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    Structured Review

    Qiagen subconfluent cells
    A . HAS3 inhibition decreased HAS3 protein production in HCT116 cells. Both HCT116 control and HAS3 scrambled cell lysates demonstrated intense HAS3 protein bands, whereas the HAS3 band was undetectable in the HAS3 silenced lysates. HAS3 protein was detected by Western blot analysis. <t>Subconfluent</t> cells were and incubated in lysis buffer for 60 minutes on ice. Protein samples (20ug) were separated by electrophoresis and transferred Hybond-ECL nitrocellulose membrane (Amersham Biosciences, Pascataway, NJ). The membrane was blocked overnight, incubated with primary antibody, washed, incubated with a peroxidase-conjugated secondary antibody, washed, and developed with a chemiluminescent detection kit. B . HAS3 inhibition decreased HAS3 protein detection by immunocytochemistry in cell culture. HAS3 (red fluorescence) was easily detectable in both the HCT116 control cells and the HAS3 scrambled cells, but was minimally detectable in the HAS3 silenced cells. Beta-actin control (green fluorescence) was similar among all cell lines. Cells in 24-well plates were fixed and then permeabilized, blocked, incubated with primary antibody, washed, incubated with secondary antibody and FITC conjugated phalloidin, then viewed using a Zeiss Observer. A1 microscope at 100× magnification.
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    1) Product Images from "Inhibition of Hyaluronan Synthase-3 Decreases Subcutaneous Colon Cancer Growth by Increasing Apoptosis"

    Article Title: Inhibition of Hyaluronan Synthase-3 Decreases Subcutaneous Colon Cancer Growth by Increasing Apoptosis

    Journal: Anti-Cancer Agents in Medicinal Chemistry

    doi:

    A . HAS3 inhibition decreased HAS3 protein production in HCT116 cells. Both HCT116 control and HAS3 scrambled cell lysates demonstrated intense HAS3 protein bands, whereas the HAS3 band was undetectable in the HAS3 silenced lysates. HAS3 protein was detected by Western blot analysis. Subconfluent cells were and incubated in lysis buffer for 60 minutes on ice. Protein samples (20ug) were separated by electrophoresis and transferred Hybond-ECL nitrocellulose membrane (Amersham Biosciences, Pascataway, NJ). The membrane was blocked overnight, incubated with primary antibody, washed, incubated with a peroxidase-conjugated secondary antibody, washed, and developed with a chemiluminescent detection kit. B . HAS3 inhibition decreased HAS3 protein detection by immunocytochemistry in cell culture. HAS3 (red fluorescence) was easily detectable in both the HCT116 control cells and the HAS3 scrambled cells, but was minimally detectable in the HAS3 silenced cells. Beta-actin control (green fluorescence) was similar among all cell lines. Cells in 24-well plates were fixed and then permeabilized, blocked, incubated with primary antibody, washed, incubated with secondary antibody and FITC conjugated phalloidin, then viewed using a Zeiss Observer. A1 microscope at 100× magnification.
    Figure Legend Snippet: A . HAS3 inhibition decreased HAS3 protein production in HCT116 cells. Both HCT116 control and HAS3 scrambled cell lysates demonstrated intense HAS3 protein bands, whereas the HAS3 band was undetectable in the HAS3 silenced lysates. HAS3 protein was detected by Western blot analysis. Subconfluent cells were and incubated in lysis buffer for 60 minutes on ice. Protein samples (20ug) were separated by electrophoresis and transferred Hybond-ECL nitrocellulose membrane (Amersham Biosciences, Pascataway, NJ). The membrane was blocked overnight, incubated with primary antibody, washed, incubated with a peroxidase-conjugated secondary antibody, washed, and developed with a chemiluminescent detection kit. B . HAS3 inhibition decreased HAS3 protein detection by immunocytochemistry in cell culture. HAS3 (red fluorescence) was easily detectable in both the HCT116 control cells and the HAS3 scrambled cells, but was minimally detectable in the HAS3 silenced cells. Beta-actin control (green fluorescence) was similar among all cell lines. Cells in 24-well plates were fixed and then permeabilized, blocked, incubated with primary antibody, washed, incubated with secondary antibody and FITC conjugated phalloidin, then viewed using a Zeiss Observer. A1 microscope at 100× magnification.

    Techniques Used: Inhibition, Western Blot, Incubation, Lysis, Electrophoresis, Immunocytochemistry, Cell Culture, Fluorescence, Microscopy

    Related Articles

    Clone Assay:

    Article Title: Altered VEGF mRNA Stability following Treatments with Immunosuppressive Agents
    Article Snippet: .. Subconfluent cells were first preincubated with 5.0 μg/ml actinomycin D for 1 h; cells were then treated with immunosuppressive agents for different time intervals (0–6 h) in the presence of actinomycin D. Total RNA was prepared using the RNeasy isolation kit (Qiagen), and cDNA was synthesized using cloned avian myeloblastosis virus first-strand synthesis kit (Invitrogen). .. To analyze VEGF expression, real-time PCR was performed using the Assays-on-Demand Gene Expression product (TaqMan, Mammalian Gene Collection probes) according to the manufacturer's instructions (Applied Biosystems).

    Luciferase:

    Article Title: Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast cancer
    Article Snippet: siRNA oligo sequences (HIF-1α, 5-UCAAGUUGCUGGUCAUCAG; HIF-2α, 5-ACUGCUAUCAAAGAUGCUG-3; ESR1#1, GAGAAGUAUUCAAGGACAU; ESR1#2, AAUGAUGAAAGGUGGGAUA; Luciferase, CUUACGCUGAGUACUUCGA) were synthesized by Dharmacon. .. The SMARTpool siRNA oligos for ERα and nontargeting control were purchased from Dharmacon. siRNAs were transfected into subconfluent cells using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions.

    Synthesized:

    Article Title: The histone demethylase JMJD2B is regulated by estrogen receptor alpha and hypoxia and is a key mediator of estrogen induced growth
    Article Snippet: siRNAs were transfected into subconfluent cells using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. .. The target sequences for HIF-1α and HIF-2α were synthesized by Eurogentec (Eurogentec, Southampton, UK) were as follows: HIF-1α, 5-UCAAGUUGCUGGUCAUCAGdTdT-3; HIF-2α, 5-ACUGCUAUCAAAGAUGCUGdTdT-3.

    Article Title: Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast cancer
    Article Snippet: siRNA oligo sequences (HIF-1α, 5-UCAAGUUGCUGGUCAUCAG; HIF-2α, 5-ACUGCUAUCAAAGAUGCUG-3; ESR1#1, GAGAAGUAUUCAAGGACAU; ESR1#2, AAUGAUGAAAGGUGGGAUA; Luciferase, CUUACGCUGAGUACUUCGA) were synthesized by Dharmacon. .. The SMARTpool siRNA oligos for ERα and nontargeting control were purchased from Dharmacon. siRNAs were transfected into subconfluent cells using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions.

    Article Title: Altered VEGF mRNA Stability following Treatments with Immunosuppressive Agents
    Article Snippet: .. Subconfluent cells were first preincubated with 5.0 μg/ml actinomycin D for 1 h; cells were then treated with immunosuppressive agents for different time intervals (0–6 h) in the presence of actinomycin D. Total RNA was prepared using the RNeasy isolation kit (Qiagen), and cDNA was synthesized using cloned avian myeloblastosis virus first-strand synthesis kit (Invitrogen). .. To analyze VEGF expression, real-time PCR was performed using the Assays-on-Demand Gene Expression product (TaqMan, Mammalian Gene Collection probes) according to the manufacturer's instructions (Applied Biosystems).

    Article Title: Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe
    Article Snippet: Northern analysis Total RNA from subconfluent cells was isolated using the RNeasy extraction kit (Qiagen) and 1 μg was used to detect HCV replicon RNA and β-actin mRNA. .. Biotinylated negative sense probes complementary to HCV genome region 6648-7770 bp (NS5b) and β-actin (GenBank accession numbers AJ242654 and X00351 , respectively) were synthesized using the MEGAscript T7 kit (Ambion, Foster City, CA), biotin-11-UTP and biotin-11-CTP (PerkinElmer, Boston, MA) following the supplier's protocol.

