p2 1 reporter  (ATCC)


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    ATCC p2 1 reporter
    (A, B) Induction of <t>p2.1-reporter</t> luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.
    P2 1 Reporter, supplied by ATCC, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2 1 reporter/product/ATCC
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2 1 reporter - by Bioz Stars, 2023-11
    80/100 stars

    Images

    1) Product Images from "A pathogen-derived effector modulates host glucose metabolism by arginine GlcNAcylation of HIF-1α protein"

    Article Title: A pathogen-derived effector modulates host glucose metabolism by arginine GlcNAcylation of HIF-1α protein

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007259

    (A, B) Induction of p2.1-reporter luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.
    Figure Legend Snippet: (A, B) Induction of p2.1-reporter luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.

    Techniques Used: Luciferase, Activity Assay, Transfection, Over Expression, Expressing, Infection, Plasmid Preparation, Mutagenesis

    (A, B) Knockdown of HIF-1α by HIF-1α-shRNA-1 or HIF-1α-shRNA-2 in HCT116 cells resulted in a reduction of activity of HRE-luciferase reporter (A) or p2.1-luciferase reporter (B) under hypoxia. (C, D) When HIF-1α was knocked-down by HIF-1α-shRNA-2 in HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (C) or PGK1 (D) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.1741 and p = 0.1371, respectively). (E, F) When the HIF-1α inhibitor PX-478 was added into HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (E) or PGK1 (F) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.4379 and p = 0.1423, respectively). Data are presented as means + SEM of three independent experiments performed in triplicate.
    Figure Legend Snippet: (A, B) Knockdown of HIF-1α by HIF-1α-shRNA-1 or HIF-1α-shRNA-2 in HCT116 cells resulted in a reduction of activity of HRE-luciferase reporter (A) or p2.1-luciferase reporter (B) under hypoxia. (C, D) When HIF-1α was knocked-down by HIF-1α-shRNA-2 in HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (C) or PGK1 (D) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.1741 and p = 0.1371, respectively). (E, F) When the HIF-1α inhibitor PX-478 was added into HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (E) or PGK1 (F) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.4379 and p = 0.1423, respectively). Data are presented as means + SEM of three independent experiments performed in triplicate.

    Techniques Used: shRNA, Activity Assay, Luciferase, Expressing, Over Expression

    p2 1 reporter  (ATCC)


    Bioz Verified Symbol ATCC is a verified supplier
    Bioz Manufacturer Symbol ATCC manufactures this product  
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    Structured Review

    ATCC p2 1 reporter
    (A, B) Induction of <t>p2.1-reporter</t> luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.
    P2 1 Reporter, supplied by ATCC, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2 1 reporter/product/ATCC
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2 1 reporter - by Bioz Stars, 2023-11
    80/100 stars

    Images

    1) Product Images from "A pathogen-derived effector modulates host glucose metabolism by arginine GlcNAcylation of HIF-1α protein"

    Article Title: A pathogen-derived effector modulates host glucose metabolism by arginine GlcNAcylation of HIF-1α protein

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007259

    (A, B) Induction of p2.1-reporter luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.
    Figure Legend Snippet: (A, B) Induction of p2.1-reporter luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.

    Techniques Used: Luciferase, Activity Assay, Transfection, Over Expression, Expressing, Infection, Plasmid Preparation, Mutagenesis

    (A, B) Knockdown of HIF-1α by HIF-1α-shRNA-1 or HIF-1α-shRNA-2 in HCT116 cells resulted in a reduction of activity of HRE-luciferase reporter (A) or p2.1-luciferase reporter (B) under hypoxia. (C, D) When HIF-1α was knocked-down by HIF-1α-shRNA-2 in HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (C) or PGK1 (D) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.1741 and p = 0.1371, respectively). (E, F) When the HIF-1α inhibitor PX-478 was added into HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (E) or PGK1 (F) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.4379 and p = 0.1423, respectively). Data are presented as means + SEM of three independent experiments performed in triplicate.
    Figure Legend Snippet: (A, B) Knockdown of HIF-1α by HIF-1α-shRNA-1 or HIF-1α-shRNA-2 in HCT116 cells resulted in a reduction of activity of HRE-luciferase reporter (A) or p2.1-luciferase reporter (B) under hypoxia. (C, D) When HIF-1α was knocked-down by HIF-1α-shRNA-2 in HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (C) or PGK1 (D) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.1741 and p = 0.1371, respectively). (E, F) When the HIF-1α inhibitor PX-478 was added into HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (E) or PGK1 (F) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.4379 and p = 0.1423, respectively). Data are presented as means + SEM of three independent experiments performed in triplicate.

    Techniques Used: shRNA, Activity Assay, Luciferase, Expressing, Over Expression

    p2 1 reporter  (ATCC)


    Bioz Verified Symbol ATCC is a verified supplier
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    Structured Review

    ATCC p2 1 reporter
    Set7 inhibits HIF-1α transcriptional activity. ( A ) The overexpression of Set7 inhibits the activity of BNIP3-luc, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( B ) The overexpression of Set7 inhibited the activity of EPO-luc, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( C ) The overexpression of Set7 inhibited the activity of <t>p2.1</t> reporter, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( D ) The overexpression of Set7 inhibited the activity of VEGF-luc, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( E ) The overexpression of Set7 inhibited the activity of HRE-luc but not the mutant HRE in HEK293T cells under normoxic (20% O 2 ) or hypoxic (1% O 2 ) conditions. Luciferase activity was determined at 24 h post transfection. HRE, hypoxia responsible element; wt, wide type; mt, mutant type. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t -test).
    P2 1 Reporter, supplied by ATCC, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2 1 reporter/product/ATCC
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2 1 reporter - by Bioz Stars, 2023-11
    80/100 stars

    Images

    1) Product Images from "Repression of hypoxia-inducible factor α signaling by Set7-mediated methylation"

    Article Title: Repression of hypoxia-inducible factor α signaling by Set7-mediated methylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv379

