mission plko 1 puro non mammalian shrna control plasmid dna  (Millipore)

 
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    Name:
    MISSION pLKO 1 puro Non Mammalian shRNA Control Plasmid DNA
    Description:

    Catalog Number:
    shc002
    Price:
    None
    Applications:
    MISSION(R) pLKO.1-puro non-mammalian shRNA control plasmid DNA has been used:. to transduce RLE-6TN cells. for the validation of Oct4-green fluorescent protein (GFP) mouse embryonic fibroblasts (MEFs). as a negative control for RNA knockdown
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    Millipore mission plko 1 puro non mammalian shrna control plasmid dna
    MISSION pLKO 1 puro Non Mammalian shRNA Control Plasmid DNA

    https://www.bioz.com/result/mission plko 1 puro non mammalian shrna control plasmid dna/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mission plko 1 puro non mammalian shrna control plasmid dna - by Bioz Stars, 2021-03
    97/100 stars

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    Related Articles

    Transfection:

    Article Title: Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1? in Cofilin Regulation and Cytoplasmic Actin Distribution
    Article Snippet: L929 HIF-1α knock down clones were generated by lentiviral transduction with the pLKO.1-puro HIF-1α-shRNA expression vector (#TRCN0000003810, Sigma-Aldrich, St. Louis, USA). .. For generating the shControl (shC) transfected cells, a pLKO.1-puro vector was used containing a non-targeting shRNA (#SHC002, Sigma-Aldrich). .. For lentiviral transduction, viral particles were produced in HEK293T cells using the ViralPower lentiviral expression system according to the manufactureŕs instructions (Life Technologies, Paisley, UK).

    Article Title: Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR
    Article Snippet: .. Plasmid containing GFP-encoding DNA (pmaxGFP; Lonza, Basel, Switzerland) was used as positive control for transfection and the pLKO.1-puro vector containing DNA encoding for non-targeting shRNA (MISSION® shRNA SHC002; Sigma-Aldrich) was used as negative control. .. T cells were transfected using the Amaxa nucleofector kit for human T cells (Lonza) according to manufacturer's instructions.

    Plasmid Preparation:

    Article Title: Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1? in Cofilin Regulation and Cytoplasmic Actin Distribution
    Article Snippet: L929 HIF-1α knock down clones were generated by lentiviral transduction with the pLKO.1-puro HIF-1α-shRNA expression vector (#TRCN0000003810, Sigma-Aldrich, St. Louis, USA). .. For generating the shControl (shC) transfected cells, a pLKO.1-puro vector was used containing a non-targeting shRNA (#SHC002, Sigma-Aldrich). .. For lentiviral transduction, viral particles were produced in HEK293T cells using the ViralPower lentiviral expression system according to the manufactureŕs instructions (Life Technologies, Paisley, UK).

    Article Title: Interplay between HIV Entry and Transportin-SR2 Dependency
    Article Snippet: Two different HeLaP4 cell lines stably knocked down for TRN-SR2 were generated by transduction with two different lentiviral vectors expressing shR NAs targeting the TRN-SR2 mRNA, called shTR3 and shTR4 (Sigma, clone-id NM_012470.2-867s21c1 and NM_012470.2-2084s21c1). .. A control cell line was established using a control vector expressing a scrambled shRNA referred to as shSCR (Sigma, product number SHC002). ..

    Article Title: Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1? Protein under Hypoxic Conditions
    Article Snippet: Lentiviral vectors expressing shRNAs and SIRT1 and SIRT1 H363Y Lentivirus production, titer determination and transduction were carried out as previously described . .. Two shRNA against SIRT1 (clone 1, ID: NM_0 12238.3-1958s1c1 termed shSIRT1_1958 and clone 2, ID: NM_012238.3-3206s1c1 termed shSIRT1_3206) and a non-target shRNA control vector (SHC002) were obtained from Sigma (Basel, Switzerland). ..