    Article Title: Knock-down of Amphiregulin Inhibits Cellular Invasion in Inflammatory Breast Cancer
    Article Snippet: Total RNA was isolated from subconfluent cells using an RNeasy kit (Qiagen, Valenica, CA, USA) and reverse-transcribed into cDNA using the Superscript III First-Strand Synthesis kit (Invitrogen, Calsbad, CA, USA). .. Primer sets specific to approximately 100bp sequences of target genes and a control gene (PUM1) used were designed and synthesized by Invitrogen.

    Construct:

    Article Title: Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method
    Article Snippet: Subconfluent cells grown in a 100 mm dish were transfected with 5 μg DNA using the Polyfect transfection reagent using the protocol of the manufacturer (Qiagen, West Sussex, UK), however with half of the recommended PolyFect volumes. .. Co-transfections (ratio 4:1) were performed with either constructs expressing the human histamine H3 receptor together with an empty vector and chimeric G protein Gqi5, respectively.

    SYBR Green Assay:

    Article Title: Knock-down of Amphiregulin Inhibits Cellular Invasion in Inflammatory Breast Cancer
    Article Snippet: Total RNA was isolated from subconfluent cells using an RNeasy kit (Qiagen, Valenica, CA, USA) and reverse-transcribed into cDNA using the Superscript III First-Strand Synthesis kit (Invitrogen, Calsbad, CA, USA). .. Real-time RT–PCR was performed in 25 ul reactions using 96-well plates, 100 to 200 ng cDNA, and the FastStart SYBR Green Master Mix (Roche Diagnostics, Mannheim, Germany).

    Incubation:

    Article Title: Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells
    Article Snippet: Subconfluent cells were co-transfected by using Superfect (Qiagen, Hilden, Germany) according to the manufacturer's instructions (Clonetech, Palo Alto, USA) with 0.1 μg of hTLR2 (generously provided by Tularik Inc., San Francisco, USA [ ]) and dominant-negative A-Fos (kind gift of Dr. Charles Vinson, NCI, NIH, Rockville, MD) [ ], or Fra1- or Fra2-antisense (kind gift of Dr. Vladimir Berezin, Institute of Molecular Pathology, School of Medicine, Copenhagen University, Copenhagen, Denmark) [ ] expression vectors or control vector. .. Cells were incubated with R6x for 6 h.

    Article Title: Aldose reductase inhibition suppresses colon cancer cell viability by modulating miR-21 mediated PDCD4 expression
    Article Snippet: Subconfluent cells were growth-arrested in 0.1% FBS with or without the AR inhibitor, fidarestat (2 μM) or transfected with control siRNA and PDCD4 siRNA using Hiperfect transfection reagent (Qiagen). .. After 24 h, cells were treated with EGF (10 ng/ml) and incubated for 24 h. Cell viability was determined by MTT assay as described previously.

    Article Title: IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells
    Article Snippet: The next day, subconfluent cells were transfected with a plasmid encoding cDNA for GFP-tagged IGFBP-3 (IGFBP-3-GFP), IGFBP-3-His, or pEGFP-N1 using SuperFect (Qiagen) in a 1:10 ratio as previously described [ ]. .. After a 24-hour recovery in serum-containing media, cells were rinsed twice in PBS and incubated with fresh SF DMEM-H for 1 hour, then treated as indicated in the figure legends.

    Article Title: Aldose Reductase Inhibition Prevents Hypoxia-induced Increase in Hypoxia-inducible Factor-1? (HIF-1?) and Vascular Endothelial Growth Factor (VEGF) by Regulating 26 S Proteasome-mediated Protein Degradation in Human Colon Cancer Cells *
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    Amplification:

    Article Title: AQP1 Is Not Only a Water Channel: It Contributes to Cell Migration through Lin7/Beta-Catenin
    Article Snippet: Total RNA was purified from subconfluent cells using the RNeasy Mini Kit from Qiagen. .. One microgram of total RNA was reverse transcribed and amplified by Enhanced avian hs RT-PCR (Sigma) according to the manufacturer's instructions.

    Article Title: Altered VEGF mRNA Stability following Treatments with Immunosuppressive Agents
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    Article Title: HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1? in normoxia
    Article Snippet: Subconfluent cells were lysed, and RNA was purified using the RNeasy Mini Kit (Qiagen). .. Membranes were hybridized with the indicated 32 P-labelled cDNA probe, and results were visualized using a phosphorimager (Storm 840; Amersham Biosciences). cDNA probes were obtained by RT–PCR using the primers allowing the amplification of nucleotides 298–563, 3800–4438 and 327–1048, corresponding to PHD1 (accession No. ), PHD2 (accession No. ) and PHD3 (accession No. ), respectively.

    Article Title: Inhibition of Hyaluronan Synthase-3 Decreases Subcutaneous Colon Cancer Growth by Increasing Apoptosis
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    Expressing:

    Article Title: Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells
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    Article Title: EGFR-Mediated Carcinoma Cell Metastasis Mediated by Integrin ?v?5 Depends on Activation of c-Src and Cleavage of MUC1
    Article Snippet: Short hairpin RNA knockdown MUC1 and GFP control lentiviral shRNA in pLKO.1 expressing system were from Open Biosystems. .. For some experiments, subconfluent cells were transfected with MUC1 small interfering RNAs (Qiagen) using Lipofectamine2000 (Life Technologies), and migration assays were performed at 48 h after transfection.

    Article Title: AQP1 Is Not Only a Water Channel: It Contributes to Cell Migration through Lin7/Beta-Catenin
    Article Snippet: Supporting Information Expression of AQPs and intracellular localization. .. Total RNA was purified from subconfluent cells using the RNeasy Mini Kit from Qiagen.

    Article Title: Altered VEGF mRNA Stability following Treatments with Immunosuppressive Agents
    Article Snippet: Subconfluent cells were first preincubated with 5.0 μg/ml actinomycin D for 1 h; cells were then treated with immunosuppressive agents for different time intervals (0–6 h) in the presence of actinomycin D. Total RNA was prepared using the RNeasy isolation kit (Qiagen), and cDNA was synthesized using cloned avian myeloblastosis virus first-strand synthesis kit (Invitrogen). .. To analyze VEGF expression, real-time PCR was performed using the Assays-on-Demand Gene Expression product (TaqMan, Mammalian Gene Collection probes) according to the manufacturer's instructions (Applied Biosystems).

    Article Title: Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method
    Article Snippet: Subconfluent cells grown in a 100 mm dish were transfected with 5 μg DNA using the Polyfect transfection reagent using the protocol of the manufacturer (Qiagen, West Sussex, UK), however with half of the recommended PolyFect volumes. .. Co-transfections (ratio 4:1) were performed with either constructs expressing the human histamine H3 receptor together with an empty vector and chimeric G protein Gqi5, respectively.

    Article Title: Inhibition of Hyaluronan Synthase-3 Decreases Subcutaneous Colon Cancer Growth by Increasing Apoptosis
    Article Snippet: Paragraph title: Determination of HAS Expression and HA Production ... Poly(A)+ RNA was isolated from subconfluent cells using the Oligotex mRNA isolation kit (Qiagen, Valencia, CA) and quantified by Ribogreen fluorescence (Molecular Probes, Eugene, OR). mRNA templates (50 ng) were reverse transcribed with an oligo(dT) primer using the Superscript II first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA).

    Modification:

    Article Title: Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method
    Article Snippet: Cell culture and transfections Human tsA201 cells, a transformed HEK-293 cell line , were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAX, supplemented with 10% foetal bovine serum, penicillin 100 U/ml and streptomycin 100 µg/ml. .. Subconfluent cells grown in a 100 mm dish were transfected with 5 μg DNA using the Polyfect transfection reagent using the protocol of the manufacturer (Qiagen, West Sussex, UK), however with half of the recommended PolyFect volumes.

