e0554  (New England Biolabs)


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  • 99
    Name:
    Q5 Site Directed Mutagenesis Kit
    Description:
    Q5 Site Directed Mutagenesis Kit 10 rxns
    Catalog Number:
    E0554S
    Price:
    197
    Category:
    PCR Mutagenesis Kits
    Size:
    10 rxns
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    Structured Review

    New England Biolabs e0554
    Q5 Site Directed Mutagenesis Kit
    Q5 Site Directed Mutagenesis Kit 10 rxns
    https://www.bioz.com/result/e0554/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e0554 - by Bioz Stars, 2021-10
    99/100 stars

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    Related Articles

    Generated:

    Article Title: Structures of signaling complexes of lipid receptors S1PR1 and S1PR5 reveal mechanisms of activation and drug recognition
    Article Snippet: .. Site-directed mutations in both receptors were generated using Q5 site-Directed Mutagenesis kit (NEB). ..

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y507A, Y585A mutations in PPxY motifs of Bon were generated by the Q5 Site-Directed Mutagenesis Kit. pUASTattB-bon-mCherry and pUASTattB-bon-Y507A Y585A-mCherry were constructed using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, Cat# E5520S). ..

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y281A, Y350A mutations of WW domains in Yki were generated by Q5 Site-Directed Mutagenesis Kit (NEB, Cat# E0554S) using pMT-yki-HA as a template. ..

    Mutagenesis:

    Article Title: Structures of signaling complexes of lipid receptors S1PR1 and S1PR5 reveal mechanisms of activation and drug recognition
    Article Snippet: .. Site-directed mutations in both receptors were generated using Q5 site-Directed Mutagenesis kit (NEB). ..

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y507A, Y585A mutations in PPxY motifs of Bon were generated by the Q5 Site-Directed Mutagenesis Kit. pUASTattB-bon-mCherry and pUASTattB-bon-Y507A Y585A-mCherry were constructed using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, Cat# E5520S). ..

    Article Title: Regulatory regions in natural transposable element insertions drive interindividual differences in response to immune challenges in Drosophila
    Article Snippet: .. Mutagenesis of the binding sites for the predicted Caudal and DEAF-1 transcription factors was performed sequentially with the Q5 Site-Directed Mutagenesis kit (New England Biolabs) taking as a template the pGreenRabbit vector containing the FBti0019386 sequence following manufacturer´s instructions [ ]. ..

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y281A, Y350A mutations of WW domains in Yki were generated by Q5 Site-Directed Mutagenesis Kit (NEB, Cat# E0554S) using pMT-yki-HA as a template. ..

    Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation
    Article Snippet: .. HindIII and BamHI restriction sites were inserted just after the codon coding for proline 16 using Q5-insertion mutagenesis (#E0554; NEB) according to the protocol from the manufacturer. ..

    Article Title: Neuronal calmodulin levels are controlled by CAMTA transcription factors
    Article Snippet: .. To convert T05C1.4b cDNA to T05C1.4a we used the Q5 Site-Directed Mutagenesis Kit (NEB) and primers gtcatactcaacatctaATTGCGGAAAATGCATGC and catcatcaatatttacaTTATTACGATTTTGTCGCATAAAATTC. ..

    Article Title: Collagen VI regulates motor circuit plasticity and motor performance by cannabinoid modulation
    Article Snippet: .. C4 domain sequence was removed with the Q5 site-directed mutagenesis kit (NEB). ..

    Article Title: A novel mechanism of enhanced transcription activity and fidelity for influenza A viral RNA-dependent RNA polymerase
    Article Snippet: .. K235 and R239 mutations were introduced by site-direct mutagenesis kit (NEB E0554S) according to the protocol. ..

    Construct:

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y507A, Y585A mutations in PPxY motifs of Bon were generated by the Q5 Site-Directed Mutagenesis Kit. pUASTattB-bon-mCherry and pUASTattB-bon-Y507A Y585A-mCherry were constructed using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, Cat# E5520S). ..

    Clone Assay:

    Article Title: Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye
    Article Snippet: .. The Y507A, Y585A mutations in PPxY motifs of Bon were generated by the Q5 Site-Directed Mutagenesis Kit. pUASTattB-bon-mCherry and pUASTattB-bon-Y507A Y585A-mCherry were constructed using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, Cat# E5520S). ..

    Binding Assay:

    Article Title: Regulatory regions in natural transposable element insertions drive interindividual differences in response to immune challenges in Drosophila
    Article Snippet: .. Mutagenesis of the binding sites for the predicted Caudal and DEAF-1 transcription factors was performed sequentially with the Q5 Site-Directed Mutagenesis kit (New England Biolabs) taking as a template the pGreenRabbit vector containing the FBti0019386 sequence following manufacturer´s instructions [ ]. ..

    Plasmid Preparation:

    Article Title: Regulatory regions in natural transposable element insertions drive interindividual differences in response to immune challenges in Drosophila
    Article Snippet: .. Mutagenesis of the binding sites for the predicted Caudal and DEAF-1 transcription factors was performed sequentially with the Q5 Site-Directed Mutagenesis kit (New England Biolabs) taking as a template the pGreenRabbit vector containing the FBti0019386 sequence following manufacturer´s instructions [ ]. ..