    Article Title: Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR
    Article Snippet: .. Plasmid containing GFP-encoding DNA (pmaxGFP; Lonza, Basel, Switzerland) was used as positive control for transfection and the pLKO.1-puro vector containing DNA encoding for non-targeting shRNA (MISSION® shRNA SHC002; Sigma-Aldrich) was used as negative control. .. T cells were transfected using the Amaxa nucleofector kit for human T cells (Lonza) according to manufacturer's instructions.

    shRNA:

    Article Title: Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1? in Cofilin Regulation and Cytoplasmic Actin Distribution
    Article Snippet: L929 HIF-1α knock down clones were generated by lentiviral transduction with the pLKO.1-puro HIF-1α-shRNA expression vector (#TRCN0000003810, Sigma-Aldrich, St. Louis, USA). .. For generating the shControl (shC) transfected cells, a pLKO.1-puro vector was used containing a non-targeting shRNA (#SHC002, Sigma-Aldrich). .. For lentiviral transduction, viral particles were produced in HEK293T cells using the ViralPower lentiviral expression system according to the manufactureŕs instructions (Life Technologies, Paisley, UK).

    Article Title: Interplay between HIV Entry and Transportin-SR2 Dependency
    Article Snippet: Two different HeLaP4 cell lines stably knocked down for TRN-SR2 were generated by transduction with two different lentiviral vectors expressing shR NAs targeting the TRN-SR2 mRNA, called shTR3 and shTR4 (Sigma, clone-id NM_012470.2-867s21c1 and NM_012470.2-2084s21c1). .. A control cell line was established using a control vector expressing a scrambled shRNA referred to as shSCR (Sigma, product number SHC002). ..

    Article Title: The metastasis suppressor NME1 inhibits melanoma cell motility via direct transcriptional induction of the integrin beta-3 gene
    Article Snippet: .. RNA knockdown was achieved by lentiviral delivery of Mission shRNA (Sigma-Aldrich) against ITGβ3 (TRCN0000003236, ITGβ3 shRNA-a; TRCN0000318546, ITGβ3 shRNA-b; TRCN0000003235, ITGβ3 shRNA-c) or a non-targeted shRNA (SHC002, Sigma-Aldrich) as a negative control. .. For assessment of focal adhesions, 1205LU cells were transduced with a retrovirus vector encoding a DsRed-paxillin fusion protein (provided by Cai Huang, University of Kentucky).

    Article Title: Periostin Secreted by Glioblastoma Stem Cells Recruits M2 Tumor-associated Macrophages and Promotes Malignant Growth
    Article Snippet: Mice were then sacrificed and the peritoneal macrophages were harvested by washing the cavity with 1ml ice cold PBS. .. shRNAs for knockdown Lentiviral mediated shRNAs targeting POSTN (TRCN0000123055 for O55; TRCN0000123056 for O56, and TRCN0000123057 for O57) or control shRNA (SHC002) were purchased from Sigma-Aldrich. .. RT-qPCR array of secreted proteins To identify potential chemoattractants differentially expressed by GSCs, we used qPCR array (RT-qPCR analysis) to determine the expression of major secreted proteins, including most cytokines along with some secreted proteins relevant to tumorigenesis, in paired GSCs and NSTCs. qPCR primers were designed to span an intron of each target gene ( ).

    Article Title: Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1? Protein under Hypoxic Conditions
    Article Snippet: Lentiviral vectors expressing shRNAs and SIRT1 and SIRT1 H363Y Lentivirus production, titer determination and transduction were carried out as previously described . .. Two shRNA against SIRT1 (clone 1, ID: NM_0 12238.3-1958s1c1 termed shSIRT1_1958 and clone 2, ID: NM_012238.3-3206s1c1 termed shSIRT1_3206) and a non-target shRNA control vector (SHC002) were obtained from Sigma (Basel, Switzerland). ..

    Article Title: Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons
    Article Snippet: .. To silence importin α1, importin α3, or importin α4 by short hairpin RNAs (shRNAs; Sigma Mission library; ) or to express a non-mammalian shRNA control (SHC002, Sigma Mission library), we used lentiviral transduction. .. HEK 293T cells were transfected with 5 μg pRSVRev, 2 μg pMD2.g (Addgene Inc., Cambridge, MA, USA, Cat. No. 12259), 10 μg pCDNA3.GP.CCCC, and 10 μg transfer plasmid per 10 cm dish as described previously ([ ]; plasmids provided by Axel Schambach).

    Article Title: Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR
    Article Snippet: .. Plasmid containing GFP-encoding DNA (pmaxGFP; Lonza, Basel, Switzerland) was used as positive control for transfection and the pLKO.1-puro vector containing DNA encoding for non-targeting shRNA (MISSION® shRNA SHC002; Sigma-Aldrich) was used as negative control. .. T cells were transfected using the Amaxa nucleofector kit for human T cells (Lonza) according to manufacturer's instructions.