    Transformation Assay:

    Article Title: Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method
    Article Snippet: Cell culture and transfections Human tsA201 cells, a transformed HEK-293 cell line , were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAX, supplemented with 10% foetal bovine serum, penicillin 100 U/ml and streptomycin 100 µg/ml. .. Subconfluent cells grown in a 100 mm dish were transfected with 5 μg DNA using the Polyfect transfection reagent using the protocol of the manufacturer (Qiagen, West Sussex, UK), however with half of the recommended PolyFect volumes.

    Hybridization:

    Article Title: Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe
    Article Snippet: Northern analysis Total RNA from subconfluent cells was isolated using the RNeasy extraction kit (Qiagen) and 1 μg was used to detect HCV replicon RNA and β-actin mRNA. .. Northern blotting and hybridization were performed using the NorthernMax kit (Ambion) and Hybond XL nylon membranes (Amersham Biosciences, Piscataway, NJ).

    Transfection:

    Article Title: Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells
    Article Snippet: Paragraph title: Plasmids, and transient transfection procedures ... Subconfluent cells were co-transfected by using Superfect (Qiagen, Hilden, Germany) according to the manufacturer's instructions (Clonetech, Palo Alto, USA) with 0.1 μg of hTLR2 (generously provided by Tularik Inc., San Francisco, USA [ ]) and dominant-negative A-Fos (kind gift of Dr. Charles Vinson, NCI, NIH, Rockville, MD) [ ], or Fra1- or Fra2-antisense (kind gift of Dr. Vladimir Berezin, Institute of Molecular Pathology, School of Medicine, Copenhagen University, Copenhagen, Denmark) [ ] expression vectors or control vector.

    Article Title: The histone demethylase JMJD2B is regulated by estrogen receptor alpha and hypoxia and is a key mediator of estrogen induced growth
    Article Snippet: .. siRNAs were transfected into subconfluent cells using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. .. The target sequences for HIF-1α and HIF-2α were synthesized by Eurogentec (Eurogentec, Southampton, UK) were as follows: HIF-1α, 5-UCAAGUUGCUGGUCAUCAGdTdT-3; HIF-2α, 5-ACUGCUAUCAAAGAUGCUGdTdT-3.

    Article Title: Aldose reductase inhibition suppresses colon cancer cell viability by modulating miR-21 mediated PDCD4 expression
    Article Snippet: .. Subconfluent cells were growth-arrested in 0.1% FBS with or without the AR inhibitor, fidarestat (2 μM) or transfected with control siRNA and PDCD4 siRNA using Hiperfect transfection reagent (Qiagen). .. After 24 h, cells were treated with EGF (10 ng/ml) and incubated for 24 h. Cell viability was determined by MTT assay as described previously.

    Article Title: Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast cancer
    Article Snippet: .. The SMARTpool siRNA oligos for ERα and nontargeting control were purchased from Dharmacon. siRNAs were transfected into subconfluent cells using HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. .. Alternatively, reverse transfection with RNAiMAX (Invitrogen) was used according to the manufacturer’s instructions.

    Article Title: EGFR-Mediated Carcinoma Cell Metastasis Mediated by Integrin ?v?5 Depends on Activation of c-Src and Cleavage of MUC1
    Article Snippet: .. For some experiments, subconfluent cells were transfected with MUC1 small interfering RNAs (Qiagen) using Lipofectamine2000 (Life Technologies), and migration assays were performed at 48 h after transfection. .. MUC1 expression Full length and cytoplasmic domain-deleted MUC1 were generously provided by Michael Hollingsworth .

    Article Title: IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells
    Article Snippet: .. The next day, subconfluent cells were transfected with a plasmid encoding cDNA for GFP-tagged IGFBP-3 (IGFBP-3-GFP), IGFBP-3-His, or pEGFP-N1 using SuperFect (Qiagen) in a 1:10 ratio as previously described [ ]. .. After a 24-hour recovery in serum-containing media, cells were rinsed twice in PBS and incubated with fresh SF DMEM-H for 1 hour, then treated as indicated in the figure legends.

    Article Title: Aldose Reductase Inhibition Prevents Hypoxia-induced Increase in Hypoxia-inducible Factor-1? (HIF-1?) and Vascular Endothelial Growth Factor (VEGF) by Regulating 26 S Proteasome-mediated Protein Degradation in Human Colon Cancer Cells *
    Article Snippet: .. Subconfluent cells were growth-arrested in 0.1% FBS with or without the AR inhibitor fidarestat (5 μ m ) or transfected with AR siRNA or control siRNA using RNAiFect reagent (Qiagen). .. After 24 h, cells were treated with bFGF (10 ng/ml) or serum (5%) and incubated at normoxia or hypoxia conditions for 24 h. Cells incubated with the AR inhibitor or transfection reagent alone served as control.

    Article Title: Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method
    Article Snippet: .. Subconfluent cells grown in a 100 mm dish were transfected with 5 μg DNA using the Polyfect transfection reagent using the protocol of the manufacturer (Qiagen, West Sussex, UK), however with half of the recommended PolyFect volumes. .. Co-transfections (ratio 4:1) were performed with either constructs expressing the human histamine H3 receptor together with an empty vector and chimeric G protein Gqi5, respectively.

    Northern Blot:

    Article Title: HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1? in normoxia
    Article Snippet: Paragraph title: Northern blotting ... Subconfluent cells were lysed, and RNA was purified using the RNeasy Mini Kit (Qiagen).

    Article Title: Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe
    Article Snippet: .. Northern analysis Total RNA from subconfluent cells was isolated using the RNeasy extraction kit (Qiagen) and 1 μg was used to detect HCV replicon RNA and β-actin mRNA. .. Biotinylated negative sense probes complementary to HCV genome region 6648-7770 bp (NS5b) and β-actin (GenBank accession numbers AJ242654 and X00351 , respectively) were synthesized using the MEGAscript T7 kit (Ambion, Foster City, CA), biotin-11-UTP and biotin-11-CTP (PerkinElmer, Boston, MA) following the supplier's protocol.

    Cell Culture:

    Article Title: Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells
    Article Snippet: Plasmids, and transient transfection procedures HEK293 cells were cultured in 12-well plates with DMEM supplemented with 10% FCS. .. Subconfluent cells were co-transfected by using Superfect (Qiagen, Hilden, Germany) according to the manufacturer's instructions (Clonetech, Palo Alto, USA) with 0.1 μg of hTLR2 (generously provided by Tularik Inc., San Francisco, USA [ ]) and dominant-negative A-Fos (kind gift of Dr. Charles Vinson, NCI, NIH, Rockville, MD) [ ], or Fra1- or Fra2-antisense (kind gift of Dr. Vladimir Berezin, Institute of Molecular Pathology, School of Medicine, Copenhagen University, Copenhagen, Denmark) [ ] expression vectors or control vector.

    Article Title: Aldose reductase inhibition suppresses colon cancer cell viability by modulating miR-21 mediated PDCD4 expression
    Article Snippet: HT29 cells were cultured in McCoy's medium in a 96-well plate. .. Subconfluent cells were growth-arrested in 0.1% FBS with or without the AR inhibitor, fidarestat (2 μM) or transfected with control siRNA and PDCD4 siRNA using Hiperfect transfection reagent (Qiagen).

    Article Title: Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method
    Article Snippet: Paragraph title: Cell culture and transfections ... Subconfluent cells grown in a 100 mm dish were transfected with 5 μg DNA using the Polyfect transfection reagent using the protocol of the manufacturer (Qiagen, West Sussex, UK), however with half of the recommended PolyFect volumes.

    Polymerase Chain Reaction:

    Article Title: The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2
    Article Snippet: Subconfluent cells were collected, and total RNA was isolated using an RNeasy mini kit (Qiagen, Crawley, West Sussex, UK). .. PCR products were subjected to electrophoresis on a 2% agarose gel, and DNA was visualised with ethidium bromide staining.

    Article Title: Inhibition of Hyaluronan Synthase-3 Decreases Subcutaneous Colon Cancer Growth by Increasing Apoptosis
    Article Snippet: Poly(A)+ RNA was isolated from subconfluent cells using the Oligotex mRNA isolation kit (Qiagen, Valencia, CA) and quantified by Ribogreen fluorescence (Molecular Probes, Eugene, OR). mRNA templates (50 ng) were reverse transcribed with an oligo(dT) primer using the Superscript II first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA). .. A PCR oligonucleotide specific for HAS3 was utilized as previously described [ ].

