inducible ampkα2 knockdown cells Search Results


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    Thermo Fisher lipofectamine rnaimax
    Lipofectamine Rnaimax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 38787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare ampkα2 catalytic isoforms
    Ampkα2 Catalytic Isoforms, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p38 mapk
    HCSA activates the <t>p38</t> <t>MAPK</t> pathway in an AMPK-dependent fashion. A , C2C12 cells were stimulated with HCSA (10 μ m ) for the indicated times. Cell lysates were analyzed by Western blotting ( IB ) using anti-phospho-p38 MAPK antibody. Blotting with
    P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GE Healthcare control non targeting sirna
    A769662 prevents NRVM hypertrophy. a – h <t>NRVMs</t> were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of increasing concentration of A769662 (from 12.5 to 100 µM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a Representative immunoblot of AMPK Thr172 and ACC Ser79 phosphorylation. b , c Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. d Quantification of ERK Thr202/Tyr204 phosphorylation. N = 3. e Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. f Representative immunoblots of p70S6K Thr389 and ERK Thr202/Tyr204 phosphorylation. g Quantification of p70S6K Thr389 phosphorylation. N = 4. h Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 3. i – l NRVMs were transfected with control non-targeting <t>siRNA</t> or AMPKα1/α2 siRNA (50 nM) for 66 h. Then, NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 μM) in the presence or absence of A769662 (12.5 μM) for 24 h. i Representative immunoblot and quantification of total AMPKα. N = 3. j Representative immunoblot and quantification of ACC Ser79 phosphorylation. N = 3. k Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with non-targeting siRNA. Scale bar, 20 µm. N = 3. l Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with AMPKα1/α2 siRNA. Scale bar, 20 µm. N = 3. Data in ( a – l ) are mean ± s.e.m. The data were analyzed using One-way ANOVA followed by Bonferroni post-test in ( i ) and Two-way ANOVA followed by Bonferroni post-test in ( b , d , e , g , h , and j – l ). * p
    Control Non Targeting Sirna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    R&D Systems a769662
    <t>A769662</t> prevents NRVM hypertrophy. a – h NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of increasing concentration of A769662 (from 12.5 to 100 µM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a Representative immunoblot of AMPK Thr172 and ACC Ser79 phosphorylation. b , c Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. d Quantification of ERK Thr202/Tyr204 phosphorylation. N = 3. e Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. f Representative immunoblots of p70S6K Thr389 and ERK Thr202/Tyr204 phosphorylation. g Quantification of p70S6K Thr389 phosphorylation. N = 4. h Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 3. i – l NRVMs were transfected with control non-targeting siRNA or AMPKα1/α2 siRNA (50 nM) for 66 h. Then, NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 μM) in the presence or absence of A769662 (12.5 μM) for 24 h. i Representative immunoblot and quantification of total AMPKα. N = 3. j Representative immunoblot and quantification of ACC Ser79 phosphorylation. N = 3. k Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with non-targeting siRNA. Scale bar, 20 µm. N = 3. l Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with AMPKα1/α2 siRNA. Scale bar, 20 µm. N = 3. Data in ( a – l ) are mean ± s.e.m. The data were analyzed using One-way ANOVA followed by Bonferroni post-test in ( i ) and Two-way ANOVA followed by Bonferroni post-test in ( b , d , e , g , h , and j – l ). * p
    A769662, supplied by R&D Systems, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lipofectamine 2000
    <t>A769662</t> prevents NRVM hypertrophy. a – h NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of increasing concentration of A769662 (from 12.5 to 100 µM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a Representative immunoblot of AMPK Thr172 and ACC Ser79 phosphorylation. b , c Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. d Quantification of ERK Thr202/Tyr204 phosphorylation. N = 3. e Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. f Representative immunoblots of p70S6K Thr389 and ERK Thr202/Tyr204 phosphorylation. g Quantification of p70S6K Thr389 phosphorylation. N = 4. h Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 3. i – l NRVMs were transfected with control non-targeting siRNA or AMPKα1/α2 siRNA (50 nM) for 66 h. Then, NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 μM) in the presence or absence of A769662 (12.5 μM) for 24 h. i Representative immunoblot and quantification of total AMPKα. N = 3. j Representative immunoblot and quantification of ACC Ser79 phosphorylation. N = 3. k Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with non-targeting siRNA. Scale bar, 20 µm. N = 3. l Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with AMPKα1/α2 siRNA. Scale bar, 20 µm. N = 3. Data in ( a – l ) are mean ± s.e.m. The data were analyzed using One-way ANOVA followed by Bonferroni post-test in ( i ) and Two-way ANOVA followed by Bonferroni post-test in ( b , d , e , g , h , and j – l ). * p
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 377302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna
    <t>A769662</t> prevents NRVM hypertrophy. a – h NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of increasing concentration of A769662 (from 12.5 to 100 µM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a Representative immunoblot of AMPK Thr172 and ACC Ser79 phosphorylation. b , c Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. d Quantification of ERK Thr202/Tyr204 phosphorylation. N = 3. e Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. f Representative immunoblots of p70S6K Thr389 and ERK Thr202/Tyr204 phosphorylation. g Quantification of p70S6K Thr389 phosphorylation. N = 4. h Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 3. i – l NRVMs were transfected with control non-targeting siRNA or AMPKα1/α2 siRNA (50 nM) for 66 h. Then, NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 μM) in the presence or absence of A769662 (12.5 μM) for 24 h. i Representative immunoblot and quantification of total AMPKα. N = 3. j Representative immunoblot and quantification of ACC Ser79 phosphorylation. N = 3. k Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with non-targeting siRNA. Scale bar, 20 µm. N = 3. l Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with AMPKα1/α2 siRNA. Scale bar, 20 µm. N = 3. Data in ( a – l ) are mean ± s.e.m. The data were analyzed using One-way ANOVA followed by Bonferroni post-test in ( i ) and Two-way ANOVA followed by Bonferroni post-test in ( b , d , e , g , h , and j – l ). * p
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 177224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    HCSA activates the p38 MAPK pathway in an AMPK-dependent fashion. A , C2C12 cells were stimulated with HCSA (10 μ m ) for the indicated times. Cell lysates were analyzed by Western blotting ( IB ) using anti-phospho-p38 MAPK antibody. Blotting with