    Set7 inhibits HIF-1α transcriptional activity. ( A ) The overexpression of Set7 inhibits the activity of BNIP3-luc, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( B ) The overexpression of Set7 inhibited the activity of EPO-luc, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( C ) The overexpression of Set7 inhibited the activity of p2.1 reporter, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( D ) The overexpression of Set7 inhibited the activity of VEGF-luc, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( E ) The overexpression of Set7 inhibited the activity of HRE-luc but not the mutant HRE in HEK293T cells under normoxic (20% O 2 ) or hypoxic (1% O 2 ) conditions. Luciferase activity was determined at 24 h post transfection. HRE, hypoxia responsible element; wt, wide type; mt, mutant type. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t -test).
    Figure Legend Snippet: Set7 inhibits HIF-1α transcriptional activity. ( A ) The overexpression of Set7 inhibits the activity of BNIP3-luc, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( B ) The overexpression of Set7 inhibited the activity of EPO-luc, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( C ) The overexpression of Set7 inhibited the activity of p2.1 reporter, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( D ) The overexpression of Set7 inhibited the activity of VEGF-luc, a well-defined hypoxia-induced reporter, in HEK293T cells under normoxia (20% O 2 ) or hypoxia (1% O 2 ) conditions. ( E ) The overexpression of Set7 inhibited the activity of HRE-luc but not the mutant HRE in HEK293T cells under normoxic (20% O 2 ) or hypoxic (1% O 2 ) conditions. Luciferase activity was determined at 24 h post transfection. HRE, hypoxia responsible element; wt, wide type; mt, mutant type. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t -test).

    Techniques Used: Activity Assay, Over Expression, Mutagenesis, Luciferase, Transfection

    Depletion of Set7 enhances HIF-1α transactivity. ( A ) (A1) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of p2.1 reporter under hypoxic condition ( P = 0.0053 or P = 0.0011, respectively). (A2) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of EPO promoter luciferase reporter under hypoxic conditions ( P = 0.0125 or P = 0.0057, respectively). ( B ) (B1) The activity of BNIP3 promoter luciferase reporter was enhanced in Set7-null MEF cells (Set7 −/− ) compared with that in the wild-type Set7 MEF cells (Set +/+ ) under hypoxic conditions. (B2) The activity of EPO promoter luciferase reporter was enhanced in Set7-null MEF cells (Set7 −/− ) compared with that in the wild-type Set7 MEF cells (Set +/+ ) under hypoxic conditions. ( C ) (C1) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7-null MEF cells (Set7 −/− ) inhibited the activity of BNIP3 promoter luciferase reporter. (C2) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7-null MEF cells (Set7 −/− ) inhibited the activity of p2.1 reporter. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t -test).
    Figure Legend Snippet: Depletion of Set7 enhances HIF-1α transactivity. ( A ) (A1) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of p2.1 reporter under hypoxic condition ( P = 0.0053 or P = 0.0011, respectively). (A2) The knockdown of Set7 by Set7-shRNA-1 or Set7-shRNA-2 in HEK293T cells significantly enhanced the activity of EPO promoter luciferase reporter under hypoxic conditions ( P = 0.0125 or P = 0.0057, respectively). ( B ) (B1) The activity of BNIP3 promoter luciferase reporter was enhanced in Set7-null MEF cells (Set7 −/− ) compared with that in the wild-type Set7 MEF cells (Set +/+ ) under hypoxic conditions. (B2) The activity of EPO promoter luciferase reporter was enhanced in Set7-null MEF cells (Set7 −/− ) compared with that in the wild-type Set7 MEF cells (Set +/+ ) under hypoxic conditions. ( C ) (C1) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7-null MEF cells (Set7 −/− ) inhibited the activity of BNIP3 promoter luciferase reporter. (C2) The overexpression of the wild-type Set7 but not the mutant Set7 (H297A) in Set7-null MEF cells (Set7 −/− ) inhibited the activity of p2.1 reporter. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t -test).

    Techniques Used: shRNA, Activity Assay, Luciferase, Over Expression, Mutagenesis

    Inhibitory role of Set7 in the hypoxia signaling pathway is mediated by HIF-1α. ( A ) (A1) The overexpression of Set7 inhibited the activity of VEGF promoter luciferase reporter activated by the ectopic expression of HIF-1α in HEK293T cells. (A2) The overexpression of Set7 inhibited the activity of p2.1 reporter activated by the ectopic expression of HIF-1α in HEK293T. (A3) The overexpression of the mutant HIF-1α with Set7-methylated site mutated (K32R) significantly enhanced the transactivity of BNIP3 promoter luciferase reporter compared with that of the wild-type HIF-1α in HEK293T cells ( P = 0.0015). (A4) The overexpression of the mutant HIF-1α with Set7-methylated site mutated (K32R) significantly enhanced the transactivity of the HRE reporter compared with that of the wild-type HIF-1α in HEK293T cells ( P = 0.0014). ( B ) The knockdown of Set7 by Set7-shRNA-2 in RCC4 cells increased the expression of SLC2A1 (B1), PDK1 (B2), EPO (B3) or PGK1 (B4); but inhibition of HIF-1α activity by adding HIF-1α inhibitor, 2ME2 (50 ng/μl, 18 h), abolished this kind of effect under normoxia conditions, as revealed by semi-quantitative real-time PCR assays. ( C ) (C1) ChIP assays show that the knockdown of Set7 in RCC4 cells considerably enhanced the binding ability of HIF-1α to the LDHA promoter. Mouse IgG served as a control for ChIP assays. (C2) The knockdown of Set7 in RCC4 cells does not affect the binding ability of HIF-1α to the RPL13A promoter. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t -test).
    Figure Legend Snippet: Inhibitory role of Set7 in the hypoxia signaling pathway is mediated by HIF-1α. ( A ) (A1) The overexpression of Set7 inhibited the activity of VEGF promoter luciferase reporter activated by the ectopic expression of HIF-1α in HEK293T cells. (A2) The overexpression of Set7 inhibited the activity of p2.1 reporter activated by the ectopic expression of HIF-1α in HEK293T. (A3) The overexpression of the mutant HIF-1α with Set7-methylated site mutated (K32R) significantly enhanced the transactivity of BNIP3 promoter luciferase reporter compared with that of the wild-type HIF-1α in HEK293T cells ( P = 0.0015). (A4) The overexpression of the mutant HIF-1α with Set7-methylated site mutated (K32R) significantly enhanced the transactivity of the HRE reporter compared with that of the wild-type HIF-1α in HEK293T cells ( P = 0.0014). ( B ) The knockdown of Set7 by Set7-shRNA-2 in RCC4 cells increased the expression of SLC2A1 (B1), PDK1 (B2), EPO (B3) or PGK1 (B4); but inhibition of HIF-1α activity by adding HIF-1α inhibitor, 2ME2 (50 ng/μl, 18 h), abolished this kind of effect under normoxia conditions, as revealed by semi-quantitative real-time PCR assays. ( C ) (C1) ChIP assays show that the knockdown of Set7 in RCC4 cells considerably enhanced the binding ability of HIF-1α to the LDHA promoter. Mouse IgG served as a control for ChIP assays. (C2) The knockdown of Set7 in RCC4 cells does not affect the binding ability of HIF-1α to the RPL13A promoter. Data were presented as mean ± SEM of three independent experiments performed in triplicates; statistical analysis was performed using GraphPad Prism 5.0 (unpaired t -test).

