aicar  (Millipore)


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    Name:
    AICAR
    Description:
    Adenosine monophosphate protein kinase AMPK activator 5 aminoimidazole 4 carboxamide ribonucleotide AICAR modulates cellular energy It regulates lipid and glucose metabolism pro inflammatory responses cytokine production cell proliferation and apoptosis AICAR improves ischemia or reperfusion injury and kidney fibrosis in rats It provides protection against acute tubular necrosis
    Catalog Number:
    a9978
    Price:
    None
    Applications:
    AICAR has been used to provide protection against cisplatin-induced acute kidney injury through the JAK (tyrosine-protein kinase )/STAT (signal transducer and activator of transcription )/SOCS (suppressor of cytokine signaling) pathway. It also has been used in the assay of FapR (malonyl-CoA responsive bacterial transcriptional factor )-NLuc (nanoLuciferase) biosensor activity.
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    Structured Review

    Millipore aicar
    AICAR
    Adenosine monophosphate protein kinase AMPK activator 5 aminoimidazole 4 carboxamide ribonucleotide AICAR modulates cellular energy It regulates lipid and glucose metabolism pro inflammatory responses cytokine production cell proliferation and apoptosis AICAR improves ischemia or reperfusion injury and kidney fibrosis in rats It provides protection against acute tubular necrosis
    https://www.bioz.com/result/aicar/product/Millipore
    Average 99 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    aicar - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "AICAR inhibits NFκB DNA binding independently of AMPK to attenuate LPS-triggered inflammatory responses in human macrophages"

    Article Title: AICAR inhibits NFκB DNA binding independently of AMPK to attenuate LPS-triggered inflammatory responses in human macrophages

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26102-3

    Stimulus specific regulation of transcriptional responses by AICAR. ( A ) mRNA Expression of SOCS3 in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. ( B ) mRNA Expression of CCL18 in macrophages treated for 24 h with 20 ng/ml IL-4, 1 mM AICAR and 0.5 µM ABT-702. ( C ) mRNA Expression of SLC2A1 in macrophages treated for 24 h with 1 mM DMOG, 1 mM AICAR, and 0.5 µM ABT-702. ( D ) Western blot analysis of STAT3 phosphorylation in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. *p
    Figure Legend Snippet: Stimulus specific regulation of transcriptional responses by AICAR. ( A ) mRNA Expression of SOCS3 in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. ( B ) mRNA Expression of CCL18 in macrophages treated for 24 h with 20 ng/ml IL-4, 1 mM AICAR and 0.5 µM ABT-702. ( C ) mRNA Expression of SLC2A1 in macrophages treated for 24 h with 1 mM DMOG, 1 mM AICAR, and 0.5 µM ABT-702. ( D ) Western blot analysis of STAT3 phosphorylation in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. *p

    Techniques Used: Expressing, Western Blot

    AICAR suppresses LPS transcriptional response. ( A , B ) mRNA expression of TNFα and IL-6 in macrophages treated for 3 h with 100 ng/ml LPS and indicated concentrations of AICAR ( A ) or with 100 ng/ml LPS, 0.1 mM AICAR and 0.5 µM ABT-702 ( B ). ( C ) mRNA expression of LPS-induced genes in macrophages treated for 1 h with 100 ng/ml LPS and 1 mM AICAR. ( D ) Cytokine secretion into culture medium of macrophages treated for 24 h with 100 ng/ml LPS and 1 mM AICAR. *p
    Figure Legend Snippet: AICAR suppresses LPS transcriptional response. ( A , B ) mRNA expression of TNFα and IL-6 in macrophages treated for 3 h with 100 ng/ml LPS and indicated concentrations of AICAR ( A ) or with 100 ng/ml LPS, 0.1 mM AICAR and 0.5 µM ABT-702 ( B ). ( C ) mRNA expression of LPS-induced genes in macrophages treated for 1 h with 100 ng/ml LPS and 1 mM AICAR. ( D ) Cytokine secretion into culture medium of macrophages treated for 24 h with 100 ng/ml LPS and 1 mM AICAR. *p

    Techniques Used: Expressing

    2) Product Images from "Convergence of IPMK and LKB1-AMPK Signaling Pathways on Metformin Action"

    Article Title: Convergence of IPMK and LKB1-AMPK Signaling Pathways on Metformin Action