    Journal: The Journal of Biological Chemistry

    Article Title: The Glutamate Agonist Homocysteine Sulfinic Acid Stimulates Glucose Uptake through the Calcium-dependent AMPK-p38 MAPK-Protein Kinase C ? Pathway in Skeletal Muscle Cells *

    doi: 10.1074/jbc.M110.149328

    Figure Lengend Snippet: HCSA activates the p38 MAPK pathway in an AMPK-dependent fashion. A , C2C12 cells were stimulated with HCSA (10 μ m ) for the indicated times. Cell lysates were analyzed by Western blotting ( IB ) using anti-phospho-p38 MAPK antibody. Blotting with

    Article Snippet: To determine the hierarchy between PKCζ and p38 MAPK, we assessed the effect of p38 MAPK knockdown on PKCζ phosphorylation and observed that knockdown of p38 MAPK blocked HCSA-induced PKCζ phosphorylation, indicating that p38 MAPK plays a role as a PKCζ upstream ( C ).

    Techniques: Western Blot

    Inhibition of AMPK and p38 MAPK inhibits HCSA-mediated glucose uptake. Differentiated C2C12 cells were incubated in 60-mm dishes for 1 h with either HCSA (10 μ m ) or insulin (100 n m ) alone in the presence of either compound C or SB203580 and then

    Journal: The Journal of Biological Chemistry

    Article Title: The Glutamate Agonist Homocysteine Sulfinic Acid Stimulates Glucose Uptake through the Calcium-dependent AMPK-p38 MAPK-Protein Kinase C ? Pathway in Skeletal Muscle Cells *

    doi: 10.1074/jbc.M110.149328

    Figure Lengend Snippet: Inhibition of AMPK and p38 MAPK inhibits HCSA-mediated glucose uptake. Differentiated C2C12 cells were incubated in 60-mm dishes for 1 h with either HCSA (10 μ m ) or insulin (100 n m ) alone in the presence of either compound C or SB203580 and then

    Article Snippet: To determine the hierarchy between PKCζ and p38 MAPK, we assessed the effect of p38 MAPK knockdown on PKCζ phosphorylation and observed that knockdown of p38 MAPK blocked HCSA-induced PKCζ phosphorylation, indicating that p38 MAPK plays a role as a PKCζ upstream ( C ).