    Techniques Used: Over Expression, Activity Assay, Luciferase, Expressing, Mutagenesis, Methylation, shRNA, Inhibition, Real-time Polymerase Chain Reaction, Binding Assay

    p2 1 reporter  (ATCC)


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    ATCC p2 1 reporter
    P2 1 Reporter, supplied by ATCC, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC p2 1 reporter
    EAF2 inhibits HIF-1α transactivity. (A) The promoter activities of four well-defined hypoxia-induced genes were suppressed by overexpression of EAF2 in HEK293T cells. (A1) Epo promoter activity was suppressed by overexpression of EAF2. (A2) VEGF promoter activity was suppressed by overexpression of EAF2. (A3) Under normoxic conditions (21% O2), the induction of the BNIP3 promoter reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of BNIP3 promoter reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (A4) Under normoxic conditions (21% O2), the induction of the <t>p2.1</t> reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of the p2.1 reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (B) The HIF reporter activity was increased in EAF2 knockout (EAF2−/−) mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs (EAF2+/+) under normoxic and hypoxic conditions. (B1) VEGF promoter reporter activity under normoxic and hypoxic conditions. (B2) Epo promoter reporter activity under normoxic and hypoxic conditions. (B3) p2.1 reporter activity under normoxic and hypoxic conditions. (B4) HIF reporter (HRE) activity under hypoxic conditions. WT, wild-type HRE reporter; MT, mutated HRE reporter. (C) The truncated mutant of EAF2 (EAF2 III-VI) without the ability to bind to HIF-1α did not suppress HIF-1α transactivity. (C1) Compared to overexpression of full-length EAF2, overexpression of the truncated EAF2 III-VI mutant in HEK293T cells did not suppress p2.1 promoter luciferase reporter activity under hypoxic conditions. (C2) Compared to overexpression of full-length EAF2, overexpression of EAF2 III-VI in EAF2-null MEFs (EAF2−/−) did not suppress p2.1 promoter luciferase reporter activity. (D) EAF2 has no effect on HIF-2α transactivity. (D1) Overexpression of EAF2 in HEK293T cells did not affect PAI-1 promoter luciferase reporter activity under hypoxic conditions. (D2) Overexpression of EAF2 in HEK293T cells did not affect SOD2 promoter luciferase reporter activity under hypoxic conditions. (D3) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α induced PAI-1 promoter luciferase reporter activity under normoxic conditions. (D4) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α-induced SOD2 promoter luciferase reporter activity under normoxic conditions. (E) The promoter activities of two HIF-1α downstream genes (p2.1 and BNIP3) were enhanced by knockdown of EAF2 in HEK293T cells, but the promoter activity of one HIF-2α downstream gene (SOD2) was not affected by knockdown of EAF2 in HEK293T cells under hypoxic conditions. (E1) p2.1 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E2) BNIP3 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E3) SOD2 promoter activity was not affected by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. pTK-Renilla was used as an internal control, and the luciferase activity was determined 18 to 24 h after transfection.
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    1) Product Images from "EAF2 Suppresses Hypoxia-Induced Factor 1α Transcriptional Activity by Disrupting Its Interaction with Coactivator CBP/p300"

    Article Title: EAF2 Suppresses Hypoxia-Induced Factor 1α Transcriptional Activity by Disrupting Its Interaction with Coactivator CBP/p300