    Expressing:

    Article Title: Interplay between HIV Entry and Transportin-SR2 Dependency
    Article Snippet: Two different HeLaP4 cell lines stably knocked down for TRN-SR2 were generated by transduction with two different lentiviral vectors expressing shR NAs targeting the TRN-SR2 mRNA, called shTR3 and shTR4 (Sigma, clone-id NM_012470.2-867s21c1 and NM_012470.2-2084s21c1). .. A control cell line was established using a control vector expressing a scrambled shRNA referred to as shSCR (Sigma, product number SHC002). ..

    Negative Control:

    Article Title: The metastasis suppressor NME1 inhibits melanoma cell motility via direct transcriptional induction of the integrin beta-3 gene
    Article Snippet: .. RNA knockdown was achieved by lentiviral delivery of Mission shRNA (Sigma-Aldrich) against ITGβ3 (TRCN0000003236, ITGβ3 shRNA-a; TRCN0000318546, ITGβ3 shRNA-b; TRCN0000003235, ITGβ3 shRNA-c) or a non-targeted shRNA (SHC002, Sigma-Aldrich) as a negative control. .. For assessment of focal adhesions, 1205LU cells were transduced with a retrovirus vector encoding a DsRed-paxillin fusion protein (provided by Cai Huang, University of Kentucky).

    Article Title: Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR
    Article Snippet: .. Plasmid containing GFP-encoding DNA (pmaxGFP; Lonza, Basel, Switzerland) was used as positive control for transfection and the pLKO.1-puro vector containing DNA encoding for non-targeting shRNA (MISSION® shRNA SHC002; Sigma-Aldrich) was used as negative control. .. T cells were transfected using the Amaxa nucleofector kit for human T cells (Lonza) according to manufacturer's instructions.

    Transduction:

    Article Title: Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons
    Article Snippet: .. To silence importin α1, importin α3, or importin α4 by short hairpin RNAs (shRNAs; Sigma Mission library; ) or to express a non-mammalian shRNA control (SHC002, Sigma Mission library), we used lentiviral transduction. .. HEK 293T cells were transfected with 5 μg pRSVRev, 2 μg pMD2.g (Addgene Inc., Cambridge, MA, USA, Cat. No. 12259), 10 μg pCDNA3.GP.CCCC, and 10 μg transfer plasmid per 10 cm dish as described previously ([ ]; plasmids provided by Axel Schambach).

    Positive Control:

    Article Title: Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR
    Article Snippet: .. Plasmid containing GFP-encoding DNA (pmaxGFP; Lonza, Basel, Switzerland) was used as positive control for transfection and the pLKO.1-puro vector containing DNA encoding for non-targeting shRNA (MISSION® shRNA SHC002; Sigma-Aldrich) was used as negative control. .. T cells were transfected using the Amaxa nucleofector kit for human T cells (Lonza) according to manufacturer's instructions.