    Sequencing:

    Article Title: Regulatory regions in natural transposable element insertions drive interindividual differences in response to immune challenges in Drosophila
    Article Snippet: .. Mutagenesis of the binding sites for the predicted Caudal and DEAF-1 transcription factors was performed sequentially with the Q5 Site-Directed Mutagenesis kit (New England Biolabs) taking as a template the pGreenRabbit vector containing the FBti0019386 sequence following manufacturer´s instructions [ ]. ..

    Article Title: Collagen VI regulates motor circuit plasticity and motor performance by cannabinoid modulation
    Article Snippet: .. C4 domain sequence was removed with the Q5 site-directed mutagenesis kit (NEB). ..

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  • 99
    New England Biolabs pcr mutagenesis
    Phosphomimetic mutant of FOXO1 Ser-329 abrogates S. gordonii -mediated antagonism of FOXO1 activation. ( A ) TIGK cells were transiently transfected dually with FOXO1, FOXO1S329E, or FOXO1S329A, along with the FOXO promoter–luciferase reporter plasmid, or a constitutively expressing Renilla luciferase reporter. Cells were challenged with P. gingivalis ( Pg ) and/or S. gordonii ( Sg ) at MOI:50 for each strain for 15 min. Control cells were noninfected (NI). FOXO luciferase was normalized to the level of Renilla luciferase. ( B ) TIGK cells were transiently transfected with FOXO1, FOXO1S329E, or FOXO1S329A and challenged with bacteria as in A for 24 h. <t>ZEB2</t> mRNA levels were measured by <t>qRT-PCR.</t> Data were normalized to GAPDH mRNA and are expressed relative to noninfected (NI) controls. Quantitative data represent three independent experiments with three replicates. Error bars represent the SEM. * P > 0.05, ** P
    Pcr Mutagenesis, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr mutagenesis/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs wwtr1
    Cholesterol Depletion of Hepatocytes Decreases TAZ in a LATS2-Dependent Manner (A) LATS2 kinase assay, using rTAZ as substrate, of extracts of livers of mice fed the NAFLD diet containing 0.2%, 0.5%, or 1.25% cholesterol for 16 weeks. (B) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from AML12 cells incubated as follows: left blot, 4 h with MG132 plus vehicle (Veh) or liposomes; right blot, 18 h with liposomes and then 4 h with MG132 plus vehicle or Lipo-Chol. (C) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from primary human hepatocytes incubated for 8 h with vehicle or Lipo-Chol, with MG132 included during the last 4 h. (D) TAZ and YAP immunoblots of siScr-treated or siLats2-treated AML12 cells that were incubated for 24 h with vehicle or liposomes. (E) HA-TAZ immunoblot of HA-WT-human TAZ- or HA-S117A-human TAZ-transfected AML12 cells that were incubated for 24 h with vehicle, or liposomes for 16 h and then Lipo-Chol for 8 h. (F-J) The following parameters were measured in <t>Wwtr1</t> fl/fl mice fed the NAFLD diet containing 0.2% cholesterol for 16 weeks, with AAV8-TBG-Cre plus either AAV8-TBG-HA-WT-hTAZ or AAV8-TBG-HA-S117A-hTAZ injected at the 8-week time point (n = 10 mice/group; means ± SEM; *p
    Wwtr1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wwtr1/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    wwtr1 - by Bioz Stars, 2021-10
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    Phosphomimetic mutant of FOXO1 Ser-329 abrogates S. gordonii -mediated antagonism of FOXO1 activation. ( A ) TIGK cells were transiently transfected dually with FOXO1, FOXO1S329E, or FOXO1S329A, along with the FOXO promoter–luciferase reporter plasmid, or a constitutively expressing Renilla luciferase reporter. Cells were challenged with P. gingivalis ( Pg ) and/or S. gordonii ( Sg ) at MOI:50 for each strain for 15 min. Control cells were noninfected (NI). FOXO luciferase was normalized to the level of Renilla luciferase. ( B ) TIGK cells were transiently transfected with FOXO1, FOXO1S329E, or FOXO1S329A and challenged with bacteria as in A for 24 h. ZEB2 mRNA levels were measured by qRT-PCR. Data were normalized to GAPDH mRNA and are expressed relative to noninfected (NI) controls. Quantitative data represent three independent experiments with three replicates. Error bars represent the SEM. * P > 0.05, ** P

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

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis

    doi: 10.1073/pnas.1900101116

    Figure Lengend Snippet: Phosphomimetic mutant of FOXO1 Ser-329 abrogates S. gordonii -mediated antagonism of FOXO1 activation. ( A ) TIGK cells were transiently transfected dually with FOXO1, FOXO1S329E, or FOXO1S329A, along with the FOXO promoter–luciferase reporter plasmid, or a constitutively expressing Renilla luciferase reporter. Cells were challenged with P. gingivalis ( Pg ) and/or S. gordonii ( Sg ) at MOI:50 for each strain for 15 min. Control cells were noninfected (NI). FOXO luciferase was normalized to the level of Renilla luciferase. ( B ) TIGK cells were transiently transfected with FOXO1, FOXO1S329E, or FOXO1S329A and challenged with bacteria as in A for 24 h. ZEB2 mRNA levels were measured by qRT-PCR. Data were normalized to GAPDH mRNA and are expressed relative to noninfected (NI) controls. Quantitative data represent three independent experiments with three replicates. Error bars represent the SEM. * P > 0.05, ** P

    Article Snippet: A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB).