    MTT Assay:

    Article Title: Aldose reductase inhibition suppresses colon cancer cell viability by modulating miR-21 mediated PDCD4 expression
    Article Snippet: Subconfluent cells were growth-arrested in 0.1% FBS with or without the AR inhibitor, fidarestat (2 μM) or transfected with control siRNA and PDCD4 siRNA using Hiperfect transfection reagent (Qiagen). .. After 24 h, cells were treated with EGF (10 ng/ml) and incubated for 24 h. Cell viability was determined by MTT assay as described previously.

    Fluorescence:

    Article Title: IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells
    Article Snippet: Paragraph title: J. Fluorescence Microscopy ... The next day, subconfluent cells were transfected with a plasmid encoding cDNA for GFP-tagged IGFBP-3 (IGFBP-3-GFP), IGFBP-3-His, or pEGFP-N1 using SuperFect (Qiagen) in a 1:10 ratio as previously described [ ].

    Article Title: Inhibition of Hyaluronan Synthase-3 Decreases Subcutaneous Colon Cancer Growth by Increasing Apoptosis
    Article Snippet: .. Poly(A)+ RNA was isolated from subconfluent cells using the Oligotex mRNA isolation kit (Qiagen, Valencia, CA) and quantified by Ribogreen fluorescence (Molecular Probes, Eugene, OR). mRNA templates (50 ng) were reverse transcribed with an oligo(dT) primer using the Superscript II first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA). .. A PCR oligonucleotide specific for HAS3 was utilized as previously described [ ].

    Isolation:

    Article Title: Altered VEGF mRNA Stability following Treatments with Immunosuppressive Agents
    Article Snippet: .. Subconfluent cells were first preincubated with 5.0 μg/ml actinomycin D for 1 h; cells were then treated with immunosuppressive agents for different time intervals (0–6 h) in the presence of actinomycin D. Total RNA was prepared using the RNeasy isolation kit (Qiagen), and cDNA was synthesized using cloned avian myeloblastosis virus first-strand synthesis kit (Invitrogen). .. To analyze VEGF expression, real-time PCR was performed using the Assays-on-Demand Gene Expression product (TaqMan, Mammalian Gene Collection probes) according to the manufacturer's instructions (Applied Biosystems).

    Article Title: Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe
    Article Snippet: .. Northern analysis Total RNA from subconfluent cells was isolated using the RNeasy extraction kit (Qiagen) and 1 μg was used to detect HCV replicon RNA and β-actin mRNA. .. Biotinylated negative sense probes complementary to HCV genome region 6648-7770 bp (NS5b) and β-actin (GenBank accession numbers AJ242654 and X00351 , respectively) were synthesized using the MEGAscript T7 kit (Ambion, Foster City, CA), biotin-11-UTP and biotin-11-CTP (PerkinElmer, Boston, MA) following the supplier's protocol.

    Article Title: The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2
    Article Snippet: .. Subconfluent cells were collected, and total RNA was isolated using an RNeasy mini kit (Qiagen, Crawley, West Sussex, UK). .. Following extraction, RNA was treated with DNase I (Ambion, Huntingdon, Cambridgeshire, UK). cDNA synthesis was carried out using 10 μg of total RNA in a 50 μl reaction containing 50 mM Tris HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 500 ng random hexamers, 1 μg oligo (dT)15 primer, 400 μM dNTPs, 40 units of RNase inhibitor, and 400 units of MMLV reverse transcriptase (Promega, Southampton, UK).

    Article Title: Inhibition of Hyaluronan Synthase-3 Decreases Subcutaneous Colon Cancer Growth by Increasing Apoptosis
    Article Snippet: .. Poly(A)+ RNA was isolated from subconfluent cells using the Oligotex mRNA isolation kit (Qiagen, Valencia, CA) and quantified by Ribogreen fluorescence (Molecular Probes, Eugene, OR). mRNA templates (50 ng) were reverse transcribed with an oligo(dT) primer using the Superscript II first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA). .. A PCR oligonucleotide specific for HAS3 was utilized as previously described [ ].

    Microscopy:

    Article Title: IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells
    Article Snippet: Paragraph title: J. Fluorescence Microscopy ... The next day, subconfluent cells were transfected with a plasmid encoding cDNA for GFP-tagged IGFBP-3 (IGFBP-3-GFP), IGFBP-3-His, or pEGFP-N1 using SuperFect (Qiagen) in a 1:10 ratio as previously described [ ].

    Purification:

    Article Title: AQP1 Is Not Only a Water Channel: It Contributes to Cell Migration through Lin7/Beta-Catenin
    Article Snippet: .. Total RNA was purified from subconfluent cells using the RNeasy Mini Kit from Qiagen. .. One microgram of total RNA was reverse transcribed and amplified by Enhanced avian hs RT-PCR (Sigma) according to the manufacturer's instructions.

    Article Title: HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1? in normoxia
    Article Snippet: .. Subconfluent cells were lysed, and RNA was purified using the RNeasy Mini Kit (Qiagen). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: AQP1 Is Not Only a Water Channel: It Contributes to Cell Migration through Lin7/Beta-Catenin
    Article Snippet: Panel A. RT-PCR of AQP1 in WM115 and HMEC-1 cells. .. Total RNA was purified from subconfluent cells using the RNeasy Mini Kit from Qiagen.

    Article Title: HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1? in normoxia
    Article Snippet: Subconfluent cells were lysed, and RNA was purified using the RNeasy Mini Kit (Qiagen). .. Membranes were hybridized with the indicated 32 P-labelled cDNA probe, and results were visualized using a phosphorimager (Storm 840; Amersham Biosciences). cDNA probes were obtained by RT–PCR using the primers allowing the amplification of nucleotides 298–563, 3800–4438 and 327–1048, corresponding to PHD1 (accession No. ), PHD2 (accession No. ) and PHD3 (accession No. ), respectively.

    Article Title: The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2
    Article Snippet: Paragraph title: Reverse transcriptase-polymerase chain reaction (RT-PCR) ... Subconfluent cells were collected, and total RNA was isolated using an RNeasy mini kit (Qiagen, Crawley, West Sussex, UK).

    Article Title: Inhibition of Hyaluronan Synthase-3 Decreases Subcutaneous Colon Cancer Growth by Increasing Apoptosis
    Article Snippet: HAS3 isozyme expression was assessed by semi-quantitative RT-PCR as previously described [ ]. .. Poly(A)+ RNA was isolated from subconfluent cells using the Oligotex mRNA isolation kit (Qiagen, Valencia, CA) and quantified by Ribogreen fluorescence (Molecular Probes, Eugene, OR). mRNA templates (50 ng) were reverse transcribed with an oligo(dT) primer using the Superscript II first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA).

    Quantitative RT-PCR:

    Article Title: Knock-down of Amphiregulin Inhibits Cellular Invasion in Inflammatory Breast Cancer
    Article Snippet: Paragraph title: Real-time RT-PCR ... Total RNA was isolated from subconfluent cells using an RNeasy kit (Qiagen, Valenica, CA, USA) and reverse-transcribed into cDNA using the Superscript III First-Strand Synthesis kit (Invitrogen, Calsbad, CA, USA).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2
    Article Snippet: Subconfluent cells were collected, and total RNA was isolated using an RNeasy mini kit (Qiagen, Crawley, West Sussex, UK). .. FAAH transcripts were detected using the primers 5′-GAG GCT TCC GTG TCC TCT C-3′ (forward) and 5′-CCT ATG TCA TAC CCA TGG GC-3′ (reverse) to amplify a 138 bp product ; glyceraldehyde-3-phosphate dehydrogenase transcripts were detected using the primers 5′-CTT CAC CAC CAT GGA GAA GGC-3′ (forward) and 5′-GGC ATG GAC TGT GGT CAT GAG-3′ (reverse) to amplify a 238 bp product, as a control.