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00718-13

    EAF2 inhibits HIF-1α transactivity. (A) The promoter activities of four well-defined hypoxia-induced genes were suppressed by overexpression of EAF2 in HEK293T cells. (A1) Epo promoter activity was suppressed by overexpression of EAF2. (A2) VEGF promoter activity was suppressed by overexpression of EAF2. (A3) Under normoxic conditions (21% O2), the induction of the BNIP3 promoter reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of BNIP3 promoter reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (A4) Under normoxic conditions (21% O2), the induction of the p2.1 reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of the p2.1 reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (B) The HIF reporter activity was increased in EAF2 knockout (EAF2−/−) mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs (EAF2+/+) under normoxic and hypoxic conditions. (B1) VEGF promoter reporter activity under normoxic and hypoxic conditions. (B2) Epo promoter reporter activity under normoxic and hypoxic conditions. (B3) p2.1 reporter activity under normoxic and hypoxic conditions. (B4) HIF reporter (HRE) activity under hypoxic conditions. WT, wild-type HRE reporter; MT, mutated HRE reporter. (C) The truncated mutant of EAF2 (EAF2 III-VI) without the ability to bind to HIF-1α did not suppress HIF-1α transactivity. (C1) Compared to overexpression of full-length EAF2, overexpression of the truncated EAF2 III-VI mutant in HEK293T cells did not suppress p2.1 promoter luciferase reporter activity under hypoxic conditions. (C2) Compared to overexpression of full-length EAF2, overexpression of EAF2 III-VI in EAF2-null MEFs (EAF2−/−) did not suppress p2.1 promoter luciferase reporter activity. (D) EAF2 has no effect on HIF-2α transactivity. (D1) Overexpression of EAF2 in HEK293T cells did not affect PAI-1 promoter luciferase reporter activity under hypoxic conditions. (D2) Overexpression of EAF2 in HEK293T cells did not affect SOD2 promoter luciferase reporter activity under hypoxic conditions. (D3) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α induced PAI-1 promoter luciferase reporter activity under normoxic conditions. (D4) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α-induced SOD2 promoter luciferase reporter activity under normoxic conditions. (E) The promoter activities of two HIF-1α downstream genes (p2.1 and BNIP3) were enhanced by knockdown of EAF2 in HEK293T cells, but the promoter activity of one HIF-2α downstream gene (SOD2) was not affected by knockdown of EAF2 in HEK293T cells under hypoxic conditions. (E1) p2.1 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E2) BNIP3 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E3) SOD2 promoter activity was not affected by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. pTK-Renilla was used as an internal control, and the luciferase activity was determined 18 to 24 h after transfection.
    Figure Legend Snippet: EAF2 inhibits HIF-1α transactivity. (A) The promoter activities of four well-defined hypoxia-induced genes were suppressed by overexpression of EAF2 in HEK293T cells. (A1) Epo promoter activity was suppressed by overexpression of EAF2. (A2) VEGF promoter activity was suppressed by overexpression of EAF2. (A3) Under normoxic conditions (21% O2), the induction of the BNIP3 promoter reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of BNIP3 promoter reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (A4) Under normoxic conditions (21% O2), the induction of the p2.1 reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of the p2.1 reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (B) The HIF reporter activity was increased in EAF2 knockout (EAF2−/−) mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs (EAF2+/+) under normoxic and hypoxic conditions. (B1) VEGF promoter reporter activity under normoxic and hypoxic conditions. (B2) Epo promoter reporter activity under normoxic and hypoxic conditions. (B3) p2.1 reporter activity under normoxic and hypoxic conditions. (B4) HIF reporter (HRE) activity under hypoxic conditions. WT, wild-type HRE reporter; MT, mutated HRE reporter. (C) The truncated mutant of EAF2 (EAF2 III-VI) without the ability to bind to HIF-1α did not suppress HIF-1α transactivity. (C1) Compared to overexpression of full-length EAF2, overexpression of the truncated EAF2 III-VI mutant in HEK293T cells did not suppress p2.1 promoter luciferase reporter activity under hypoxic conditions. (C2) Compared to overexpression of full-length EAF2, overexpression of EAF2 III-VI in EAF2-null MEFs (EAF2−/−) did not suppress p2.1 promoter luciferase reporter activity. (D) EAF2 has no effect on HIF-2α transactivity. (D1) Overexpression of EAF2 in HEK293T cells did not affect PAI-1 promoter luciferase reporter activity under hypoxic conditions. (D2) Overexpression of EAF2 in HEK293T cells did not affect SOD2 promoter luciferase reporter activity under hypoxic conditions. (D3) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α induced PAI-1 promoter luciferase reporter activity under normoxic conditions. (D4) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α-induced SOD2 promoter luciferase reporter activity under normoxic conditions. (E) The promoter activities of two HIF-1α downstream genes (p2.1 and BNIP3) were enhanced by knockdown of EAF2 in HEK293T cells, but the promoter activity of one HIF-2α downstream gene (SOD2) was not affected by knockdown of EAF2 in HEK293T cells under hypoxic conditions. (E1) p2.1 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E2) BNIP3 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E3) SOD2 promoter activity was not affected by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. pTK-Renilla was used as an internal control, and the luciferase activity was determined 18 to 24 h after transfection.

    Techniques Used: Over Expression, Activity Assay, Knock-Out, Mutagenesis, Luciferase, Transfection

    The repression of HIF-1α by EAF2 is independent of FIH-1. (A) The transcriptional activities of HIF-1α C-TAD (aa 786 to 826) and the N803A mutant were suppressed by EAF2 overexpression. (B) Coimmunoprecipitation assays showed that EAF2 could not interact with FIH-1 when the two proteins were overexpressed in HEK293T cells. (C) EAF2 suppressed the transcriptional activity of the p2.1 reporter (C1) and the BNIP3 promoter reporter (C2) induced by the HIF-1α PP mutant (P402A P564A) and PPN mutant (P402A P564A N803A) in HEK293T cells under normoxic conditions. (D) EAF2 suppressed the transcriptional activity of the p2.1 reporter (D1) and the BNIP3 promoter reporter (D2) induced by HIF-1α PP and PPN mutants in HepG2 cells under normoxic conditions.
    Figure Legend Snippet: The repression of HIF-1α by EAF2 is independent of FIH-1. (A) The transcriptional activities of HIF-1α C-TAD (aa 786 to 826) and the N803A mutant were suppressed by EAF2 overexpression. (B) Coimmunoprecipitation assays showed that EAF2 could not interact with FIH-1 when the two proteins were overexpressed in HEK293T cells. (C) EAF2 suppressed the transcriptional activity of the p2.1 reporter (C1) and the BNIP3 promoter reporter (C2) induced by the HIF-1α PP mutant (P402A P564A) and PPN mutant (P402A P564A N803A) in HEK293T cells under normoxic conditions. (D) EAF2 suppressed the transcriptional activity of the p2.1 reporter (D1) and the BNIP3 promoter reporter (D2) induced by HIF-1α PP and PPN mutants in HepG2 cells under normoxic conditions.