    Techniques: Mutagenesis, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Impact of dual species challenge on ZEB2 mRNA and associated phenotypic properties. ( A and B ) ZEB2 mRNA levels measured by qRT-PCR in TIGK cells infected with P. gingivalis 33277 ( Pg ) alone or together with F. nucleatum ( Fn ) or with oral streptococcal species. Sc , S. cristatus ; Sg , S. gordonii ; So , S. oralis ; Ss , S. sanguinis . Monoinfection was MOI:100 for 24 h. Dual species infection was MOI:100 for each strain for 24 h. ( C ) Quantitative analysis of TIGK migration through matrigel-coated transwells. TIGK cells were transiently transfected with siRNA to ZEB2 (siZEB2) or scrambled siRNA (siControl) ( Left ) or nontransfected ( Right ). TIGKs were challenged with P. gingivalis 33277 and/or S. gordonii at MOI:50 for each strain for 24 h. Control cells were not infected (NI). Data are presented as the mean number of cells invading through the transwell. ( D ) ZEB2 was silenced with siRNA ( Left ), and TIGKs were challenged with bacteria as in C for 2 h. IL-6 mRNA levels were measured by qRT-PCR. Data were normalized to GAPDH mRNA and are expressed relative to noninfected (NI) controls. Quantitative data represent three independent experiments with three replicates. Error bars represent the SEM. ** P

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

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis

    doi: 10.1073/pnas.1900101116

    Figure Lengend Snippet: Impact of dual species challenge on ZEB2 mRNA and associated phenotypic properties. ( A and B ) ZEB2 mRNA levels measured by qRT-PCR in TIGK cells infected with P. gingivalis 33277 ( Pg ) alone or together with F. nucleatum ( Fn ) or with oral streptococcal species. Sc , S. cristatus ; Sg , S. gordonii ; So , S. oralis ; Ss , S. sanguinis . Monoinfection was MOI:100 for 24 h. Dual species infection was MOI:100 for each strain for 24 h. ( C ) Quantitative analysis of TIGK migration through matrigel-coated transwells. TIGK cells were transiently transfected with siRNA to ZEB2 (siZEB2) or scrambled siRNA (siControl) ( Left ) or nontransfected ( Right ). TIGKs were challenged with P. gingivalis 33277 and/or S. gordonii at MOI:50 for each strain for 24 h. Control cells were not infected (NI). Data are presented as the mean number of cells invading through the transwell. ( D ) ZEB2 was silenced with siRNA ( Left ), and TIGKs were challenged with bacteria as in C for 2 h. IL-6 mRNA levels were measured by qRT-PCR. Data were normalized to GAPDH mRNA and are expressed relative to noninfected (NI) controls. Quantitative data represent three independent experiments with three replicates. Error bars represent the SEM. ** P

    Article Snippet: A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB).

    Techniques: Quantitative RT-PCR, Infection, Migration, Transfection

    P. gingivalis up-regulates transcription factors controlling EMT. ( A and B ) TIGK cells were infected with P. gingivalis 33277 at the times and MOIs indicated. ZEB2 ( A ) or TWIST1/2 ( B ) mRNA levels were measured by qRT-PCR. Data were normalized to GAPDH mRNA and are expressed relative to noninfected (NI) controls. ( C ) Fluorescent confocal microscopy of TIGK cells infected with P. gingivalis 33277 ( Pg ) at the MOI indicated for 24 h. Control cells were noninfected (NI). Cells were fixed and probed with ZEB2 antibodies (green). Actin (red) was stained with Texas Red-phalloidin, and nuclei (blue) were stained with DAPI. Cells were imaged at magnification 63×. Shown are merged images of projections of z-stacks ( Left ) and Pearson’s correlation coefficient of ZEB2 with nuclei ( Right ) obtained with Volocity software. ( D ) TIGK cells were infected with P. gingivalis strains at MOI:100 for 24 h. ZEB2 mRNA levels were determined as in A . ( E ) ZEB2 mRNA levels in different cell types following P. gingivalis 33277 infection for 24 h. Quantitative data represent three independent experiments with three replicates. Error bars represent the SEM. * P

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

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis

    doi: 10.1073/pnas.1900101116

    Figure Lengend Snippet: P. gingivalis up-regulates transcription factors controlling EMT. ( A and B ) TIGK cells were infected with P. gingivalis 33277 at the times and MOIs indicated. ZEB2 ( A ) or TWIST1/2 ( B ) mRNA levels were measured by qRT-PCR. Data were normalized to GAPDH mRNA and are expressed relative to noninfected (NI) controls. ( C ) Fluorescent confocal microscopy of TIGK cells infected with P. gingivalis 33277 ( Pg ) at the MOI indicated for 24 h. Control cells were noninfected (NI). Cells were fixed and probed with ZEB2 antibodies (green). Actin (red) was stained with Texas Red-phalloidin, and nuclei (blue) were stained with DAPI. Cells were imaged at magnification 63×. Shown are merged images of projections of z-stacks ( Left ) and Pearson’s correlation coefficient of ZEB2 with nuclei ( Right ) obtained with Volocity software. ( D ) TIGK cells were infected with P. gingivalis strains at MOI:100 for 24 h. ZEB2 mRNA levels were determined as in A . ( E ) ZEB2 mRNA levels in different cell types following P. gingivalis 33277 infection for 24 h. Quantitative data represent three independent experiments with three replicates. Error bars represent the SEM. * P

    Article Snippet: A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB).