    Plasmid Preparation:

    Article Title: Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells
    Article Snippet: .. Subconfluent cells were co-transfected by using Superfect (Qiagen, Hilden, Germany) according to the manufacturer's instructions (Clonetech, Palo Alto, USA) with 0.1 μg of hTLR2 (generously provided by Tularik Inc., San Francisco, USA [ ]) and dominant-negative A-Fos (kind gift of Dr. Charles Vinson, NCI, NIH, Rockville, MD) [ ], or Fra1- or Fra2-antisense (kind gift of Dr. Vladimir Berezin, Institute of Molecular Pathology, School of Medicine, Copenhagen University, Copenhagen, Denmark) [ ] expression vectors or control vector. .. Cells were incubated with R6x for 6 h.

    Article Title: IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells
    Article Snippet: .. The next day, subconfluent cells were transfected with a plasmid encoding cDNA for GFP-tagged IGFBP-3 (IGFBP-3-GFP), IGFBP-3-His, or pEGFP-N1 using SuperFect (Qiagen) in a 1:10 ratio as previously described [ ]. .. After a 24-hour recovery in serum-containing media, cells were rinsed twice in PBS and incubated with fresh SF DMEM-H for 1 hour, then treated as indicated in the figure legends.

    Article Title: Identification of Histamine H3 Receptor Ligands Using a New Crystal Structure Fragment-based Method
    Article Snippet: Subconfluent cells grown in a 100 mm dish were transfected with 5 μg DNA using the Polyfect transfection reagent using the protocol of the manufacturer (Qiagen, West Sussex, UK), however with half of the recommended PolyFect volumes. .. Co-transfections (ratio 4:1) were performed with either constructs expressing the human histamine H3 receptor together with an empty vector and chimeric G protein Gqi5, respectively.

    Dominant Negative Mutation:

    Article Title: Streptococcus pneumoniae induced c-Jun-N-terminal kinase- and AP-1 -dependent IL-8 release by lung epithelial BEAS-2B cells
    Article Snippet: .. Subconfluent cells were co-transfected by using Superfect (Qiagen, Hilden, Germany) according to the manufacturer's instructions (Clonetech, Palo Alto, USA) with 0.1 μg of hTLR2 (generously provided by Tularik Inc., San Francisco, USA [ ]) and dominant-negative A-Fos (kind gift of Dr. Charles Vinson, NCI, NIH, Rockville, MD) [ ], or Fra1- or Fra2-antisense (kind gift of Dr. Vladimir Berezin, Institute of Molecular Pathology, School of Medicine, Copenhagen University, Copenhagen, Denmark) [ ] expression vectors or control vector. .. Cells were incubated with R6x for 6 h.

    Real-time Polymerase Chain Reaction:

    Article Title: Altered VEGF mRNA Stability following Treatments with Immunosuppressive Agents
    Article Snippet: Subconfluent cells were first preincubated with 5.0 μg/ml actinomycin D for 1 h; cells were then treated with immunosuppressive agents for different time intervals (0–6 h) in the presence of actinomycin D. Total RNA was prepared using the RNeasy isolation kit (Qiagen), and cDNA was synthesized using cloned avian myeloblastosis virus first-strand synthesis kit (Invitrogen). .. To analyze VEGF expression, real-time PCR was performed using the Assays-on-Demand Gene Expression product (TaqMan, Mammalian Gene Collection probes) according to the manufacturer's instructions (Applied Biosystems).

    shRNA:

    Article Title: EGFR-Mediated Carcinoma Cell Metastasis Mediated by Integrin ?v?5 Depends on Activation of c-Src and Cleavage of MUC1
    Article Snippet: Paragraph title: Short hairpin RNA knockdown ... For some experiments, subconfluent cells were transfected with MUC1 small interfering RNAs (Qiagen) using Lipofectamine2000 (Life Technologies), and migration assays were performed at 48 h after transfection.

    Agarose Gel Electrophoresis:

    Article Title: The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2
    Article Snippet: Subconfluent cells were collected, and total RNA was isolated using an RNeasy mini kit (Qiagen, Crawley, West Sussex, UK). .. PCR products were subjected to electrophoresis on a 2% agarose gel, and DNA was visualised with ethidium bromide staining.

    Electrophoresis:

    Article Title: The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2
    Article Snippet: Subconfluent cells were collected, and total RNA was isolated using an RNeasy mini kit (Qiagen, Crawley, West Sussex, UK). .. PCR products were subjected to electrophoresis on a 2% agarose gel, and DNA was visualised with ethidium bromide staining.

    Produced:

    Article Title: EGFR-Mediated Carcinoma Cell Metastasis Mediated by Integrin ?v?5 Depends on Activation of c-Src and Cleavage of MUC1
    Article Snippet: Lentiviruses were produced in 293FT cells using FuGene transfection. .. For some experiments, subconfluent cells were transfected with MUC1 small interfering RNAs (Qiagen) using Lipofectamine2000 (Life Technologies), and migration assays were performed at 48 h after transfection.

    Migration:

    Article Title: EGFR-Mediated Carcinoma Cell Metastasis Mediated by Integrin ?v?5 Depends on Activation of c-Src and Cleavage of MUC1
    Article Snippet: .. For some experiments, subconfluent cells were transfected with MUC1 small interfering RNAs (Qiagen) using Lipofectamine2000 (Life Technologies), and migration assays were performed at 48 h after transfection. .. MUC1 expression Full length and cytoplasmic domain-deleted MUC1 were generously provided by Michael Hollingsworth .

    Staining:

    Article Title: IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells
    Article Snippet: The next day, subconfluent cells were transfected with a plasmid encoding cDNA for GFP-tagged IGFBP-3 (IGFBP-3-GFP), IGFBP-3-His, or pEGFP-N1 using SuperFect (Qiagen) in a 1:10 ratio as previously described [ ]. .. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific).

    Article Title: The endogenous cannabinoid, anandamide, induces cell death in colorectal carcinoma cells: a possible role for cyclooxygenase 2
    Article Snippet: Subconfluent cells were collected, and total RNA was isolated using an RNeasy mini kit (Qiagen, Crawley, West Sussex, UK). .. PCR products were subjected to electrophoresis on a 2% agarose gel, and DNA was visualised with ethidium bromide staining.

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    Qiagen stable expressing eyfp tubulin cell line cv 1 subconfluent cv 1 cells
    Microtubule destabilizing effect of IC261 in interphase cells. <t>CV-1</t> cells expressing <t>EYFP-tubulin</t> were cultured in a flow-through chamber, treated with DMSO (0.1%), 50 µM IC261 or 10 µM taxol +50 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, movie S2). Here representative cells are shown for time points “−29 min”, “0 min”, “10 min” und “30 min” ( row 1–3 ). Treatment with IC261 induced the depolymerization of microtubules within a few minutes ( row 2 ) while in cells treated with 10 µM taxol 30 min prior to treatment with IC261 at time point “0 min” the IC261 microtubule destabilizing effect of IC261 could be blocked ( row 3 ).
    Stable Expressing Eyfp Tubulin Cell Line Cv 1 Subconfluent Cv 1 Cells, supplied by Qiagen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen confocal laser scanning microscopy subconfluent trail sensitive hela cells
    A–D Differential subcellular localisation and lost proapoptotic potential of the novel <t>TRAIL</t> variants. ( A ) Confocal laser scanning microscopy revealed a prominent cytoplasmatic localisation of all GFP-tagged TRAIL variants. The endoplasmatic reticulum was marked with the endoplasmatic reticulum-specific fluorescence dye concanavalin A Alexa-594. Yellow staining in ‘merge’ indicates colocalisation of all GFP-tagged TRAIL variants and the endoplasmatic reticulum. Of note, GFP-tagged TRAIL- γ additionally showed a prominent fluorescence of both the cell surface and the nuclear membrane, which was not observed for the other TRAIL variants. <t>HeLa</t> cells known to be sensitive to TRAIL-mediated apoptosis ( Harper et al , 2001 ) were transfected with GFP constructs containing the different TRAIL isoforms or the empty vector. ( B ) Phase contrast microscopy of the transfected cells showed prominent clusters of apoptotic cells (arrows) in cultures of GFP- TRAIL- α transfected cells but not after transfection of other TRAIL variants or the vector control. ( C ) DAPI staining of nuclei permitted an identification of nuclei with fragmented or condensed chromatin, indicative for apoptotic cells (arrows), which could easily be distinguished from cells with intact nuclei under UV light. ( D ) Visual quantification of apoptosis was done by counting all GFP-positive cells as well as all GFP-positive cells with fragmented/condensed nuclei. The percentage of apoptotic cells was calculated as the ratio between the number of GFP-positive cells with fragmented or condensed nuclei and all GFP-positive cells. (Continued on next page.)
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    Qiagen irf7 subconfluent hbe cells
    Validation of the microarray data at the mRNA level. <t>HBE</t> cells were transfected with the all-star control siRNA, or two independent siRNA reagents that target <t>IRF7</t> (siRNA #1, siRNA #2). The cells were allowed to recover, and then exposed to HRV for 24 h. Gene expression levels were measured by real time RT-qPCR. The data for siRNA #1 and siRNA #2 are expressed as percent mRNA response relative to the all-star control. Data are mean ± SEM from 4 experiments. Asterisks show significant differences compared to the all star control siRNA treatment. Hashmarks (#) indicate significant differences between siRNA#1 and siRNA#2
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    Qiagen subconfluent mc3t3 cells
    C/EBPβ-LAP*-mediated repression of the Ric-8B promoter in osteoblastic cells is impaired in the R3L mutant protein unable to interact with SWI/SNF. <t>Subconfluent</t> <t>MC3T3</t> cells were cotransfected with p2.1Ric-8B-Luc together with increasing amounts
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    Microtubule destabilizing effect of IC261 in interphase cells. CV-1 cells expressing EYFP-tubulin were cultured in a flow-through chamber, treated with DMSO (0.1%), 50 µM IC261 or 10 µM taxol +50 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, movie S2). Here representative cells are shown for time points “−29 min”, “0 min”, “10 min” und “30 min” ( row 1–3 ). Treatment with IC261 induced the depolymerization of microtubules within a few minutes ( row 2 ) while in cells treated with 10 µM taxol 30 min prior to treatment with IC261 at time point “0 min” the IC261 microtubule destabilizing effect of IC261 could be blocked ( row 3 ).