    Techniques Used: Mutagenesis, Over Expression, Activity Assay

    EAF2 protects against hypoxia-induced cell death and inhibits cellular uptake of glucose under hypoxic conditions. (A, left) Under hypoxic conditions (1% O2 for 24 h), EAF2+/+ MEFs had a significantly higher cell survival rate than EAF2−/− MEFs (P = 0.0005). (Right) Under hypoxic conditions (1% O2 for 24 h), overexpression of full-length EAF2 in EAF2−/− MEFs enhanced the cell survival rate (P = 0.0014), but overexpression of domains III to VI of EAF2 did not do so (P = 0.4095). Cells were counted using a Bio-Rad cell counter after trypan blue staining. (B1) The representative pictures of EAF2+/+ and EAF2−/− MEFs stained with Hoechst 33342 after treatment by normoxia (21% O2) or hypoxia (1% O2) for 24 h. The dying cells are marked by arrows. (B2) Quantitative analysis for Hoechst staining. EAF2−/− MEFs had a significantly higher dead cell ratio than EAF2+/+ MEFs (P = 0.0104). Data are means and SEM representing nine randomly selected fields of three wells in a six-well plate. (C1) EAF2−/− MEFs had significantly higher intracellular glucose levels than EAF2+/+ MEFs. The glucose level was measured by a glucose assay kit (BioVision). (C2) Knockdown of EAF2 in HepG2 cells caused an increase in cellular uptake of glucose under hypoxia (P = 0.0048 and P = 0.0139). (D) Inhibition of p300 activity abolished the suppressive role of EAF2 on p2.1 promoter luciferase activity in EAF2−/− MEFs (P = 0.9370 versus P = 0.0047). Anacardic acid (AA) was added to the culture medium (50 μM) for 18-h treatment. Data are means and SEM of three independent experiments performed in triplicate. (E) Anacardic acid treatment (50 μM) abrogated the effect of EAF2 on cellular glucose uptake. Without anacardic acid treatment, EAF2−/− MEFs had higher intracellular glucose levels than EAF2+/+ MEFs (green curve versus orange curve). With anacardic acid treatment, the intracellular glucose levels in both EAF2−/− MEFs and EAF2+/+ MEFs dropped to the same level as that of EAF2+/+ MEFs without anacardic acid (AA) treatment (purple, dark green, and orange curves versus green curve). Glucose uptake was measured by FACS, following 1.5 h exposure to 2-NBDG (100 μM).
    Figure Legend Snippet: EAF2 protects against hypoxia-induced cell death and inhibits cellular uptake of glucose under hypoxic conditions. (A, left) Under hypoxic conditions (1% O2 for 24 h), EAF2+/+ MEFs had a significantly higher cell survival rate than EAF2−/− MEFs (P = 0.0005). (Right) Under hypoxic conditions (1% O2 for 24 h), overexpression of full-length EAF2 in EAF2−/− MEFs enhanced the cell survival rate (P = 0.0014), but overexpression of domains III to VI of EAF2 did not do so (P = 0.4095). Cells were counted using a Bio-Rad cell counter after trypan blue staining. (B1) The representative pictures of EAF2+/+ and EAF2−/− MEFs stained with Hoechst 33342 after treatment by normoxia (21% O2) or hypoxia (1% O2) for 24 h. The dying cells are marked by arrows. (B2) Quantitative analysis for Hoechst staining. EAF2−/− MEFs had a significantly higher dead cell ratio than EAF2+/+ MEFs (P = 0.0104). Data are means and SEM representing nine randomly selected fields of three wells in a six-well plate. (C1) EAF2−/− MEFs had significantly higher intracellular glucose levels than EAF2+/+ MEFs. The glucose level was measured by a glucose assay kit (BioVision). (C2) Knockdown of EAF2 in HepG2 cells caused an increase in cellular uptake of glucose under hypoxia (P = 0.0048 and P = 0.0139). (D) Inhibition of p300 activity abolished the suppressive role of EAF2 on p2.1 promoter luciferase activity in EAF2−/− MEFs (P = 0.9370 versus P = 0.0047). Anacardic acid (AA) was added to the culture medium (50 μM) for 18-h treatment. Data are means and SEM of three independent experiments performed in triplicate. (E) Anacardic acid treatment (50 μM) abrogated the effect of EAF2 on cellular glucose uptake. Without anacardic acid treatment, EAF2−/− MEFs had higher intracellular glucose levels than EAF2+/+ MEFs (green curve versus orange curve). With anacardic acid treatment, the intracellular glucose levels in both EAF2−/− MEFs and EAF2+/+ MEFs dropped to the same level as that of EAF2+/+ MEFs without anacardic acid (AA) treatment (purple, dark green, and orange curves versus green curve). Glucose uptake was measured by FACS, following 1.5 h exposure to 2-NBDG (100 μM).

    Techniques Used: Over Expression, Staining, Glucose Assay, Inhibition, Activity Assay, Luciferase

    p2 1 reporter  (ATCC)


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    ATCC p2 1 reporter
    EAF2 inhibits HIF-1α transactivity. (A) The promoter activities of four well-defined hypoxia-induced genes were suppressed by overexpression of EAF2 in HEK293T cells. (A1) Epo promoter activity was suppressed by overexpression of EAF2. (A2) VEGF promoter activity was suppressed by overexpression of EAF2. (A3) Under normoxic conditions (21% O2), the induction of the BNIP3 promoter reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of BNIP3 promoter reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (A4) Under normoxic conditions (21% O2), the induction of the <t>p2.1</t> reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of the p2.1 reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (B) The HIF reporter activity was increased in EAF2 knockout (EAF2−/−) mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs (EAF2+/+) under normoxic and hypoxic conditions. (B1) VEGF promoter reporter activity under normoxic and hypoxic conditions. (B2) Epo promoter reporter activity under normoxic and hypoxic conditions. (B3) p2.1 reporter activity under normoxic and hypoxic conditions. (B4) HIF reporter (HRE) activity under hypoxic conditions. WT, wild-type HRE reporter; MT, mutated HRE reporter. (C) The truncated mutant of EAF2 (EAF2 III-VI) without the ability to bind to HIF-1α did not suppress HIF-1α transactivity. (C1) Compared to overexpression of full-length EAF2, overexpression of the truncated EAF2 III-VI mutant in HEK293T cells did not suppress p2.1 promoter luciferase reporter activity under hypoxic conditions. (C2) Compared to overexpression of full-length EAF2, overexpression of EAF2 III-VI in EAF2-null MEFs (EAF2−/−) did not suppress p2.1 promoter luciferase reporter activity. (D) EAF2 has no effect on HIF-2α transactivity. (D1) Overexpression of EAF2 in HEK293T cells did not affect PAI-1 promoter luciferase reporter activity under hypoxic conditions. (D2) Overexpression of EAF2 in HEK293T cells did not affect SOD2 promoter luciferase reporter activity under hypoxic conditions. (D3) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α induced PAI-1 promoter luciferase reporter activity under normoxic conditions. (D4) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α-induced SOD2 promoter luciferase reporter activity under normoxic conditions. (E) The promoter activities of two HIF-1α downstream genes (p2.1 and BNIP3) were enhanced by knockdown of EAF2 in HEK293T cells, but the promoter activity of one HIF-2α downstream gene (SOD2) was not affected by knockdown of EAF2 in HEK293T cells under hypoxic conditions. (E1) p2.1 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E2) BNIP3 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E3) SOD2 promoter activity was not affected by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. pTK-Renilla was used as an internal control, and the luciferase activity was determined 18 to 24 h after transfection.
    P2 1 Reporter, supplied by ATCC, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "EAF2 Suppresses Hypoxia-Induced Factor 1α Transcriptional Activity by Disrupting Its Interaction with Coactivator CBP/p300"