    Techniques: Infection, Quantitative RT-PCR, Confocal Microscopy, Staining, Software

    ZEB2 responses to P. gingivalis are controlled by β-catenin and FOXO1 pathways. ( A ) TIGK cells were transiently transfected with siRNA to β-catenin or scrambled siRNA (siControl) and infected with P. gingivalis 33277 for 24 h at the MOI indicated. ZEB2 mRNA levels were measured by qRT-PCR. Data were normalized to GAPDH mRNA and are expressed relative to noninfected (NI) controls. ( B ) TIGK cells were transiently transfected with plasmid expressing full-length β-catenin, a Δ151 truncation derivative, or with empty vector. Cells were challenged with P. gingivalis and ZEB2 mRNA measured as described in A . ( C ) TIGK cells were challenged with P. gingivalis strains for 24 h at the MOI indicated. WT, P. gingivalis 33277; ΔrgpA/B, deletion mutant of the rgpA and rgpB arginine gingipain genes; Δkgp, deletion mutant of the kgp lysine gingipain gene; ΔrgpAB Δkgp, triple gingipain deletion mutant; TLCK, WT preincubated with the protease inhibitor TLCK (100 µM, 2 h). ZEB2 mRNA was measured as described in A . ( D and E ) TIGK cells were transiently transfected with siRNA to TCF7L2/TCF7L3/TCF7, or TCF7L1 ( D ), FOXO1 or FOXO3 ( E ), or control scrambled siRNA. Cells were challenged with P. gingivalis , and ZEB2 mRNA was measured as described in A . ( F ) TIGK cells were transiently transfected with siRNA to FOXO1 or control scrambled siRNA. Cells were challenged with P. gingivalis 33277 and/or S. gordonii at MOI:50 for each strain for 24 h. Quantitative analysis of TIGK migration through matrigel-coated transwells is presented as the mean number of migrated cells. ( G ) TIGK cells were challenged with P. gingivalis 33277 MOI:100 for 24 h, or left uninfected (NI), and subjected to chromatin immunoprecipitation (ChIP) using anti-FOXO1 IgG, anti-TCF7L1 IgG, or preimmune IgG. The precipitated DNA was subsequently analyzed by end point PCR and by qPCR with primers to the ZEB2 promoter region or the GAPDH promoter as a control. qPCR was expressed relative to the input DNA. ( H ) Luciferase assay for ZEB2 promoter activity in TIGKs challenged with P. gingivalis 33277 MOI:100 for 30 min, or left uninfected (NI). Cells were transiently transfected with a ZEB2 promoter–luciferase reporter plasmid, or a constitutively expressing Renilla luciferase reporter. Derivatives of the ZEB2 promoter included serial deletions and site-specific mutations (denoted X) in the FOXO1 binding sites. FOXO luciferase activity was normalized to the level of Renilla luciferase. Quantitative data represent three independent experiments with three replicates. Error bars represent the SEM. * P > 0.05, ** P

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

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis

    doi: 10.1073/pnas.1900101116

    Figure Lengend Snippet: ZEB2 responses to P. gingivalis are controlled by β-catenin and FOXO1 pathways. ( A ) TIGK cells were transiently transfected with siRNA to β-catenin or scrambled siRNA (siControl) and infected with P. gingivalis 33277 for 24 h at the MOI indicated. ZEB2 mRNA levels were measured by qRT-PCR. Data were normalized to GAPDH mRNA and are expressed relative to noninfected (NI) controls. ( B ) TIGK cells were transiently transfected with plasmid expressing full-length β-catenin, a Δ151 truncation derivative, or with empty vector. Cells were challenged with P. gingivalis and ZEB2 mRNA measured as described in A . ( C ) TIGK cells were challenged with P. gingivalis strains for 24 h at the MOI indicated. WT, P. gingivalis 33277; ΔrgpA/B, deletion mutant of the rgpA and rgpB arginine gingipain genes; Δkgp, deletion mutant of the kgp lysine gingipain gene; ΔrgpAB Δkgp, triple gingipain deletion mutant; TLCK, WT preincubated with the protease inhibitor TLCK (100 µM, 2 h). ZEB2 mRNA was measured as described in A . ( D and E ) TIGK cells were transiently transfected with siRNA to TCF7L2/TCF7L3/TCF7, or TCF7L1 ( D ), FOXO1 or FOXO3 ( E ), or control scrambled siRNA. Cells were challenged with P. gingivalis , and ZEB2 mRNA was measured as described in A . ( F ) TIGK cells were transiently transfected with siRNA to FOXO1 or control scrambled siRNA. Cells were challenged with P. gingivalis 33277 and/or S. gordonii at MOI:50 for each strain for 24 h. Quantitative analysis of TIGK migration through matrigel-coated transwells is presented as the mean number of migrated cells. ( G ) TIGK cells were challenged with P. gingivalis 33277 MOI:100 for 24 h, or left uninfected (NI), and subjected to chromatin immunoprecipitation (ChIP) using anti-FOXO1 IgG, anti-TCF7L1 IgG, or preimmune IgG. The precipitated DNA was subsequently analyzed by end point PCR and by qPCR with primers to the ZEB2 promoter region or the GAPDH promoter as a control. qPCR was expressed relative to the input DNA. ( H ) Luciferase assay for ZEB2 promoter activity in TIGKs challenged with P. gingivalis 33277 MOI:100 for 30 min, or left uninfected (NI). Cells were transiently transfected with a ZEB2 promoter–luciferase reporter plasmid, or a constitutively expressing Renilla luciferase reporter. Derivatives of the ZEB2 promoter included serial deletions and site-specific mutations (denoted X) in the FOXO1 binding sites. FOXO luciferase activity was normalized to the level of Renilla luciferase. Quantitative data represent three independent experiments with three replicates. Error bars represent the SEM. * P > 0.05, ** P