    Techniques: Inhibition, Incubation

    HCSA stimulates PKCζ through the p38 MAPK pathway. A , C2C12 cells were stimulated with HCSA (10 μ m ) for the indicated times. Cell lysates were analyzed by Western blotting ( IB ) using anti-phospho-PKCζ and anti-PKCζ antibodies.

    Journal: The Journal of Biological Chemistry

    Article Title: The Glutamate Agonist Homocysteine Sulfinic Acid Stimulates Glucose Uptake through the Calcium-dependent AMPK-p38 MAPK-Protein Kinase C ? Pathway in Skeletal Muscle Cells *

    doi: 10.1074/jbc.M110.149328

    Figure Lengend Snippet: HCSA stimulates PKCζ through the p38 MAPK pathway. A , C2C12 cells were stimulated with HCSA (10 μ m ) for the indicated times. Cell lysates were analyzed by Western blotting ( IB ) using anti-phospho-PKCζ and anti-PKCζ antibodies.

    Article Snippet: To determine the hierarchy between PKCζ and p38 MAPK, we assessed the effect of p38 MAPK knockdown on PKCζ phosphorylation and observed that knockdown of p38 MAPK blocked HCSA-induced PKCζ phosphorylation, indicating that p38 MAPK plays a role as a PKCζ upstream ( C ).

    Techniques: Western Blot

    A769662 prevents NRVM hypertrophy. a – h NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of increasing concentration of A769662 (from 12.5 to 100 µM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a Representative immunoblot of AMPK Thr172 and ACC Ser79 phosphorylation. b , c Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. d Quantification of ERK Thr202/Tyr204 phosphorylation. N = 3. e Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. f Representative immunoblots of p70S6K Thr389 and ERK Thr202/Tyr204 phosphorylation. g Quantification of p70S6K Thr389 phosphorylation. N = 4. h Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 3. i – l NRVMs were transfected with control non-targeting siRNA or AMPKα1/α2 siRNA (50 nM) for 66 h. Then, NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 μM) in the presence or absence of A769662 (12.5 μM) for 24 h. i Representative immunoblot and quantification of total AMPKα. N = 3. j Representative immunoblot and quantification of ACC Ser79 phosphorylation. N = 3. k Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with non-targeting siRNA. Scale bar, 20 µm. N = 3. l Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with AMPKα1/α2 siRNA. Scale bar, 20 µm. N = 3. Data in ( a – l ) are mean ± s.e.m. The data were analyzed using One-way ANOVA followed by Bonferroni post-test in ( i ) and Two-way ANOVA followed by Bonferroni post-test in ( b , d , e , g , h , and j – l ). * p

    Journal: Nature Communications

    Article Title: AMPK activation counteracts cardiac hypertrophy by reducing O-GlcNAcylation

    doi: 10.1038/s41467-017-02795-4

    Figure Lengend Snippet: A769662 prevents NRVM hypertrophy. a – h NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of increasing concentration of A769662 (from 12.5 to 100 µM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a Representative immunoblot of AMPK Thr172 and ACC Ser79 phosphorylation. b , c Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. d Quantification of ERK Thr202/Tyr204 phosphorylation. N = 3. e Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. f Representative immunoblots of p70S6K Thr389 and ERK Thr202/Tyr204 phosphorylation. g Quantification of p70S6K Thr389 phosphorylation. N = 4. h Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 3. i – l NRVMs were transfected with control non-targeting siRNA or AMPKα1/α2 siRNA (50 nM) for 66 h. Then, NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 μM) in the presence or absence of A769662 (12.5 μM) for 24 h. i Representative immunoblot and quantification of total AMPKα. N = 3. j Representative immunoblot and quantification of ACC Ser79 phosphorylation. N = 3. k Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with non-targeting siRNA. Scale bar, 20 µm. N = 3. l Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with AMPKα1/α2 siRNA. Scale bar, 20 µm. N = 3. Data in ( a – l ) are mean ± s.e.m. The data were analyzed using One-way ANOVA followed by Bonferroni post-test in ( i ) and Two-way ANOVA followed by Bonferroni post-test in ( b , d , e , g , h , and j – l ). * p