    Article Title: EAF2 Suppresses Hypoxia-Induced Factor 1α Transcriptional Activity by Disrupting Its Interaction with Coactivator CBP/p300

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00718-13

    EAF2 inhibits HIF-1α transactivity. (A) The promoter activities of four well-defined hypoxia-induced genes were suppressed by overexpression of EAF2 in HEK293T cells. (A1) Epo promoter activity was suppressed by overexpression of EAF2. (A2) VEGF promoter activity was suppressed by overexpression of EAF2. (A3) Under normoxic conditions (21% O2), the induction of the BNIP3 promoter reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of BNIP3 promoter reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (A4) Under normoxic conditions (21% O2), the induction of the p2.1 reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of the p2.1 reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (B) The HIF reporter activity was increased in EAF2 knockout (EAF2−/−) mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs (EAF2+/+) under normoxic and hypoxic conditions. (B1) VEGF promoter reporter activity under normoxic and hypoxic conditions. (B2) Epo promoter reporter activity under normoxic and hypoxic conditions. (B3) p2.1 reporter activity under normoxic and hypoxic conditions. (B4) HIF reporter (HRE) activity under hypoxic conditions. WT, wild-type HRE reporter; MT, mutated HRE reporter. (C) The truncated mutant of EAF2 (EAF2 III-VI) without the ability to bind to HIF-1α did not suppress HIF-1α transactivity. (C1) Compared to overexpression of full-length EAF2, overexpression of the truncated EAF2 III-VI mutant in HEK293T cells did not suppress p2.1 promoter luciferase reporter activity under hypoxic conditions. (C2) Compared to overexpression of full-length EAF2, overexpression of EAF2 III-VI in EAF2-null MEFs (EAF2−/−) did not suppress p2.1 promoter luciferase reporter activity. (D) EAF2 has no effect on HIF-2α transactivity. (D1) Overexpression of EAF2 in HEK293T cells did not affect PAI-1 promoter luciferase reporter activity under hypoxic conditions. (D2) Overexpression of EAF2 in HEK293T cells did not affect SOD2 promoter luciferase reporter activity under hypoxic conditions. (D3) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α induced PAI-1 promoter luciferase reporter activity under normoxic conditions. (D4) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α-induced SOD2 promoter luciferase reporter activity under normoxic conditions. (E) The promoter activities of two HIF-1α downstream genes (p2.1 and BNIP3) were enhanced by knockdown of EAF2 in HEK293T cells, but the promoter activity of one HIF-2α downstream gene (SOD2) was not affected by knockdown of EAF2 in HEK293T cells under hypoxic conditions. (E1) p2.1 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E2) BNIP3 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E3) SOD2 promoter activity was not affected by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. pTK-Renilla was used as an internal control, and the luciferase activity was determined 18 to 24 h after transfection.
    Figure Legend Snippet: EAF2 inhibits HIF-1α transactivity. (A) The promoter activities of four well-defined hypoxia-induced genes were suppressed by overexpression of EAF2 in HEK293T cells. (A1) Epo promoter activity was suppressed by overexpression of EAF2. (A2) VEGF promoter activity was suppressed by overexpression of EAF2. (A3) Under normoxic conditions (21% O2), the induction of the BNIP3 promoter reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of BNIP3 promoter reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (A4) Under normoxic conditions (21% O2), the induction of the p2.1 reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of the p2.1 reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (B) The HIF reporter activity was increased in EAF2 knockout (EAF2−/−) mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs (EAF2+/+) under normoxic and hypoxic conditions. (B1) VEGF promoter reporter activity under normoxic and hypoxic conditions. (B2) Epo promoter reporter activity under normoxic and hypoxic conditions. (B3) p2.1 reporter activity under normoxic and hypoxic conditions. (B4) HIF reporter (HRE) activity under hypoxic conditions. WT, wild-type HRE reporter; MT, mutated HRE reporter. (C) The truncated mutant of EAF2 (EAF2 III-VI) without the ability to bind to HIF-1α did not suppress HIF-1α transactivity. (C1) Compared to overexpression of full-length EAF2, overexpression of the truncated EAF2 III-VI mutant in HEK293T cells did not suppress p2.1 promoter luciferase reporter activity under hypoxic conditions. (C2) Compared to overexpression of full-length EAF2, overexpression of EAF2 III-VI in EAF2-null MEFs (EAF2−/−) did not suppress p2.1 promoter luciferase reporter activity. (D) EAF2 has no effect on HIF-2α transactivity. (D1) Overexpression of EAF2 in HEK293T cells did not affect PAI-1 promoter luciferase reporter activity under hypoxic conditions. (D2) Overexpression of EAF2 in HEK293T cells did not affect SOD2 promoter luciferase reporter activity under hypoxic conditions. (D3) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α induced PAI-1 promoter luciferase reporter activity under normoxic conditions. (D4) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α-induced SOD2 promoter luciferase reporter activity under normoxic conditions. (E) The promoter activities of two HIF-1α downstream genes (p2.1 and BNIP3) were enhanced by knockdown of EAF2 in HEK293T cells, but the promoter activity of one HIF-2α downstream gene (SOD2) was not affected by knockdown of EAF2 in HEK293T cells under hypoxic conditions. (E1) p2.1 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E2) BNIP3 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E3) SOD2 promoter activity was not affected by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. pTK-Renilla was used as an internal control, and the luciferase activity was determined 18 to 24 h after transfection.