    Article Snippet: A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB).

    Techniques: Transfection, Infection, Quantitative RT-PCR, Plasmid Preparation, Expressing, Mutagenesis, Protease Inhibitor, Migration, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Binding Assay

    LSD1 is required for EGF-induced migration of cells with an active AKT-GSK3β-Snail axis. (A) 10x bright field images of crystal violet stained HT29 cells after 48H migration through transwell insert. WT, LSD1 KO, 10 μM GSK690693 or 3 μM corin cells were co-treated with 100 ng/mL EGF for 48H. (B) Quantification of migration normalized to migration counts for untreated cells. Results are represented as mean ± SD. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. All significant comparisons are shown. adj. p-value; ****

    Journal: Molecular cancer research : MCR

    Article Title: Lysine-specific demethylase 1 mediates AKT activity and promotes epithelial-mesenchymal transition in PIK3CA mutant colorectal cancer

    doi: 10.1158/1541-7786.MCR-19-0748

    Figure Lengend Snippet: LSD1 is required for EGF-induced migration of cells with an active AKT-GSK3β-Snail axis. (A) 10x bright field images of crystal violet stained HT29 cells after 48H migration through transwell insert. WT, LSD1 KO, 10 μM GSK690693 or 3 μM corin cells were co-treated with 100 ng/mL EGF for 48H. (B) Quantification of migration normalized to migration counts for untreated cells. Results are represented as mean ± SD. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. All significant comparisons are shown. adj. p-value; ****

    Article Snippet: Mutagenesis was performed according to manufacturer’s protocol (NEB, E0554) using HA-LSD1 plasmid as template, and confirmed via Sanger sequencing by Eurofins Scientific.

    Techniques: Migration, Staining

    Gastrointestinal cell lines with mutant PIK3CA are sensitive to LSD1 KD. (A) SW480, LoVo, HT29, AGS, HCT116 and RKO cell growth over a five-day time course determined by the CellTiter-Glo Luminescent Cell Viability Assay. N=4. Graph depicts mean + SD. Statistical analyses are performed using 2way ANOVA and Sidaks multiple comparisons test with all statistically significant comparisons shown. adj. p-value; **

    Journal: Molecular cancer research : MCR

    Article Title: Lysine-specific demethylase 1 mediates AKT activity and promotes epithelial-mesenchymal transition in PIK3CA mutant colorectal cancer

    doi: 10.1158/1541-7786.MCR-19-0748

    Figure Lengend Snippet: Gastrointestinal cell lines with mutant PIK3CA are sensitive to LSD1 KD. (A) SW480, LoVo, HT29, AGS, HCT116 and RKO cell growth over a five-day time course determined by the CellTiter-Glo Luminescent Cell Viability Assay. N=4. Graph depicts mean + SD. Statistical analyses are performed using 2way ANOVA and Sidaks multiple comparisons test with all statistically significant comparisons shown. adj. p-value; **

    Article Snippet: Mutagenesis was performed according to manufacturer’s protocol (NEB, E0554) using HA-LSD1 plasmid as template, and confirmed via Sanger sequencing by Eurofins Scientific.