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    Millipore non targeting shrna
    HIF-1α stabilisation regulates cell area. (A) Schematic drawing of HIF-1 and its regulation in normoxia and hypoxia. The stability of the HIF-1α subunit is regulated by prolyl-4-hydroxylase domain (PHD) enzymes in an oxygen dependent manner. Following hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognises HIF-1α subunits and targets them for rapid ubiquitination and proteasomal degradation under normoxic conditions. Hypoxia impairs the hydroxylation, which results in HIF-1α stabilisation, nuclear accumulation, heterodimerisation with HIF-1β and subsequent hypoxia-inducible gene expression. (B) Inhibition of PHDs by DMOG causes HIF-1α stabilisation under normoxic conditions. L929 cells were treated with 1 mM DMOG for 48 hrs and total extracts were analysed by immunoblot with the respective antibodies. (C) The cell area of DMOG treated cells increased significantly. The cell area of single cells was measured and was calculated as fold change compared to 20% O 2. (D) Two HIF-1α knock down cell clones (c1, c2) and a non-target control <t>shRNA</t> cell clone <t>(shC)</t> were obtained via stable transduction of specific sh-plasmids. The HIF-1α knock down was verified in western blot experiments of cell lysates from cells incubated at 20% O 2 or 1% O 2 . β-tubulin was used as a loading control. (E) HIF-1α knock down cell clones (c1, c2) do not respond to hypoxia with an increase in cell area. The cell area of single cells was measured and was calculated as fold change compared to the cell clone cultivated at 20% O 2 . (F) Flow cytometry analysis of cell volume after incubation in normoxia and hypoxia. shC, c1 and c2 cells were harvested after 24 hrs of normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Single cell suspension was prepared by enzymatic digestion. Note that hypoxia increases the cell volume independently of the HIF-1α knock down.
    Non Targeting Shrna, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/non targeting shrna/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    non targeting shrna - by Bioz Stars, 2021-03
    97/100 stars
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    97
    Millipore control shrna plasmid
    Phenotypes of RA-FLSs with depletion of α9 cultured in 3D-micromass system under stimulation with PDGF or TNF-α. RA-FLSs treated with <t>shRNA</t> either for <t>ITGA9</t> or the control (Con) were cultured in a 3D-micromass system in the media supplemented with PDGF (PDGF-BB, 50 ng/ml) or TNF-α (20 ng/ml) and the phenotypes were analyzed. ( A ) Frozen sections from the resultant architectures were costained with Alexa Fluor 488–phalloidin (green) and DAPI (blue). Representative data from three independent experiments are shown. Scale bars, 50 μm. ( B ) Protein amounts of MMP-1, MMP-3, and IL-6 in the culture supernatants collected at the end of the study were determined by ELISA and expressed as mean ± SE ( n = 5). Statistical analyses were performed by a paired t test. * /# p
    Control Shrna Plasmid, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/control shrna plasmid/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    control shrna plasmid - by Bioz Stars, 2021-03
    97/100 stars
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    Image Search Results


    HIF-1α stabilisation regulates cell area. (A) Schematic drawing of HIF-1 and its regulation in normoxia and hypoxia. The stability of the HIF-1α subunit is regulated by prolyl-4-hydroxylase domain (PHD) enzymes in an oxygen dependent manner. Following hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognises HIF-1α subunits and targets them for rapid ubiquitination and proteasomal degradation under normoxic conditions. Hypoxia impairs the hydroxylation, which results in HIF-1α stabilisation, nuclear accumulation, heterodimerisation with HIF-1β and subsequent hypoxia-inducible gene expression. (B) Inhibition of PHDs by DMOG causes HIF-1α stabilisation under normoxic conditions. L929 cells were treated with 1 mM DMOG for 48 hrs and total extracts were analysed by immunoblot with the respective antibodies. (C) The cell area of DMOG treated cells increased significantly. The cell area of single cells was measured and was calculated as fold change compared to 20% O 2. (D) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were obtained via stable transduction of specific sh-plasmids. The HIF-1α knock down was verified in western blot experiments of cell lysates from cells incubated at 20% O 2 or 1% O 2 . β-tubulin was used as a loading control. (E) HIF-1α knock down cell clones (c1, c2) do not respond to hypoxia with an increase in cell area. The cell area of single cells was measured and was calculated as fold change compared to the cell clone cultivated at 20% O 2 . (F) Flow cytometry analysis of cell volume after incubation in normoxia and hypoxia. shC, c1 and c2 cells were harvested after 24 hrs of normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Single cell suspension was prepared by enzymatic digestion. Note that hypoxia increases the cell volume independently of the HIF-1α knock down.

    Journal: PLoS ONE

    Article Title: Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1? in Cofilin Regulation and Cytoplasmic Actin Distribution