    Article Snippet: AMPK knockdown by siRNA transfection NRVMs were transfected (at a confluence of 40–50%) with either control non-targeting siRNA (ON-TARGETplus Non-targeting siRNA, D-001810-01 from GE Healthcare, 50 nM) or pooled siRNA targeting both AMPKα1 and AMPKα2 catalytic isoforms (AMPKα1/α2 siRNA) (ON-TARGETplus Smart pool siRNAs, L-100623, and L-091373 from GE Healthcare, 50 nM) using lipofectamine RNAimax transfect reagent (Invitrogen) according to the manufacturer’s protocol.

    Techniques: Concentration Assay, Immunostaining, Activity Assay, Luciferase, Western Blot, Transfection

    A769662 prevents NRVM hypertrophy. a – h NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of increasing concentration of A769662 (from 12.5 to 100 µM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a Representative immunoblot of AMPK Thr172 and ACC Ser79 phosphorylation. b , c Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. d Quantification of ERK Thr202/Tyr204 phosphorylation. N = 3. e Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. f Representative immunoblots of p70S6K Thr389 and ERK Thr202/Tyr204 phosphorylation. g Quantification of p70S6K Thr389 phosphorylation. N = 4. h Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 3. i – l NRVMs were transfected with control non-targeting siRNA or AMPKα1/α2 siRNA (50 nM) for 66 h. Then, NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 μM) in the presence or absence of A769662 (12.5 μM) for 24 h. i Representative immunoblot and quantification of total AMPKα. N = 3. j Representative immunoblot and quantification of ACC Ser79 phosphorylation. N = 3. k Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with non-targeting siRNA. Scale bar, 20 µm. N = 3. l Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with AMPKα1/α2 siRNA. Scale bar, 20 µm. N = 3. Data in ( a – l ) are mean ± s.e.m. The data were analyzed using One-way ANOVA followed by Bonferroni post-test in ( i ) and Two-way ANOVA followed by Bonferroni post-test in ( b , d , e , g , h , and j – l ). * p

    Journal: Nature Communications

    Article Title: AMPK activation counteracts cardiac hypertrophy by reducing O-GlcNAcylation

    doi: 10.1038/s41467-017-02795-4

    Figure Lengend Snippet: A769662 prevents NRVM hypertrophy. a – h NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of increasing concentration of A769662 (from 12.5 to 100 µM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a Representative immunoblot of AMPK Thr172 and ACC Ser79 phosphorylation. b , c Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. d Quantification of ERK Thr202/Tyr204 phosphorylation. N = 3. e Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. f Representative immunoblots of p70S6K Thr389 and ERK Thr202/Tyr204 phosphorylation. g Quantification of p70S6K Thr389 phosphorylation. N = 4. h Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 3. i – l NRVMs were transfected with control non-targeting siRNA or AMPKα1/α2 siRNA (50 nM) for 66 h. Then, NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 μM) in the presence or absence of A769662 (12.5 μM) for 24 h. i Representative immunoblot and quantification of total AMPKα. N = 3. j Representative immunoblot and quantification of ACC Ser79 phosphorylation. N = 3. k Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with non-targeting siRNA. Scale bar, 20 µm. N = 3. l Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining of NRVMs transfected with AMPKα1/α2 siRNA. Scale bar, 20 µm. N = 3. Data in ( a – l ) are mean ± s.e.m. The data were analyzed using One-way ANOVA followed by Bonferroni post-test in ( i ) and Two-way ANOVA followed by Bonferroni post-test in ( b , d , e , g , h , and j – l ). * p

    Article Snippet: After 66 h of transfection, medium was replaced and cells were treated for 24 h with pharmacological agents (PE [20 µM] or AngII [100 nM]) to induce hypertrophy and with different concentrations of A769662 as described in figure legends.