    Journal: PLoS ONE

    Article Title: Microtubules Depolymerization Caused by the CK1 Inhibitor IC261 May Be Not Mediated by CK1 Blockage

    doi: 10.1371/journal.pone.0100090

    Figure Lengend Snippet: Microtubule destabilizing effect of IC261 in interphase cells. CV-1 cells expressing EYFP-tubulin were cultured in a flow-through chamber, treated with DMSO (0.1%), 50 µM IC261 or 10 µM taxol +50 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, movie S2). Here representative cells are shown for time points “−29 min”, “0 min”, “10 min” und “30 min” ( row 1–3 ). Treatment with IC261 induced the depolymerization of microtubules within a few minutes ( row 2 ) while in cells treated with 10 µM taxol 30 min prior to treatment with IC261 at time point “0 min” the IC261 microtubule destabilizing effect of IC261 could be blocked ( row 3 ).

    Article Snippet: Generation of Stable Expressing EYFP-tubulin Cell Line CV-1 Subconfluent CV-1 cells were transfected with Effectene (Qiagen) according to the company’s manual with a vector containing EYFP-tubulin under the control of the CMV promoter (pEYFP-Tub; Clontech).

    Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence, Microscopy, Sequencing

    Microtubule depolymerization by IC261 treatment is reversible. ( A ) CV-1 cells expressing EYFP-tubulin were treated at time point “0 min” with 3.2 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, movie S5 ). The spindle apparatus of the representative cell shown here was dissolved within 8 min. At time point “10 min” IC261 was removed by exchange of media. Within a few minutes spindle MTs were built up again (“15 min”) and 20 min after removal a morphologically unimpaired spindle apparatus had been developed (“30 min”). After 2 h the cell proceeded into anaphase and cytokinesis (“155 min”). ( B ) Densitometric analysis of grey values. For quantitative analysis the relative mean intensity of EYFP-tubulin fluorescence signal in a defined region of interest (ROI) around the spindle apparatus and in the cytoplasm was measured by the software CellR. Due to IC261 treatment at time point “0 min” (arrow up) the relative intensity immediately decreased due to MT depolymerization and subsequent removal of IC261 at time point “10 min” (arrow down) lead to a reconstruction of microtubules.

    Journal: PLoS ONE

    Article Title: Microtubules Depolymerization Caused by the CK1 Inhibitor IC261 May Be Not Mediated by CK1 Blockage

    doi: 10.1371/journal.pone.0100090

    Figure Lengend Snippet: Microtubule depolymerization by IC261 treatment is reversible. ( A ) CV-1 cells expressing EYFP-tubulin were treated at time point “0 min” with 3.2 µM IC261 and observed by time-resolved fluorescence microscopy (see video sequence, movie S5 ). The spindle apparatus of the representative cell shown here was dissolved within 8 min. At time point “10 min” IC261 was removed by exchange of media. Within a few minutes spindle MTs were built up again (“15 min”) and 20 min after removal a morphologically unimpaired spindle apparatus had been developed (“30 min”). After 2 h the cell proceeded into anaphase and cytokinesis (“155 min”). ( B ) Densitometric analysis of grey values. For quantitative analysis the relative mean intensity of EYFP-tubulin fluorescence signal in a defined region of interest (ROI) around the spindle apparatus and in the cytoplasm was measured by the software CellR. Due to IC261 treatment at time point “0 min” (arrow up) the relative intensity immediately decreased due to MT depolymerization and subsequent removal of IC261 at time point “10 min” (arrow down) lead to a reconstruction of microtubules.

    Article Snippet: Generation of Stable Expressing EYFP-tubulin Cell Line CV-1 Subconfluent CV-1 cells were transfected with Effectene (Qiagen) according to the company’s manual with a vector containing EYFP-tubulin under the control of the CMV promoter (pEYFP-Tub; Clontech).

    Techniques: Expressing, Fluorescence, Microscopy, Sequencing, Software

    Microtubule destabilizing effect of IC261 in mitotic cells. CV-1 cells expressing EYFP-tubulin were cultured in a flow-through chamber and observed by time-resolved fluorescence microscopy. At time point “0 min” cells were treated with DMSO (0.125%), IC261 (1 µM, 3.2 µM, 50 µM), 10 µM taxol or 0.4 µM nocodazole. Here exemplary cells are shown for indicated time points (see video sequence, movies S3 and S4 ). Treatment with low concentrations of IC261 induced a depolymerization of spindle microtubules within a few minutes ( row 2–3 ) in a concentration dependent manner and interestingly by nocodazole treatment a similar phenotype could be observed ( row 6 ). Cells entering mitosis during IC261 treatment had spindle poles and microtubule nucleating centers, but could not form a spindle ( row 4 , arrows indicate spindle poles). Treatment with 50 µM IC261 induced the complete depolymerization of microtubules within a few minutes (3–5 min, row 5 ). When cells were treated with 10 µM taxol during time period “−10 min” to “0 min” prior to treatment with taxol+IC261 at time point “0 min” the MT depolymerizing effect of IC261 could be blocked ( row 7 ). When cells were first treated for 10 min with IC261 resulting in a complete dissolution of the spindle apparatus, and subsequently treated with taxol+IC261 tubulin could re-polymerize at the spindle poles (arrows) and in other MT nucleation centers within the cell ( row 8 ).