    Techniques: Mutagenesis, Cell Viability Assay

    LSD1 regulates phosphorylation of AKT in CRC. (A) Box and whisker plot of fragments per kilobase of transcript per million mapped reads (FPKM) expression values for LSD1 across different TCGA datasets. Box limits are set at the third and first quartile range with central line at the median, with whiskers depicting 1.5 times the interquartile range. Datapoints outside this range are represented at outliers (black dots). Black and blue outline indicates data for WT and PIK3CA mutant tumors, respectively. Red and purple fill represent significant increase and decrease in LSD1 expression, respectively, between PIK3CA mutant and PIK3CA WT tumors. The numbers under the box plots are the number of samples used to generate the plots. Western blot analysis of empty vector (shEV) or LSD1 KD in (B) SW480 or (C) HT29 cells. Arrow head indicates correct position of pT308-AKT band. Western blot quantified by densitometric analysis and normalized to β-actin and shEV. Results are represented as mean ± SD. (N=3). Significance was determined by two-tailed Student’s t-test. LSD1 CRISPR KO clones with or without LSD1 OE plasmid (HA-LSD1) in (D) SW480 or (E) HT29 cells analyzed by western blot. (F) EV, LSD1 KD or LSD1 OE cells treated w/ 250 μM H 2 O 2 for 1H. (G) Brightfield and immunofluorescence images of EV or LSD1 KD HT29 cells under untreated or H 2 O 2 treated conditions. A field was selected in the H 2 O 2 treated shLSD1 cells to facilitate direct comparison of LSD1 deficient and LSD1 proficient cells. White arrow indicates cells with LSD1 expression and orange arrow indicates cells deficient in LSD1. (p-value; ***

    Journal: Molecular cancer research : MCR

    Article Title: Lysine-specific demethylase 1 mediates AKT activity and promotes epithelial-mesenchymal transition in PIK3CA mutant colorectal cancer

    doi: 10.1158/1541-7786.MCR-19-0748

    Figure Lengend Snippet: LSD1 regulates phosphorylation of AKT in CRC. (A) Box and whisker plot of fragments per kilobase of transcript per million mapped reads (FPKM) expression values for LSD1 across different TCGA datasets. Box limits are set at the third and first quartile range with central line at the median, with whiskers depicting 1.5 times the interquartile range. Datapoints outside this range are represented at outliers (black dots). Black and blue outline indicates data for WT and PIK3CA mutant tumors, respectively. Red and purple fill represent significant increase and decrease in LSD1 expression, respectively, between PIK3CA mutant and PIK3CA WT tumors. The numbers under the box plots are the number of samples used to generate the plots. Western blot analysis of empty vector (shEV) or LSD1 KD in (B) SW480 or (C) HT29 cells. Arrow head indicates correct position of pT308-AKT band. Western blot quantified by densitometric analysis and normalized to β-actin and shEV. Results are represented as mean ± SD. (N=3). Significance was determined by two-tailed Student’s t-test. LSD1 CRISPR KO clones with or without LSD1 OE plasmid (HA-LSD1) in (D) SW480 or (E) HT29 cells analyzed by western blot. (F) EV, LSD1 KD or LSD1 OE cells treated w/ 250 μM H 2 O 2 for 1H. (G) Brightfield and immunofluorescence images of EV or LSD1 KD HT29 cells under untreated or H 2 O 2 treated conditions. A field was selected in the H 2 O 2 treated shLSD1 cells to facilitate direct comparison of LSD1 deficient and LSD1 proficient cells. White arrow indicates cells with LSD1 expression and orange arrow indicates cells deficient in LSD1. (p-value; ***

    Article Snippet: Mutagenesis was performed according to manufacturer’s protocol (NEB, E0554) using HA-LSD1 plasmid as template, and confirmed via Sanger sequencing by Eurofins Scientific.

    Techniques: Whisker Assay, Expressing, Mutagenesis, Western Blot, Plasmid Preparation, Two Tailed Test, CRISPR, Clone Assay, Immunofluorescence, Significance Assay

    LSD1 regulates AKT activation via scaffolding of the CoREST complex on chromatin. (A) Chromatin affinity assay performed in shEV or shLSD1 with whole cell extract (WCE) or chromatin bound fraction. Western blots were quantified by densitometric analysis and normalized to loading control and shEV fraction. Significance determined by 2way ANOVA with Sidak’s multiple comparisons test (N=3). Graphs depict mean ± SD (adj. p-value; **

    Journal: Molecular cancer research : MCR

    Article Title: Lysine-specific demethylase 1 mediates AKT activity and promotes epithelial-mesenchymal transition in PIK3CA mutant colorectal cancer

    doi: 10.1158/1541-7786.MCR-19-0748

    Figure Lengend Snippet: LSD1 regulates AKT activation via scaffolding of the CoREST complex on chromatin. (A) Chromatin affinity assay performed in shEV or shLSD1 with whole cell extract (WCE) or chromatin bound fraction. Western blots were quantified by densitometric analysis and normalized to loading control and shEV fraction. Significance determined by 2way ANOVA with Sidak’s multiple comparisons test (N=3). Graphs depict mean ± SD (adj. p-value; **

    Article Snippet: Mutagenesis was performed according to manufacturer’s protocol (NEB, E0554) using HA-LSD1 plasmid as template, and confirmed via Sanger sequencing by Eurofins Scientific.