    doi: 10.1371/journal.pone.0069128

    Figure Lengend Snippet: HIF-1α stabilisation regulates cell area. (A) Schematic drawing of HIF-1 and its regulation in normoxia and hypoxia. The stability of the HIF-1α subunit is regulated by prolyl-4-hydroxylase domain (PHD) enzymes in an oxygen dependent manner. Following hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognises HIF-1α subunits and targets them for rapid ubiquitination and proteasomal degradation under normoxic conditions. Hypoxia impairs the hydroxylation, which results in HIF-1α stabilisation, nuclear accumulation, heterodimerisation with HIF-1β and subsequent hypoxia-inducible gene expression. (B) Inhibition of PHDs by DMOG causes HIF-1α stabilisation under normoxic conditions. L929 cells were treated with 1 mM DMOG for 48 hrs and total extracts were analysed by immunoblot with the respective antibodies. (C) The cell area of DMOG treated cells increased significantly. The cell area of single cells was measured and was calculated as fold change compared to 20% O 2. (D) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were obtained via stable transduction of specific sh-plasmids. The HIF-1α knock down was verified in western blot experiments of cell lysates from cells incubated at 20% O 2 or 1% O 2 . β-tubulin was used as a loading control. (E) HIF-1α knock down cell clones (c1, c2) do not respond to hypoxia with an increase in cell area. The cell area of single cells was measured and was calculated as fold change compared to the cell clone cultivated at 20% O 2 . (F) Flow cytometry analysis of cell volume after incubation in normoxia and hypoxia. shC, c1 and c2 cells were harvested after 24 hrs of normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Single cell suspension was prepared by enzymatic digestion. Note that hypoxia increases the cell volume independently of the HIF-1α knock down.

    Article Snippet: For generating the shControl (shC) transfected cells, a pLKO.1-puro vector was used containing a non-targeting shRNA (#SHC002, Sigma-Aldrich).

    Techniques: Expressing, Inhibition, Clone Assay, shRNA, Transduction, Western Blot, Incubation, Flow Cytometry, Cytometry

    Depletion of HIF-1α alters cell motility. (A) Wounding assay of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were grown in normoxia and hypoxia and experimental wounds were caused by scratching cell monolayers with a pipet tip. Images were taken at the indicated time points and the cell free area was determined. Hypoxia delayed wound healing. Note that knocking down HIF-1α slowed wound closure down even more (dashed red line). (B) Single cell migration of the HIF-1α knock down cell clones c1 and c2 and the shC cells. Cells were incubated at 20% O 2 and 1% O 2 for 24 hrs. Images were taken over a time period of 360 min and cell movement was analysed. shC cells showed a reduced migration under hypoxic condition, however this effect was not seen in the HIF-1α knock down clones c1 and c2. Numbers within the bars indicate the number of cells analysed. ** p

    Journal: PLoS ONE

    Article Title: Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1? in Cofilin Regulation and Cytoplasmic Actin Distribution

    doi: 10.1371/journal.pone.0069128

    Figure Lengend Snippet: Depletion of HIF-1α alters cell motility. (A) Wounding assay of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were grown in normoxia and hypoxia and experimental wounds were caused by scratching cell monolayers with a pipet tip. Images were taken at the indicated time points and the cell free area was determined. Hypoxia delayed wound healing. Note that knocking down HIF-1α slowed wound closure down even more (dashed red line). (B) Single cell migration of the HIF-1α knock down cell clones c1 and c2 and the shC cells. Cells were incubated at 20% O 2 and 1% O 2 for 24 hrs. Images were taken over a time period of 360 min and cell movement was analysed. shC cells showed a reduced migration under hypoxic condition, however this effect was not seen in the HIF-1α knock down clones c1 and c2. Numbers within the bars indicate the number of cells analysed. ** p

    Article Snippet: For generating the shControl (shC) transfected cells, a pLKO.1-puro vector was used containing a non-targeting shRNA (#SHC002, Sigma-Aldrich).

    Techniques: Clone Assay, shRNA, Migration, Incubation

    Depletion of HIF-1α does not affect the number of focal contacts and cell spreading. (A) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were stained for vinculin 24 hrs after normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Counting vinculin positive focal contacts showed a higher number of focal contacts in hypoxia in all three cell lines. (B) Flow cytometry analysis of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA cell clone (shC) after 24 hrs of normoxia or hypoxia stained with integrin β1 antibodies. (C) Wt, shC, and the HIF-1α knock down cell clones c1 and c2 cells were lysed after 24 hrs of normoxia (20% O 2 ) or hypoxia (1% O 2 ). Cell extracts were analysed by Western blots. Note that vinculin and integrin β1 levels are not changed in hypoxia. (D) Cell spreading of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were incubated at 20% O 2 and 1% O 2 , trypsinised and replated for 20 min. Cells were fixed and stained with phalloidin-FITC and divided into three categories (a: round, barely spread; b: in the course of spreading; c: well spread). The percentage of cells in each category was determined. All three cell lines spread faster under hypoxic conditions. Numbers within the bars indicate the number of cells analysed. ** p