    Journal: PLoS ONE

    Article Title: Microtubules Depolymerization Caused by the CK1 Inhibitor IC261 May Be Not Mediated by CK1 Blockage

    doi: 10.1371/journal.pone.0100090

    Figure Lengend Snippet: Microtubule destabilizing effect of IC261 in mitotic cells. CV-1 cells expressing EYFP-tubulin were cultured in a flow-through chamber and observed by time-resolved fluorescence microscopy. At time point “0 min” cells were treated with DMSO (0.125%), IC261 (1 µM, 3.2 µM, 50 µM), 10 µM taxol or 0.4 µM nocodazole. Here exemplary cells are shown for indicated time points (see video sequence, movies S3 and S4 ). Treatment with low concentrations of IC261 induced a depolymerization of spindle microtubules within a few minutes ( row 2–3 ) in a concentration dependent manner and interestingly by nocodazole treatment a similar phenotype could be observed ( row 6 ). Cells entering mitosis during IC261 treatment had spindle poles and microtubule nucleating centers, but could not form a spindle ( row 4 , arrows indicate spindle poles). Treatment with 50 µM IC261 induced the complete depolymerization of microtubules within a few minutes (3–5 min, row 5 ). When cells were treated with 10 µM taxol during time period “−10 min” to “0 min” prior to treatment with taxol+IC261 at time point “0 min” the MT depolymerizing effect of IC261 could be blocked ( row 7 ). When cells were first treated for 10 min with IC261 resulting in a complete dissolution of the spindle apparatus, and subsequently treated with taxol+IC261 tubulin could re-polymerize at the spindle poles (arrows) and in other MT nucleation centers within the cell ( row 8 ).

    Article Snippet: Generation of Stable Expressing EYFP-tubulin Cell Line CV-1 Subconfluent CV-1 cells were transfected with Effectene (Qiagen) according to the company’s manual with a vector containing EYFP-tubulin under the control of the CMV promoter (pEYFP-Tub; Clontech).

    Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence, Microscopy, Sequencing, Concentration Assay

    A–D Differential subcellular localisation and lost proapoptotic potential of the novel TRAIL variants. ( A ) Confocal laser scanning microscopy revealed a prominent cytoplasmatic localisation of all GFP-tagged TRAIL variants. The endoplasmatic reticulum was marked with the endoplasmatic reticulum-specific fluorescence dye concanavalin A Alexa-594. Yellow staining in ‘merge’ indicates colocalisation of all GFP-tagged TRAIL variants and the endoplasmatic reticulum. Of note, GFP-tagged TRAIL- γ additionally showed a prominent fluorescence of both the cell surface and the nuclear membrane, which was not observed for the other TRAIL variants. HeLa cells known to be sensitive to TRAIL-mediated apoptosis ( Harper et al , 2001 ) were transfected with GFP constructs containing the different TRAIL isoforms or the empty vector. ( B ) Phase contrast microscopy of the transfected cells showed prominent clusters of apoptotic cells (arrows) in cultures of GFP- TRAIL- α transfected cells but not after transfection of other TRAIL variants or the vector control. ( C ) DAPI staining of nuclei permitted an identification of nuclei with fragmented or condensed chromatin, indicative for apoptotic cells (arrows), which could easily be distinguished from cells with intact nuclei under UV light. ( D ) Visual quantification of apoptosis was done by counting all GFP-positive cells as well as all GFP-positive cells with fragmented/condensed nuclei. The percentage of apoptotic cells was calculated as the ratio between the number of GFP-positive cells with fragmented or condensed nuclei and all GFP-positive cells. (Continued on next page.)

    Journal: British Journal of Cancer

    Article Title: TRAIL-β and TRAIL-γ: two novel splice variants of the human TNF-related apoptosis-inducing ligand (TRAIL) without apoptotic potential

    doi: 10.1038/sj.bjc.6600772

    Figure Lengend Snippet: A–D Differential subcellular localisation and lost proapoptotic potential of the novel TRAIL variants. ( A ) Confocal laser scanning microscopy revealed a prominent cytoplasmatic localisation of all GFP-tagged TRAIL variants. The endoplasmatic reticulum was marked with the endoplasmatic reticulum-specific fluorescence dye concanavalin A Alexa-594. Yellow staining in ‘merge’ indicates colocalisation of all GFP-tagged TRAIL variants and the endoplasmatic reticulum. Of note, GFP-tagged TRAIL- γ additionally showed a prominent fluorescence of both the cell surface and the nuclear membrane, which was not observed for the other TRAIL variants. HeLa cells known to be sensitive to TRAIL-mediated apoptosis ( Harper et al , 2001 ) were transfected with GFP constructs containing the different TRAIL isoforms or the empty vector. ( B ) Phase contrast microscopy of the transfected cells showed prominent clusters of apoptotic cells (arrows) in cultures of GFP- TRAIL- α transfected cells but not after transfection of other TRAIL variants or the vector control. ( C ) DAPI staining of nuclei permitted an identification of nuclei with fragmented or condensed chromatin, indicative for apoptotic cells (arrows), which could easily be distinguished from cells with intact nuclei under UV light. ( D ) Visual quantification of apoptosis was done by counting all GFP-positive cells as well as all GFP-positive cells with fragmented/condensed nuclei. The percentage of apoptotic cells was calculated as the ratio between the number of GFP-positive cells with fragmented or condensed nuclei and all GFP-positive cells. (Continued on next page.)

    Article Snippet: Transfection of cultured cells, assessment of cell viability and apoptosis as well as analysis of subcellular localisation by confocal laser scanning microscopy Subconfluent TRAIL-sensitive HeLa cells were transiently transfected in 24-wells using polyfect transfection reagent, as described by the manufacturer (Qiagen).

    Techniques: Confocal Laser Scanning Microscopy, Fluorescence, Staining, Transfection, Construct, Plasmid Preparation, Microscopy, Polyacrylamide Gel Electrophoresis

    Continued E–G This visual apoptosis assay ( n = 3) further confimed the loss of proapoptotic potential for GFP-TRAIL- β and GFP-TRAIL- γ , which also became evident ( E ) from an increase of surviving cells in our MTT assays ( n = 6). ( F ) Immunodetection of the caspase-3-dependent ‘death substrate’ PARP showed enhancend cleavage only after ectopic expression of GFP-TRAIL- α . To ensure the detection of this cleavage product, protein lysates from TRAIL-treated control cells were separated on the same gel. The data presented are the mean ±s.d. from three independent transfections. ( G ) To show that the recombinant TRAIL variants are expressed on protein level, whole protein lysates of transfected HeLa cells were separated by SDS gels and immunodetection of GFP fusion proteins with anti-GFP-IgG was carried out after blotting on nitrocellulose membrane.

    Journal: British Journal of Cancer

    Article Title: TRAIL-β and TRAIL-γ: two novel splice variants of the human TNF-related apoptosis-inducing ligand (TRAIL) without apoptotic potential

    doi: 10.1038/sj.bjc.6600772

    Figure Lengend Snippet: Continued E–G This visual apoptosis assay ( n = 3) further confimed the loss of proapoptotic potential for GFP-TRAIL- β and GFP-TRAIL- γ , which also became evident ( E ) from an increase of surviving cells in our MTT assays ( n = 6). ( F ) Immunodetection of the caspase-3-dependent ‘death substrate’ PARP showed enhancend cleavage only after ectopic expression of GFP-TRAIL- α . To ensure the detection of this cleavage product, protein lysates from TRAIL-treated control cells were separated on the same gel. The data presented are the mean ±s.d. from three independent transfections. ( G ) To show that the recombinant TRAIL variants are expressed on protein level, whole protein lysates of transfected HeLa cells were separated by SDS gels and immunodetection of GFP fusion proteins with anti-GFP-IgG was carried out after blotting on nitrocellulose membrane.

    Article Snippet: Transfection of cultured cells, assessment of cell viability and apoptosis as well as analysis of subcellular localisation by confocal laser scanning microscopy Subconfluent TRAIL-sensitive HeLa cells were transiently transfected in 24-wells using polyfect transfection reagent, as described by the manufacturer (Qiagen).