    Techniques: Activation Assay, Scaffolding, Western Blot

    LSD1 regulates Snail stability via AKT in PIK3CA C2 domain mutant cancer cells. (A) Western blot analysis of HT29 or (B) AGS cells treated with DMSO or 10 μM GSK690693 for 48H. (C) Real-time PCR analysis of SNAI1 RNA expression levels after 48H DMSO or 10 μM GSK690693 treatment in HT29 and AGS cells. Expression was normalized to Gapdh and DMSO. Results are represented as mean ± SD. Significance determined by 2way ANOVA with Sidak’s multiple comparisons test. ns – not significant. (D) Proportion of total PIK3CA mutations occurring in the different domains indicated across various cancer types in the TCGA pancancer datasets. (E) shEV and shLSD1 HT29 cells treated with DMSO or 10 μM MG-132 for 4H and analyzed by western blot. (F) Real-time PCR analysis of LSD1 and SNAI1 RNA expression levels in shEV and shLSD1 HT29 cells as in (C). adj. p-value; ****

    Journal: Molecular cancer research : MCR

    Article Title: Lysine-specific demethylase 1 mediates AKT activity and promotes epithelial-mesenchymal transition in PIK3CA mutant colorectal cancer

    doi: 10.1158/1541-7786.MCR-19-0748

    Figure Lengend Snippet: LSD1 regulates Snail stability via AKT in PIK3CA C2 domain mutant cancer cells. (A) Western blot analysis of HT29 or (B) AGS cells treated with DMSO or 10 μM GSK690693 for 48H. (C) Real-time PCR analysis of SNAI1 RNA expression levels after 48H DMSO or 10 μM GSK690693 treatment in HT29 and AGS cells. Expression was normalized to Gapdh and DMSO. Results are represented as mean ± SD. Significance determined by 2way ANOVA with Sidak’s multiple comparisons test. ns – not significant. (D) Proportion of total PIK3CA mutations occurring in the different domains indicated across various cancer types in the TCGA pancancer datasets. (E) shEV and shLSD1 HT29 cells treated with DMSO or 10 μM MG-132 for 4H and analyzed by western blot. (F) Real-time PCR analysis of LSD1 and SNAI1 RNA expression levels in shEV and shLSD1 HT29 cells as in (C). adj. p-value; ****

    Article Snippet: Mutagenesis was performed according to manufacturer’s protocol (NEB, E0554) using HA-LSD1 plasmid as template, and confirmed via Sanger sequencing by Eurofins Scientific.

    Techniques: Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Expressing

    LSD1 catalytic activity is dispensable for regulation of gene expression and activation of AKT. (A) Metageneplot and heatmap depicting ChIP-seq of LSD1 in WT (N=2) or LSD1 KO (N=1) SW480 cells at gene enrichment sites genome-wide. Average plots and heatmaps depicting: (B) LSD1 enrichment peak overlap with DNase-seq peaks in SW480, and (C) H3K4me2 ChIP-seq signal at TSS enrichment sites genome-wide in shEV and shLSD1 SW480 cells (N=3). (A-C) Values are derived from CPM (counts per million) normalized reads. (E) Differentially expressed genes (DEGs) from RNA-seq (log2FC > =1 FDR

    Journal: Molecular cancer research : MCR

    Article Title: Lysine-specific demethylase 1 mediates AKT activity and promotes epithelial-mesenchymal transition in PIK3CA mutant colorectal cancer

    doi: 10.1158/1541-7786.MCR-19-0748

    Figure Lengend Snippet: LSD1 catalytic activity is dispensable for regulation of gene expression and activation of AKT. (A) Metageneplot and heatmap depicting ChIP-seq of LSD1 in WT (N=2) or LSD1 KO (N=1) SW480 cells at gene enrichment sites genome-wide. Average plots and heatmaps depicting: (B) LSD1 enrichment peak overlap with DNase-seq peaks in SW480, and (C) H3K4me2 ChIP-seq signal at TSS enrichment sites genome-wide in shEV and shLSD1 SW480 cells (N=3). (A-C) Values are derived from CPM (counts per million) normalized reads. (E) Differentially expressed genes (DEGs) from RNA-seq (log2FC > =1 FDR

    Article Snippet: Mutagenesis was performed according to manufacturer’s protocol (NEB, E0554) using HA-LSD1 plasmid as template, and confirmed via Sanger sequencing by Eurofins Scientific.

    Techniques: Activity Assay, Expressing, Activation Assay, Chromatin Immunoprecipitation, Genome Wide, Derivative Assay, RNA Sequencing Assay

    HP-PRRSV with deletion mutant NSP2 impairs PGE2 production.  (A)  Strategy for the construction of the recombinant cDNA clone. The plasmid containing assembled genome of HV (pcDNA3.1-HV) was treated with Spe I and Afl II to get 439-6478 bp of the HV deleted mutant pcDNA3.1-HV [pcDNA3.1-HV (Δ439-6478)], and then the Q5 ®  Site-Directed Mutagenesis Kit was used to generated deleted mutations. Finally, using HiFi DNA Assembly Master Mix, pcDNA-HV-Δ500-596 [Δ500-596], pcDNA-HV-Δ658-777 [Δ658-777], and pcDNA-HV-Δ500-596/658-777 [Δ56]) were constructed.  (B)  Representative images of cytopathic effects induced by wt HP-PRRSV strain HV and the mutant viruses at 48 hours post-infection.  (C, D)  Supernatants were collected at indicated times post virus infection, and titrated for viruses using TCID 50  method.  (E, F)  Microglia were infected with HP-PRRSV strain HV and the mutant viruses at an MOI of 0.1. COX-2 expression was analyzed by real-time PCR  (E)  and PGE2 production was measured by ELISA  (F)  at 12 and 24 hours post-infection. Data are representative of three independent experiments (mean ± SEM). Statistical analysis was performed by Student’s  t -test. *,  P