    Journal: PLoS ONE

    Article Title: Hypoxia Modulates Fibroblastic Architecture, Adhesion and Migration: A Role for HIF-1? in Cofilin Regulation and Cytoplasmic Actin Distribution

    doi: 10.1371/journal.pone.0069128

    Figure Lengend Snippet: Depletion of HIF-1α does not affect the number of focal contacts and cell spreading. (A) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were stained for vinculin 24 hrs after normoxic (20% O 2 ) or hypoxic (1% O 2 ) incubation. Counting vinculin positive focal contacts showed a higher number of focal contacts in hypoxia in all three cell lines. (B) Flow cytometry analysis of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA cell clone (shC) after 24 hrs of normoxia or hypoxia stained with integrin β1 antibodies. (C) Wt, shC, and the HIF-1α knock down cell clones c1 and c2 cells were lysed after 24 hrs of normoxia (20% O 2 ) or hypoxia (1% O 2 ). Cell extracts were analysed by Western blots. Note that vinculin and integrin β1 levels are not changed in hypoxia. (D) Cell spreading of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were incubated at 20% O 2 and 1% O 2 , trypsinised and replated for 20 min. Cells were fixed and stained with phalloidin-FITC and divided into three categories (a: round, barely spread; b: in the course of spreading; c: well spread). The percentage of cells in each category was determined. All three cell lines spread faster under hypoxic conditions. Numbers within the bars indicate the number of cells analysed. ** p

    Article Snippet: For generating the shControl (shC) transfected cells, a pLKO.1-puro vector was used containing a non-targeting shRNA (#SHC002, Sigma-Aldrich).

    Techniques: Clone Assay, shRNA, Staining, Incubation, Flow Cytometry, Cytometry, Western Blot

    Presence of hCCX-CKR is associated with suppression of chemotaxis towards CXCL10 in T cells (A) Silencing hCCX-CKR using vectors containing DNA encoding for short hairpin RNA targeting hCCX-CKR resulted in a down-regulation of hCCX-CKR expression by freshly

    Journal: British Journal of Pharmacology

    Article Title: Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR

    doi: 10.1111/bph.12042

    Figure Lengend Snippet: Presence of hCCX-CKR is associated with suppression of chemotaxis towards CXCL10 in T cells (A) Silencing hCCX-CKR using vectors containing DNA encoding for short hairpin RNA targeting hCCX-CKR resulted in a down-regulation of hCCX-CKR expression by freshly

    Article Snippet: Plasmid containing GFP-encoding DNA (pmaxGFP; Lonza, Basel, Switzerland) was used as positive control for transfection and the pLKO.1-puro vector containing DNA encoding for non-targeting shRNA (MISSION® shRNA SHC002; Sigma-Aldrich) was used as negative control.

    Techniques: Chemotaxis Assay, shRNA, Expressing

    NME1 suppresses motile and invasive potential of WM1158 melanoma cells by upregulating ITGβ3 expression. (a) Expression of NME1 and ITGβ3 proteins was measured by immunoblot analysis in cells receiving the indicated combinations of forced NME1 expression and shRNAs directed to ITGβ3. shRNA treatments consisted a non-targeting control shRNA (−) or one of two shRNAs specific for ITGβ3 (“a” or “b”). (b) Representative images of wound/scratch assays were acquired 24 h after wound induction in cells receiving the indicated combinations of forced NME1 expression and shRNA-mediated silencing of ITGβ3. Dotted boxes depict borders of original wounds. (c) Graph provides a quantitative analysis of individual cells migrating into wounds. Closed circles and error bars represent means (+/− SEM) derived from 3– 4 independent experiments. Asterisks denote means that are significantly different (*p≤0.05 by ANOVA with Holm-Sidak pairwise testing). (d) Expression of ITGβ3 mRNA was measured in cells used for measurement of single cell invasion activity in 3-dimensional sphere assays after the indicated combination of forced NME1 expression and shRNA sequences (-, non-targeting control; ITGβ3-directed shRNAs “c” and “a”). The shRNA sequence “c” was employed instead of sequence “b” used in panel a, as sequence “b” strongly disrupted spheroid formation. Cultures were co-infected with a lentiviral vector for expression of eGFP for enhanced imaging of invading cells. *, comparison between vector- and NME1-treated; †, denotes significant silencing of ITGβ3 RNA in absence of forced NME1 expression (black bars); ‡, denotes significant silencing of ITGβ3 RNA in presence of forced NME1 expression (white bars). p