    Techniques: Concentration Assay, Immunostaining, Activity Assay, Luciferase, Western Blot, Transfection

    AICAr and phenformin mimick A769662 effects. a – e NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or not of AICAr (from 0.06 to 1 mM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a , b Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. c Representative immunoblots of AMPK Thr172 , ACC Ser79 , ERK Thr202/Tyr204 and p70S6K Thr389 phosphorylation. d Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. e Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 5. f – j NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or not of phenformin (from 0.01 to 1 mM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. f , g Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. h Representative immunoblots of AMPK Thr172 , ACC Ser79 , ERK Thr202/Tyr204 , and p70S6K Thr389 phosphorylation. i Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. j Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 4. Data in ( a – j ) are mean ± s.e.m. The data in ( b , d , e , g , and i , j ) were analyzed using Two-way ANOVA followed by Bonferroni post-test. * p

    Journal: Nature Communications

    Article Title: AMPK activation counteracts cardiac hypertrophy by reducing O-GlcNAcylation

    doi: 10.1038/s41467-017-02795-4

    Figure Lengend Snippet: AICAr and phenformin mimick A769662 effects. a – e NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or not of AICAr (from 0.06 to 1 mM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. a , b Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. c Representative immunoblots of AMPK Thr172 , ACC Ser79 , ERK Thr202/Tyr204 and p70S6K Thr389 phosphorylation. d Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. e Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 5. f – j NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or not of phenformin (from 0.01 to 1 mM) for 24 h except for ERK1/2 phosphorylation which has been evaluated after 1 h. f , g Representative images and quantification of cardiomyocyte area evaluated after α-actinin immunostaining. Scale bar, 20 µm. N = 3. h Representative immunoblots of AMPK Thr172 , ACC Ser79 , ERK Thr202/Tyr204 , and p70S6K Thr389 phosphorylation. i Evaluation of NFAT transcriptional activity by luciferase activity. N = 3. j Amino acids incorporation into proteins measured by [ 14 C]-phenylalanine incorporation. N = 4. Data in ( a – j ) are mean ± s.e.m. The data in ( b , d , e , g , and i , j ) were analyzed using Two-way ANOVA followed by Bonferroni post-test. * p

    Article Snippet: After 66 h of transfection, medium was replaced and cells were treated for 24 h with pharmacological agents (PE [20 µM] or AngII [100 nM]) to induce hypertrophy and with different concentrations of A769662 as described in figure legends.

    Techniques: Immunostaining, Western Blot, Activity Assay, Luciferase

    Glucosamine or PUGNAc prevents the anti-hypertrophic action of AMPK. a – d NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of A769662 (12.5 µM), phenformin (phen, 0.03 mM), AICAr (0.25 mM), PUGNAc (50 µM) and/or glucosamine (GlcN, 5 mM) for 24 h. a Representative immunoblot of protein O-GlcNAcylation levels and ACC Ser79 phosphorylation in GlcN experiments. b Effect of GlcN on the anti-hypertrophic action of AMPK activators. N = 3. c Representative immunoblot of protein O-GlcNAcylation levels and ACC Ser79 phosphorylation in PUGNAc experiments. d Effect of PUGNAc on the anti-hypertrophic action of AMPK activators. N = 3. e Quantification of cardiomyocyte area of NRVMs treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or not of Azaserine (Aza, 5 µM), DON (20 µM) and/or glucosamine (GlcN, 5 mM) for 24 h. N = 3. Data in ( b , d and e ) are mean ± s.e.m. The data were analyzed using Two-way ANOVA followed by Bonferroni post-test in ( b , d and e ). * p

    Journal: Nature Communications

    Article Title: AMPK activation counteracts cardiac hypertrophy by reducing O-GlcNAcylation

    doi: 10.1038/s41467-017-02795-4

    Figure Lengend Snippet: Glucosamine or PUGNAc prevents the anti-hypertrophic action of AMPK. a – d NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or absence of A769662 (12.5 µM), phenformin (phen, 0.03 mM), AICAr (0.25 mM), PUGNAc (50 µM) and/or glucosamine (GlcN, 5 mM) for 24 h. a Representative immunoblot of protein O-GlcNAcylation levels and ACC Ser79 phosphorylation in GlcN experiments. b Effect of GlcN on the anti-hypertrophic action of AMPK activators. N = 3. c Representative immunoblot of protein O-GlcNAcylation levels and ACC Ser79 phosphorylation in PUGNAc experiments. d Effect of PUGNAc on the anti-hypertrophic action of AMPK activators. N = 3. e Quantification of cardiomyocyte area of NRVMs treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or not of Azaserine (Aza, 5 µM), DON (20 µM) and/or glucosamine (GlcN, 5 mM) for 24 h. N = 3. Data in ( b , d and e ) are mean ± s.e.m. The data were analyzed using Two-way ANOVA followed by Bonferroni post-test in ( b , d and e ). * p