    Journal: Frontiers in Immunology

    Article Title: NSP2 Is Important for Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus to Trigger High Fever-Related COX-2-PGE2 Pathway in Pigs

    doi: 10.3389/fimmu.2021.657071

    Figure Lengend Snippet: HP-PRRSV with deletion mutant NSP2 impairs PGE2 production. (A) Strategy for the construction of the recombinant cDNA clone. The plasmid containing assembled genome of HV (pcDNA3.1-HV) was treated with Spe I and Afl II to get 439-6478 bp of the HV deleted mutant pcDNA3.1-HV [pcDNA3.1-HV (Δ439-6478)], and then the Q5 ® Site-Directed Mutagenesis Kit was used to generated deleted mutations. Finally, using HiFi DNA Assembly Master Mix, pcDNA-HV-Δ500-596 [Δ500-596], pcDNA-HV-Δ658-777 [Δ658-777], and pcDNA-HV-Δ500-596/658-777 [Δ56]) were constructed. (B) Representative images of cytopathic effects induced by wt HP-PRRSV strain HV and the mutant viruses at 48 hours post-infection. (C, D) Supernatants were collected at indicated times post virus infection, and titrated for viruses using TCID 50 method. (E, F) Microglia were infected with HP-PRRSV strain HV and the mutant viruses at an MOI of 0.1. COX-2 expression was analyzed by real-time PCR (E) and PGE2 production was measured by ELISA (F) at 12 and 24 hours post-infection. Data are representative of three independent experiments (mean ± SEM). Statistical analysis was performed by Student’s t -test. *, P

    Article Snippet: The Q5® Site-Directed Mutagenesis Kit was used to generate deleted or point mutations [F2 (Δ500-596), F2 (Δ658-777), and F2 (Δ500-596/658-777)].

    Techniques: Mutagenesis, Recombinant, Plasmid Preparation, Generated, Construct, Infection, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Cholesterol Depletion of Hepatocytes Decreases TAZ in a LATS2-Dependent Manner (A) LATS2 kinase assay, using rTAZ as substrate, of extracts of livers of mice fed the NAFLD diet containing 0.2%, 0.5%, or 1.25% cholesterol for 16 weeks. (B) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from AML12 cells incubated as follows: left blot, 4 h with MG132 plus vehicle (Veh) or liposomes; right blot, 18 h with liposomes and then 4 h with MG132 plus vehicle or Lipo-Chol. (C) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from primary human hepatocytes incubated for 8 h with vehicle or Lipo-Chol, with MG132 included during the last 4 h. (D) TAZ and YAP immunoblots of siScr-treated or siLats2-treated AML12 cells that were incubated for 24 h with vehicle or liposomes. (E) HA-TAZ immunoblot of HA-WT-human TAZ- or HA-S117A-human TAZ-transfected AML12 cells that were incubated for 24 h with vehicle, or liposomes for 16 h and then Lipo-Chol for 8 h. (F-J) The following parameters were measured in Wwtr1 fl/fl mice fed the NAFLD diet containing 0.2% cholesterol for 16 weeks, with AAV8-TBG-Cre plus either AAV8-TBG-HA-WT-hTAZ or AAV8-TBG-HA-S117A-hTAZ injected at the 8-week time point (n = 10 mice/group; means ± SEM; *p

    Journal: Cell metabolism

    Article Title: Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Nonalcoholic Steatohepatitis

    doi: 10.1016/j.cmet.2020.03.010

    Figure Lengend Snippet: Cholesterol Depletion of Hepatocytes Decreases TAZ in a LATS2-Dependent Manner (A) LATS2 kinase assay, using rTAZ as substrate, of extracts of livers of mice fed the NAFLD diet containing 0.2%, 0.5%, or 1.25% cholesterol for 16 weeks. (B) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from AML12 cells incubated as follows: left blot, 4 h with MG132 plus vehicle (Veh) or liposomes; right blot, 18 h with liposomes and then 4 h with MG132 plus vehicle or Lipo-Chol. (C) Immunoblots of phospho- and total TAZ in TAZ immunoprecipitates from primary human hepatocytes incubated for 8 h with vehicle or Lipo-Chol, with MG132 included during the last 4 h. (D) TAZ and YAP immunoblots of siScr-treated or siLats2-treated AML12 cells that were incubated for 24 h with vehicle or liposomes. (E) HA-TAZ immunoblot of HA-WT-human TAZ- or HA-S117A-human TAZ-transfected AML12 cells that were incubated for 24 h with vehicle, or liposomes for 16 h and then Lipo-Chol for 8 h. (F-J) The following parameters were measured in Wwtr1 fl/fl mice fed the NAFLD diet containing 0.2% cholesterol for 16 weeks, with AAV8-TBG-Cre plus either AAV8-TBG-HA-WT-hTAZ or AAV8-TBG-HA-S117A-hTAZ injected at the 8-week time point (n = 10 mice/group; means ± SEM; *p

    Article Snippet: The kit to carry out mutagenesis of WWTR1 was from New England Biolabs (#E0554S), and the primers are listed in .

    Techniques: Kinase Assay, Mouse Assay, Western Blot, Incubation, Transfection, Injection