    Techniques: Apoptosis Assay, MTT Assay, Immunodetection, Expressing, Transfection, Recombinant

    Validation of the microarray data at the mRNA level. HBE cells were transfected with the all-star control siRNA, or two independent siRNA reagents that target IRF7 (siRNA #1, siRNA #2). The cells were allowed to recover, and then exposed to HRV for 24 h. Gene expression levels were measured by real time RT-qPCR. The data for siRNA #1 and siRNA #2 are expressed as percent mRNA response relative to the all-star control. Data are mean ± SEM from 4 experiments. Asterisks show significant differences compared to the all star control siRNA treatment. Hashmarks (#) indicate significant differences between siRNA#1 and siRNA#2

    Journal: BMC Genomics

    Article Title: Interferon regulatory factor 7 regulates airway epithelial cell responses to human rhinovirus infection

    doi: 10.1186/s12864-016-2405-z

    Figure Lengend Snippet: Validation of the microarray data at the mRNA level. HBE cells were transfected with the all-star control siRNA, or two independent siRNA reagents that target IRF7 (siRNA #1, siRNA #2). The cells were allowed to recover, and then exposed to HRV for 24 h. Gene expression levels were measured by real time RT-qPCR. The data for siRNA #1 and siRNA #2 are expressed as percent mRNA response relative to the all-star control. Data are mean ± SEM from 4 experiments. Asterisks show significant differences compared to the all star control siRNA treatment. Hashmarks (#) indicate significant differences between siRNA#1 and siRNA#2

    Article Snippet: siRNA knockdown of IRF7 Subconfluent HBE cells were transfected with either 10 nM of a siRNA targeted to IRF7 or to a nontargeting siRNA, all-star control (Qiagen, Toronto, ON) for 24 h at 37 °C using Lipofectamine RNAiMAX (Life Technologies) in BEGM without antibiotics.

    Techniques: Microarray, Transfection, Expressing, Quantitative RT-PCR

    Validation of the microarray data at the protein level. HBE cells were transfected with the all-star control siRNA, or two independent siRNA reagents that target IRF7 (siRNA #1, siRNA #2). The cells were allowed to recover, and then exposed to HRV for 24 h. Protein levels for CCL5, CXCL5, CXCL10 were measured by ELISA and are shown as mean ± SEM from 4 experiments. Asterisks show significant differences compared to the all star control siRNA treatment. Expression of viperin protein was assessed by western blot. Data are representative of 4 such experiments

    Journal: BMC Genomics

    Article Title: Interferon regulatory factor 7 regulates airway epithelial cell responses to human rhinovirus infection

    doi: 10.1186/s12864-016-2405-z

    Figure Lengend Snippet: Validation of the microarray data at the protein level. HBE cells were transfected with the all-star control siRNA, or two independent siRNA reagents that target IRF7 (siRNA #1, siRNA #2). The cells were allowed to recover, and then exposed to HRV for 24 h. Protein levels for CCL5, CXCL5, CXCL10 were measured by ELISA and are shown as mean ± SEM from 4 experiments. Asterisks show significant differences compared to the all star control siRNA treatment. Expression of viperin protein was assessed by western blot. Data are representative of 4 such experiments

    Article Snippet: siRNA knockdown of IRF7 Subconfluent HBE cells were transfected with either 10 nM of a siRNA targeted to IRF7 or to a nontargeting siRNA, all-star control (Qiagen, Toronto, ON) for 24 h at 37 °C using Lipofectamine RNAiMAX (Life Technologies) in BEGM without antibiotics.

    Techniques: Microarray, Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

    Knockdown of IRF7 protein in HBE cells. HBE cells were transfected with the transfection reagent only (lipid reagent alone), the all-star control siRNA (control siRNA), or two independent siRNAs that target IRF7 (siRNA #1, siRNA #2). The cells were allowed to recover, and then exposed to HRV. Protein expression was measured by Western Blot. Data are representative of n = 4

    Journal: BMC Genomics

    Article Title: Interferon regulatory factor 7 regulates airway epithelial cell responses to human rhinovirus infection

    doi: 10.1186/s12864-016-2405-z

    Figure Lengend Snippet: Knockdown of IRF7 protein in HBE cells. HBE cells were transfected with the transfection reagent only (lipid reagent alone), the all-star control siRNA (control siRNA), or two independent siRNAs that target IRF7 (siRNA #1, siRNA #2). The cells were allowed to recover, and then exposed to HRV. Protein expression was measured by Western Blot. Data are representative of n = 4

    Article Snippet: siRNA knockdown of IRF7 Subconfluent HBE cells were transfected with either 10 nM of a siRNA targeted to IRF7 or to a nontargeting siRNA, all-star control (Qiagen, Toronto, ON) for 24 h at 37 °C using Lipofectamine RNAiMAX (Life Technologies) in BEGM without antibiotics.

    Techniques: Transfection, Expressing, Western Blot

    Knockdown of IRF7 perturbs HBE cells responses to HRV. HBE cells from five donors were transfected with siRNA that targets IRF7 (siRNA #1) or the non-silencing all-star control, allowed to recover, and then exposed to HRV for 24 h. Gene expression was profiled on microarrays. The data was compared between HRV-induced cells treated with IRF7-siRNA #1 versus the all-star control. Genes with negative values on the horizontal axis were decreased by IRF7 knockdown, whereas those with positive values were increased. The black triangles show the location of genes from the IRF7 network. The dashed horizontal line indicates FDR

    Journal: BMC Genomics

    Article Title: Interferon regulatory factor 7 regulates airway epithelial cell responses to human rhinovirus infection

    doi: 10.1186/s12864-016-2405-z

    Figure Lengend Snippet: Knockdown of IRF7 perturbs HBE cells responses to HRV. HBE cells from five donors were transfected with siRNA that targets IRF7 (siRNA #1) or the non-silencing all-star control, allowed to recover, and then exposed to HRV for 24 h. Gene expression was profiled on microarrays. The data was compared between HRV-induced cells treated with IRF7-siRNA #1 versus the all-star control. Genes with negative values on the horizontal axis were decreased by IRF7 knockdown, whereas those with positive values were increased. The black triangles show the location of genes from the IRF7 network. The dashed horizontal line indicates FDR

    Article Snippet: siRNA knockdown of IRF7 Subconfluent HBE cells were transfected with either 10 nM of a siRNA targeted to IRF7 or to a nontargeting siRNA, all-star control (Qiagen, Toronto, ON) for 24 h at 37 °C using Lipofectamine RNAiMAX (Life Technologies) in BEGM without antibiotics.

    Techniques: Transfection, Expressing

    IRF7 is a putative molecular driver of HBE responses to HRV. Differentially expressed genes were identified in HRV-induced HBE cells (Fig. 2 ), and then the wiring diagram of the IRF7 gene network was reconstructed from these data employing experimentally supported findings from published studies. Solid and dashed lines indicate direct and indirect interactions respectively. Genes colored red were upregulated in the response and genes colored green were downregulated

    Journal: BMC Genomics

    Article Title: Interferon regulatory factor 7 regulates airway epithelial cell responses to human rhinovirus infection

    doi: 10.1186/s12864-016-2405-z

    Figure Lengend Snippet: IRF7 is a putative molecular driver of HBE responses to HRV. Differentially expressed genes were identified in HRV-induced HBE cells (Fig. 2 ), and then the wiring diagram of the IRF7 gene network was reconstructed from these data employing experimentally supported findings from published studies. Solid and dashed lines indicate direct and indirect interactions respectively. Genes colored red were upregulated in the response and genes colored green were downregulated

    Article Snippet: siRNA knockdown of IRF7 Subconfluent HBE cells were transfected with either 10 nM of a siRNA targeted to IRF7 or to a nontargeting siRNA, all-star control (Qiagen, Toronto, ON) for 24 h at 37 °C using Lipofectamine RNAiMAX (Life Technologies) in BEGM without antibiotics.

    Techniques:

    C/EBPβ-LAP*-mediated repression of the Ric-8B promoter in osteoblastic cells is impaired in the R3L mutant protein unable to interact with SWI/SNF. Subconfluent MC3T3 cells were cotransfected with p2.1Ric-8B-Luc together with increasing amounts

    Journal: Journal of cellular physiology

    Article Title: A Functional N-terminal Domain in C/EBPβ-LAP* is Required for Interacting with SWI/SNF and to Repress Ric-8B Gene Transcription in Osteoblasts

    doi: 10.1002/jcp.24595

    Figure Lengend Snippet: C/EBPβ-LAP*-mediated repression of the Ric-8B promoter in osteoblastic cells is impaired in the R3L mutant protein unable to interact with SWI/SNF. Subconfluent MC3T3 cells were cotransfected with p2.1Ric-8B-Luc together with increasing amounts

    Article Snippet: Subconfluent MC3T3 cells were transiently transfected for 24 hrs using Superfect (Qiagen), according to the manufacturer’s recommendations.

    Techniques: Mutagenesis