    Article Snippet: After 66 h of transfection, medium was replaced and cells were treated for 24 h with pharmacological agents (PE [20 µM] or AngII [100 nM]) to induce hypertrophy and with different concentrations of A769662 as described in figure legends.

    Techniques:

    AMPK activation reduces protein O-GlcNAcylation. a Schematic representation of the HBP/O-GlcNAcylation pathway. b – e NRVMs were treated with phenylephrine (PE, 20 µM) for increasing time periods (from 2 to 8 h). b Representative immunoblots of protein O-GlcNAcylation levels and GFAT protein expression. c Quantification of protein O-GlcNAcylation levels. N = 5. d Quantification of GFAT protein expression. N = 5. e Quantification of cardiomyocyte area evaluated after α-actinin immunostaining. N = 3. f – i NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or not of A769662 (12.5 µM) for 24 h. Representative immunoblot and quantification of protein O-GlcNAcylation levels. N = 3. j Representative immunoblot and quantification of GFAT Ser243 phosphorylation in NRVMs treated 1 h with A769662. N = 3. k – l NRVMs were treated with phenylephrine (PE, 20 µM) in the presence or absence of A769662 (12.5 µM) for increasing time periods (from 2 to 24 h). k Quantification of cardiomyocyte area evaluated after α-actinin immunostaining. N = 3–6. l Quantification of protein O-GlcNAcylation levels. N = 5–7. Data in ( c – l ) are mean ± s.e.m. The data were analyzed using Two-way ANOVA followed by Bonferroni post-test in ( g – i ) and ( k , l ), One-way Anova followed by Bonferroni post-test in ( c – e ) and unpaired Student’s t -test in ( j ). * p

    Journal: Nature Communications

    Article Title: AMPK activation counteracts cardiac hypertrophy by reducing O-GlcNAcylation

    doi: 10.1038/s41467-017-02795-4

    Figure Lengend Snippet: AMPK activation reduces protein O-GlcNAcylation. a Schematic representation of the HBP/O-GlcNAcylation pathway. b – e NRVMs were treated with phenylephrine (PE, 20 µM) for increasing time periods (from 2 to 8 h). b Representative immunoblots of protein O-GlcNAcylation levels and GFAT protein expression. c Quantification of protein O-GlcNAcylation levels. N = 5. d Quantification of GFAT protein expression. N = 5. e Quantification of cardiomyocyte area evaluated after α-actinin immunostaining. N = 3. f – i NRVMs were treated with (open bars) or without (solid bars) phenylephrine (PE, 20 µM) in the presence or not of A769662 (12.5 µM) for 24 h. Representative immunoblot and quantification of protein O-GlcNAcylation levels. N = 3. j Representative immunoblot and quantification of GFAT Ser243 phosphorylation in NRVMs treated 1 h with A769662. N = 3. k – l NRVMs were treated with phenylephrine (PE, 20 µM) in the presence or absence of A769662 (12.5 µM) for increasing time periods (from 2 to 24 h). k Quantification of cardiomyocyte area evaluated after α-actinin immunostaining. N = 3–6. l Quantification of protein O-GlcNAcylation levels. N = 5–7. Data in ( c – l ) are mean ± s.e.m. The data were analyzed using Two-way ANOVA followed by Bonferroni post-test in ( g – i ) and ( k , l ), One-way Anova followed by Bonferroni post-test in ( c – e ) and unpaired Student’s t -test in ( j ). * p

    Article Snippet: After 66 h of transfection, medium was replaced and cells were treated for 24 h with pharmacological agents (PE [20 µM] or AngII [100 nM]) to induce hypertrophy and with different concentrations of A769662 as described in figure legends.

    Techniques: Activation Assay, Western Blot, Expressing, Immunostaining