    Journal: Experimental cell research

    Article Title: The metastasis suppressor NME1 inhibits melanoma cell motility via direct transcriptional induction of the integrin beta-3 gene

    doi: 10.1016/j.yexcr.2018.11.010

    Figure Lengend Snippet: NME1 suppresses motile and invasive potential of WM1158 melanoma cells by upregulating ITGβ3 expression. (a) Expression of NME1 and ITGβ3 proteins was measured by immunoblot analysis in cells receiving the indicated combinations of forced NME1 expression and shRNAs directed to ITGβ3. shRNA treatments consisted a non-targeting control shRNA (−) or one of two shRNAs specific for ITGβ3 (“a” or “b”). (b) Representative images of wound/scratch assays were acquired 24 h after wound induction in cells receiving the indicated combinations of forced NME1 expression and shRNA-mediated silencing of ITGβ3. Dotted boxes depict borders of original wounds. (c) Graph provides a quantitative analysis of individual cells migrating into wounds. Closed circles and error bars represent means (+/− SEM) derived from 3– 4 independent experiments. Asterisks denote means that are significantly different (*p≤0.05 by ANOVA with Holm-Sidak pairwise testing). (d) Expression of ITGβ3 mRNA was measured in cells used for measurement of single cell invasion activity in 3-dimensional sphere assays after the indicated combination of forced NME1 expression and shRNA sequences (-, non-targeting control; ITGβ3-directed shRNAs “c” and “a”). The shRNA sequence “c” was employed instead of sequence “b” used in panel a, as sequence “b” strongly disrupted spheroid formation. Cultures were co-infected with a lentiviral vector for expression of eGFP for enhanced imaging of invading cells. *, comparison between vector- and NME1-treated; †, denotes significant silencing of ITGβ3 RNA in absence of forced NME1 expression (black bars); ‡, denotes significant silencing of ITGβ3 RNA in presence of forced NME1 expression (white bars). p

    Article Snippet: RNA knockdown was achieved by lentiviral delivery of Mission shRNA (Sigma-Aldrich) against ITGβ3 (TRCN0000003236, ITGβ3 shRNA-a; TRCN0000318546, ITGβ3 shRNA-b; TRCN0000003235, ITGβ3 shRNA-c) or a non-targeted shRNA (SHC002, Sigma-Aldrich) as a negative control.

    Techniques: Expressing, shRNA, Derivative Assay, Activity Assay, Sequencing, Infection, Plasmid Preparation, Imaging

    Phenotypes of RA-FLSs with depletion of α9 cultured in 3D-micromass system under stimulation with PDGF or TNF-α. RA-FLSs treated with shRNA either for ITGA9 or the control (Con) were cultured in a 3D-micromass system in the media supplemented with PDGF (PDGF-BB, 50 ng/ml) or TNF-α (20 ng/ml) and the phenotypes were analyzed. ( A ) Frozen sections from the resultant architectures were costained with Alexa Fluor 488–phalloidin (green) and DAPI (blue). Representative data from three independent experiments are shown. Scale bars, 50 μm. ( B ) Protein amounts of MMP-1, MMP-3, and IL-6 in the culture supernatants collected at the end of the study were determined by ELISA and expressed as mean ± SE ( n = 5). Statistical analyses were performed by a paired t test. * /# p

    Journal: The Journal of Immunology Author Choice

    Article Title: Constitutive Activation of Integrin α9 Augments Self-Directed Hyperplastic and Proinflammatory Properties of Fibroblast-like Synoviocytes of Rheumatoid Arthritis

    doi: 10.4049/jimmunol.1700941

    Figure Lengend Snippet: Phenotypes of RA-FLSs with depletion of α9 cultured in 3D-micromass system under stimulation with PDGF or TNF-α. RA-FLSs treated with shRNA either for ITGA9 or the control (Con) were cultured in a 3D-micromass system in the media supplemented with PDGF (PDGF-BB, 50 ng/ml) or TNF-α (20 ng/ml) and the phenotypes were analyzed. ( A ) Frozen sections from the resultant architectures were costained with Alexa Fluor 488–phalloidin (green) and DAPI (blue). Representative data from three independent experiments are shown. Scale bars, 50 μm. ( B ) Protein amounts of MMP-1, MMP-3, and IL-6 in the culture supernatants collected at the end of the study were determined by ELISA and expressed as mean ± SE ( n = 5). Statistical analyses were performed by a paired t test. * /# p

    Article Snippet: Lentivirus short hairpin RNA (shRNA) expression plasmids for ITGA9 (TRCN0000230788), TNC (TRCN0000057740), control shRNA plasmid (pLKO.1-puro non-mammalian shRNA control plasmid DNA), and lentiviral packaging mix were purchased from Sigma-Aldrich.