    Techniques Used: Over Expression, Activity Assay, Knock-Out, Mutagenesis, Luciferase, Transfection

    The repression of HIF-1α by EAF2 is independent of FIH-1. (A) The transcriptional activities of HIF-1α C-TAD (aa 786 to 826) and the N803A mutant were suppressed by EAF2 overexpression. (B) Coimmunoprecipitation assays showed that EAF2 could not interact with FIH-1 when the two proteins were overexpressed in HEK293T cells. (C) EAF2 suppressed the transcriptional activity of the p2.1 reporter (C1) and the BNIP3 promoter reporter (C2) induced by the HIF-1α PP mutant (P402A P564A) and PPN mutant (P402A P564A N803A) in HEK293T cells under normoxic conditions. (D) EAF2 suppressed the transcriptional activity of the p2.1 reporter (D1) and the BNIP3 promoter reporter (D2) induced by HIF-1α PP and PPN mutants in HepG2 cells under normoxic conditions.
    Figure Legend Snippet: The repression of HIF-1α by EAF2 is independent of FIH-1. (A) The transcriptional activities of HIF-1α C-TAD (aa 786 to 826) and the N803A mutant were suppressed by EAF2 overexpression. (B) Coimmunoprecipitation assays showed that EAF2 could not interact with FIH-1 when the two proteins were overexpressed in HEK293T cells. (C) EAF2 suppressed the transcriptional activity of the p2.1 reporter (C1) and the BNIP3 promoter reporter (C2) induced by the HIF-1α PP mutant (P402A P564A) and PPN mutant (P402A P564A N803A) in HEK293T cells under normoxic conditions. (D) EAF2 suppressed the transcriptional activity of the p2.1 reporter (D1) and the BNIP3 promoter reporter (D2) induced by HIF-1α PP and PPN mutants in HepG2 cells under normoxic conditions.

    Techniques Used: Mutagenesis, Over Expression, Activity Assay

    EAF2 protects against hypoxia-induced cell death and inhibits cellular uptake of glucose under hypoxic conditions. (A, left) Under hypoxic conditions (1% O2 for 24 h), EAF2+/+ MEFs had a significantly higher cell survival rate than EAF2−/− MEFs (P = 0.0005). (Right) Under hypoxic conditions (1% O2 for 24 h), overexpression of full-length EAF2 in EAF2−/− MEFs enhanced the cell survival rate (P = 0.0014), but overexpression of domains III to VI of EAF2 did not do so (P = 0.4095). Cells were counted using a Bio-Rad cell counter after trypan blue staining. (B1) The representative pictures of EAF2+/+ and EAF2−/− MEFs stained with Hoechst 33342 after treatment by normoxia (21% O2) or hypoxia (1% O2) for 24 h. The dying cells are marked by arrows. (B2) Quantitative analysis for Hoechst staining. EAF2−/− MEFs had a significantly higher dead cell ratio than EAF2+/+ MEFs (P = 0.0104). Data are means and SEM representing nine randomly selected fields of three wells in a six-well plate. (C1) EAF2−/− MEFs had significantly higher intracellular glucose levels than EAF2+/+ MEFs. The glucose level was measured by a glucose assay kit (BioVision). (C2) Knockdown of EAF2 in HepG2 cells caused an increase in cellular uptake of glucose under hypoxia (P = 0.0048 and P = 0.0139). (D) Inhibition of p300 activity abolished the suppressive role of EAF2 on p2.1 promoter luciferase activity in EAF2−/− MEFs (P = 0.9370 versus P = 0.0047). Anacardic acid (AA) was added to the culture medium (50 μM) for 18-h treatment. Data are means and SEM of three independent experiments performed in triplicate. (E) Anacardic acid treatment (50 μM) abrogated the effect of EAF2 on cellular glucose uptake. Without anacardic acid treatment, EAF2−/− MEFs had higher intracellular glucose levels than EAF2+/+ MEFs (green curve versus orange curve). With anacardic acid treatment, the intracellular glucose levels in both EAF2−/− MEFs and EAF2+/+ MEFs dropped to the same level as that of EAF2+/+ MEFs without anacardic acid (AA) treatment (purple, dark green, and orange curves versus green curve). Glucose uptake was measured by FACS, following 1.5 h exposure to 2-NBDG (100 μM).
    Figure Legend Snippet: EAF2 protects against hypoxia-induced cell death and inhibits cellular uptake of glucose under hypoxic conditions. (A, left) Under hypoxic conditions (1% O2 for 24 h), EAF2+/+ MEFs had a significantly higher cell survival rate than EAF2−/− MEFs (P = 0.0005). (Right) Under hypoxic conditions (1% O2 for 24 h), overexpression of full-length EAF2 in EAF2−/− MEFs enhanced the cell survival rate (P = 0.0014), but overexpression of domains III to VI of EAF2 did not do so (P = 0.4095). Cells were counted using a Bio-Rad cell counter after trypan blue staining. (B1) The representative pictures of EAF2+/+ and EAF2−/− MEFs stained with Hoechst 33342 after treatment by normoxia (21% O2) or hypoxia (1% O2) for 24 h. The dying cells are marked by arrows. (B2) Quantitative analysis for Hoechst staining. EAF2−/− MEFs had a significantly higher dead cell ratio than EAF2+/+ MEFs (P = 0.0104). Data are means and SEM representing nine randomly selected fields of three wells in a six-well plate. (C1) EAF2−/− MEFs had significantly higher intracellular glucose levels than EAF2+/+ MEFs. The glucose level was measured by a glucose assay kit (BioVision). (C2) Knockdown of EAF2 in HepG2 cells caused an increase in cellular uptake of glucose under hypoxia (P = 0.0048 and P = 0.0139). (D) Inhibition of p300 activity abolished the suppressive role of EAF2 on p2.1 promoter luciferase activity in EAF2−/− MEFs (P = 0.9370 versus P = 0.0047). Anacardic acid (AA) was added to the culture medium (50 μM) for 18-h treatment. Data are means and SEM of three independent experiments performed in triplicate. (E) Anacardic acid treatment (50 μM) abrogated the effect of EAF2 on cellular glucose uptake. Without anacardic acid treatment, EAF2−/− MEFs had higher intracellular glucose levels than EAF2+/+ MEFs (green curve versus orange curve). With anacardic acid treatment, the intracellular glucose levels in both EAF2−/− MEFs and EAF2+/+ MEFs dropped to the same level as that of EAF2+/+ MEFs without anacardic acid (AA) treatment (purple, dark green, and orange curves versus green curve). Glucose uptake was measured by FACS, following 1.5 h exposure to 2-NBDG (100 μM).

