 days post-infection. Bar indicates 200 μm. All assays were performed in triplicate. (A)-(C) Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with control cells with no induction treatment (B) or HuH-7 cells (C) are shown. * p

    Article Snippet: Culture supernatant (20 μl) collected from cells expressing plasmids carrying the GLuc reporter was used to determine GLuc activity by using a BioLux Gaussia Luciferase Assay Kit (New England Biolabs).

    Techniques: Transfection, Luciferase, Derivative Assay, Expressing, Quantitative RT-PCR, TaqMan microRNA Assay, Infection, Staining

    Susceptibility of Hdo cells to HCV RNA replication and involvement of miR-200a in replication A . HuH-7 (white circles), Hdo-17 (black squares), and Hdo-23 (black triangles) cells were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 2a [JFH-1, SGR-JFH1/GLuc wild (solid lines) and GND (dashed lines)] in left panel and 1b [Con1, SGR-Con1/GLuc wild (solid lines) and GND (dashed lines)] in right panel. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pRLucHCVLuc (white bars) or pRLucEMCVLuc (black bars). At 60 h after transfection, luciferase activity in cell lysates was measured by the dual-luciferase reporter assay system. The vertical axis indicates activity of firefly luciferase to that of Renilla luciferase. C . HuH-7, Hdo-17, Hdo-23, and Hec1B cells were infected with a lentivirus encoding miR-122 (white circles) or AcGFP as control (black triangles). Cells were transfected with pHH/SGR-JFH1/GLuc (solid lines) or pHH/SGR-JFH1/GLuc/GND (dashed lines) and pSV40-CLuc 48 h post-infection. Luciferase activity in the supernatant was measured by the BioLux Dual Luciferase Starter Kit at the indicated time points. Relative expression levels of secreted GLuc to CLuc in the supernatants are shown. D . HuH 5-15 cells, which are HCV genotype 1b-replicon cells, were transfected with miRNAs, miRNA inhibitors, and non-target miRNA control (miR cont) by ScreenFect A. At 3 days after transfection, HCV copies in cells were measured by qRT-PCR (grey bars). Cell viability was determined by the ATP concentration in cell lysates with the CellTiter-Glo Luminescent Cell Viability Assay (white bars). E . Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVcc (J6/JFH1, MOI=0.5). HCV copies in cells were measured by qRT-PCR at 3 days post-infection (upper graph). Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVpp (grey bars) or VSVpp (white bars). NanoLuc activity was measured at 1 day post-infection (lower graph). All assays were performed in triplicate. Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with HuH-7 cells (B) or miR cont (D) and (E) are shown. * p

    Journal: Oncotarget

    Article Title: Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation

    doi: 10.18632/oncotarget.19108

    Figure Lengend Snippet: Susceptibility of Hdo cells to HCV RNA replication and involvement of miR-200a in replication A . HuH-7 (white circles), Hdo-17 (black squares), and Hdo-23 (black triangles) cells were transfected with subgenomic luciferase reporter replicon RNAs derived from HCV genotypes 2a [JFH-1, SGR-JFH1/GLuc wild (solid lines) and GND (dashed lines)] in left panel and 1b [Con1, SGR-Con1/GLuc wild (solid lines) and GND (dashed lines)] in right panel. Luciferase activities in supernatants were measured by the BioLux Gaussia Luciferase Assay Kit at the indicated time points. B . HuH-7, Hdo-17, and Hdo-23 cells were transfected with pRLucHCVLuc (white bars) or pRLucEMCVLuc (black bars). At 60 h after transfection, luciferase activity in cell lysates was measured by the dual-luciferase reporter assay system. The vertical axis indicates activity of firefly luciferase to that of Renilla luciferase. C . HuH-7, Hdo-17, Hdo-23, and Hec1B cells were infected with a lentivirus encoding miR-122 (white circles) or AcGFP as control (black triangles). Cells were transfected with pHH/SGR-JFH1/GLuc (solid lines) or pHH/SGR-JFH1/GLuc/GND (dashed lines) and pSV40-CLuc 48 h post-infection. Luciferase activity in the supernatant was measured by the BioLux Dual Luciferase Starter Kit at the indicated time points. Relative expression levels of secreted GLuc to CLuc in the supernatants are shown. D . HuH 5-15 cells, which are HCV genotype 1b-replicon cells, were transfected with miRNAs, miRNA inhibitors, and non-target miRNA control (miR cont) by ScreenFect A. At 3 days after transfection, HCV copies in cells were measured by qRT-PCR (grey bars). Cell viability was determined by the ATP concentration in cell lysates with the CellTiter-Glo Luminescent Cell Viability Assay (white bars). E . Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVcc (J6/JFH1, MOI=0.5). HCV copies in cells were measured by qRT-PCR at 3 days post-infection (upper graph). Huh7.5.1 cells were transfected with miRNAs, miR-122 inhibitor, or miR cont by the Xfect miRNA Transfection Kit. At 1 day after transfection, cells were infected with HCVpp (grey bars) or VSVpp (white bars). NanoLuc activity was measured at 1 day post-infection (lower graph). All assays were performed in triplicate. Results are presented as means ± SEM ( n = 3). Statistically significant differences compared with HuH-7 cells (B) or miR cont (D) and (E) are shown. * p

    Article Snippet: Culture supernatant (20 μl) collected from cells expressing plasmids carrying the GLuc reporter was used to determine GLuc activity by using a BioLux Gaussia Luciferase Assay Kit (New England Biolabs).

    Techniques: Transfection, Luciferase, Derivative Assay, Activity Assay, Reporter Assay, Infection, Expressing, Quantitative RT-PCR, Concentration Assay, Cell Viability Assay