    Techniques: Cell Culture, shRNA, Enzyme-linked Immunosorbent Assay

    Involvement of α9 and Tn-C in the self-directed abnormal behavior of RA-FLSs. ( A ) Gene expression levels of ITGA9 (left) and TNC (middle) in OA- ( n = 5) and RA-FLSs ( n = 5) were determined by real-time PCR and expressed as mean ± SE. Distributions of α9 and Tn-C in 3D-cultured RA-FLSs and RA synovial tissue was determined by costaining with Alexa Fluor 594–labeled Ab to α9 (red) and Ab to Tn-C with Alexa Fluor 488–labeled secondary Ab (green) (right). Scale bars, 100 μm. ( B – E ) RA-FLSs treated with shRNA either for ITGA9 , TNC , or the control (Con) cultured in 3D micromass were analyzed the phenotypes. (B) Sections from the resultant architectures were costained with Alexa Fluor 488–phalloidin (green) and DAPI (blue). Representative data from three independent RA-FLSs are shown. Scale bars, 50 μm. (C) Cell lysates from 3D-cultured RA-FLSs were analyzed by Western blotting with Abs to pFAK (upper) and total FAK (lower). Representative data from five independent blots are shown. (D) Protein amounts of MMP-1, MMP-3, and IL-6 in the culture supernatants were determined by ELISA and indicated as mean ± SE ( n = 5). (E) Western blotting of the cell lysates with Abs to MMP-14 (left), TNFSF11 (right) and GAPDH. A representative data from five independent blots was shown. Statistical analyses were performed by a Student t test (A) and a Dunnett test using within-subject error (D). * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Constitutive Activation of Integrin α9 Augments Self-Directed Hyperplastic and Proinflammatory Properties of Fibroblast-like Synoviocytes of Rheumatoid Arthritis

    doi: 10.4049/jimmunol.1700941

    Figure Lengend Snippet: Involvement of α9 and Tn-C in the self-directed abnormal behavior of RA-FLSs. ( A ) Gene expression levels of ITGA9 (left) and TNC (middle) in OA- ( n = 5) and RA-FLSs ( n = 5) were determined by real-time PCR and expressed as mean ± SE. Distributions of α9 and Tn-C in 3D-cultured RA-FLSs and RA synovial tissue was determined by costaining with Alexa Fluor 594–labeled Ab to α9 (red) and Ab to Tn-C with Alexa Fluor 488–labeled secondary Ab (green) (right). Scale bars, 100 μm. ( B – E ) RA-FLSs treated with shRNA either for ITGA9 , TNC , or the control (Con) cultured in 3D micromass were analyzed the phenotypes. (B) Sections from the resultant architectures were costained with Alexa Fluor 488–phalloidin (green) and DAPI (blue). Representative data from three independent RA-FLSs are shown. Scale bars, 50 μm. (C) Cell lysates from 3D-cultured RA-FLSs were analyzed by Western blotting with Abs to pFAK (upper) and total FAK (lower). Representative data from five independent blots are shown. (D) Protein amounts of MMP-1, MMP-3, and IL-6 in the culture supernatants were determined by ELISA and indicated as mean ± SE ( n = 5). (E) Western blotting of the cell lysates with Abs to MMP-14 (left), TNFSF11 (right) and GAPDH. A representative data from five independent blots was shown. Statistical analyses were performed by a Student t test (A) and a Dunnett test using within-subject error (D). * p

    Article Snippet: Lentivirus short hairpin RNA (shRNA) expression plasmids for ITGA9 (TRCN0000230788), TNC (TRCN0000057740), control shRNA plasmid (pLKO.1-puro non-mammalian shRNA control plasmid DNA), and lentiviral packaging mix were purchased from Sigma-Aldrich.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Labeling, shRNA, Western Blot, Enzyme-linked Immunosorbent Assay