    Techniques Used: Over Expression, Staining, Glucose Assay, Inhibition, Activity Assay, Luciferase

    reporter gene assays plasmid p2 1  (ATCC)


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    ATCC reporter gene assays plasmid p2 1
    Reporter Gene Assays Plasmid P2 1, supplied by ATCC, 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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    ATCC p2 1 reporter
    (A, B) Induction of <t>p2.1-reporter</t> luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.
    P2 1 Reporter, supplied by ATCC, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    reporter gene assays plasmid p2 1  (ATCC)
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    ATCC reporter gene assays plasmid p2 1
    (A, B) Induction of <t>p2.1-reporter</t> luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.
    Reporter Gene Assays Plasmid P2 1, supplied by ATCC, 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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    (A, B) Induction of p2.1-reporter luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.

    Journal: PLoS Pathogens

    Article Title: A pathogen-derived effector modulates host glucose metabolism by arginine GlcNAcylation of HIF-1α protein

    doi: 10.1371/journal.ppat.1007259

    Figure Lengend Snippet: (A, B) Induction of p2.1-reporter luciferase activity (A) or HRE-reporter luciferase activity (B) by HIF-1α transfection under normoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0040 and p = 0.0003, respectively). HRE, hypoxia response element. (C, D) Induction of p2.1-reporter luciferase activity (C) or HRE-reporter luciferase activity (D) under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0387 and p = 0.0184, respectively). (E, F, G, H) Induction of LDHA (E), PDK1 (F), PGK1 (G), or SLC2A1 ( GLUT1 ) (H) expression under hypoxia was significantly enhanced by NleB overexpression in HCT116 cells ( p = 0.0020, 0.0054, 0.0001, and 0.0066, respectively). (I, J) Induction of p2.1-reporter luciferase activity (I) or HRE-reporter luciferase activity (J) by HIF-1α overexpression under normoxia in HCT116 cells was significantly enhanced by infection with the wild-type EPEC strain (EPEC E2348/69) compared with that by infection with the control EPEC strain lacking both nleE and nleB (strain SC309) but complemented with an empty plasmid (Δ nleBE +vector) ( p = 0.0094 and p = 0.0010, respectively). (K, L) Under hypoxia, the activity of p2.1-luciferase reporter (K) or HRE-luciferase reporter (L) was significantly enhanced in HCT116 by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector ( p = 0.0076 and p = 0.0299, respectively). (M) Under hypoxia, expressions of PDK1 , PGK1 , SLC2A1 , and PKM2 were significantly enhanced in HCT116 cells by infection with EPEC E2348/69 compared with those by infection with the control Δ nleBE +vector, but expression of SOD2 was not changed. (N, O) Under normoxia, in RCC4 cells, infection with EPEC E2348/69 or the mutant EPEC strain lacking both nleE and nleB (strain SC309, indicated as Δ nleBE ) complemented with a plasmid expressing wild-type NleB (Δ nleBE +pNleB) enhanced the expression of SLC2A1 (N) or PDK1 (O) significantly compared with cells infected with the mutant EPEC strain lacking escN (indicated as Δ escN ) complemented with a plasmid expressing WT NleB (Δ escN +pNleB), the control Δ nleBE +vector, or the mutant EPEC strain Δ nleBE +pNleB-DXD. Data are presented as means + SEM of three independent experiments performed in triplicate.

    Article Snippet: The p2.1 reporter was purchased from ATCC and is commonly used for monitoring HIF-1α transcriptional activity [ ].

    Techniques: Luciferase, Activity Assay, Transfection, Over Expression, Expressing, Infection, Plasmid Preparation, Mutagenesis

    (A, B) Knockdown of HIF-1α by HIF-1α-shRNA-1 or HIF-1α-shRNA-2 in HCT116 cells resulted in a reduction of activity of HRE-luciferase reporter (A) or p2.1-luciferase reporter (B) under hypoxia. (C, D) When HIF-1α was knocked-down by HIF-1α-shRNA-2 in HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (C) or PGK1 (D) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.1741 and p = 0.1371, respectively). (E, F) When the HIF-1α inhibitor PX-478 was added into HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (E) or PGK1 (F) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.4379 and p = 0.1423, respectively). Data are presented as means + SEM of three independent experiments performed in triplicate.

    Journal: PLoS Pathogens

    Article Title: A pathogen-derived effector modulates host glucose metabolism by arginine GlcNAcylation of HIF-1α protein

    doi: 10.1371/journal.ppat.1007259

    Figure Lengend Snippet: (A, B) Knockdown of HIF-1α by HIF-1α-shRNA-1 or HIF-1α-shRNA-2 in HCT116 cells resulted in a reduction of activity of HRE-luciferase reporter (A) or p2.1-luciferase reporter (B) under hypoxia. (C, D) When HIF-1α was knocked-down by HIF-1α-shRNA-2 in HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (C) or PGK1 (D) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.1741 and p = 0.1371, respectively). (E, F) When the HIF-1α inhibitor PX-478 was added into HCT116 cells, the enhancement of SLC2A1 ( GLUT1 ) (E) or PGK1 (F) expression by overexpression of GFP-NleB under hypoxia was diminished ( p = 0.4379 and p = 0.1423, respectively). Data are presented as means + SEM of three independent experiments performed in triplicate.

    Article Snippet: The p2.1 reporter was purchased from ATCC and is commonly used for monitoring HIF-1α transcriptional activity [ ].

    Techniques: shRNA, Activity Assay, Luciferase, Expressing, Over Expression