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2014-1134

    IPMK depletion attenuates LKB1-AMPK signaling in response to AMPK agonists. Cells were treated with 4mM metformin (A–C) or 2mM AICAR (D–F) for 2 hours. Cell lysates were subjected to SDS-PAGE and immunoblotting for phospho-AMPK (pAMPK)
    Figure Legend Snippet: IPMK depletion attenuates LKB1-AMPK signaling in response to AMPK agonists. Cells were treated with 4mM metformin (A–C) or 2mM AICAR (D–F) for 2 hours. Cell lysates were subjected to SDS-PAGE and immunoblotting for phospho-AMPK (pAMPK)

    Techniques Used: SDS Page

    3) Product Images from "AMP-activated protein kinase (AMPK) activation inhibits nuclear translocation of Smad4 in mesangial cells and diabetic kidneys"

    Article Title: AMP-activated protein kinase (AMPK) activation inhibits nuclear translocation of Smad4 in mesangial cells and diabetic kidneys

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00234.2014

    AICAR inhibited HG-induced protein expression of Smad4 via ubiquitin-dependent protein degradation. MMCs were treated with or without specific proteasome inhibitor lactacystin (10 μM) for 2 h and cultured under different conditions. Mean value
    Figure Legend Snippet: AICAR inhibited HG-induced protein expression of Smad4 via ubiquitin-dependent protein degradation. MMCs were treated with or without specific proteasome inhibitor lactacystin (10 μM) for 2 h and cultured under different conditions. Mean value

    Techniques Used: Expressing, Cell Culture

    4) Product Images from "Sestrin2, as a negative feedback regulator of mTOR, provides neuroprotection by activation AMPK phosphorylation in neonatal hypoxic-ischemic encephalopathy in rat pups"

    Article Title: Sestrin2, as a negative feedback regulator of mTOR, provides neuroprotection by activation AMPK phosphorylation in neonatal hypoxic-ischemic encephalopathy in rat pups

    Journal: Journal of Cerebral Blood Flow & Metabolism

    doi: 10.1177/0271678X16656201

    The effects of AMPK activator (AICAR) and inhibitor (Dorsomorphin) on infarct area and neurological function at 24 h post HIE. (a) The infarct area was significantly reduced in the siRNA sestrin2 + AICAR group compared to only siRNA sestrin2. rh-sestrin2 treatment with Dorsomorphin significantly increased infarct area when compared to rh-sestrin2 treatment group alone. Silencing sestrin2 with siRNA sestrin2 showed no effect to brain infarct area, while after hypoxic-ischemic injury, knockout sestrin2 exacerbated brain infarct area. (b–c), Righting reflex and Geotaxis showed that animal group treated with rh-sestrin2 + Dorsomorphine significantly impaired neurological function compared to rh-sestrin2 treatment group, whereas siRNA sestrin2 + AICAR showed to significantly improve neurological function when compared to siRNA sestrin2 group alone. Statistical differences between groups were analyzed using one-way ANOVA followed by Tukey multiple-comparison post hoc analysis. * versus sham; # versus vehicle or scramble siRNA. n = 6/group.
    Figure Legend Snippet: The effects of AMPK activator (AICAR) and inhibitor (Dorsomorphin) on infarct area and neurological function at 24 h post HIE. (a) The infarct area was significantly reduced in the siRNA sestrin2 + AICAR group compared to only siRNA sestrin2. rh-sestrin2 treatment with Dorsomorphin significantly increased infarct area when compared to rh-sestrin2 treatment group alone. Silencing sestrin2 with siRNA sestrin2 showed no effect to brain infarct area, while after hypoxic-ischemic injury, knockout sestrin2 exacerbated brain infarct area. (b–c), Righting reflex and Geotaxis showed that animal group treated with rh-sestrin2 + Dorsomorphine significantly impaired neurological function compared to rh-sestrin2 treatment group, whereas siRNA sestrin2 + AICAR showed to significantly improve neurological function when compared to siRNA sestrin2 group alone. Statistical differences between groups were analyzed using one-way ANOVA followed by Tukey multiple-comparison post hoc analysis. * versus sham; # versus vehicle or scramble siRNA. n = 6/group.

    Techniques Used: Knock-Out

    Representative pictures of modulation of sestrin2 on AMPK at 24 h post HIE. (a) Representative picture of Western blot data showed bands of the expression levels of proteins of interest with or without AMPK inhibitor (Dorsomorphin). (b–c) Western blot data quantification showed that animals treated with rh-sestrin2 significantly increased sestrin2 and p-AMPK expression in the brain compared to vehicle, while animal groups treated with rh-sestrin + AMPK inhibitor (Dorsomorphin) did not affect sestrin2 expression levels (b) however, it reduced p-AMPK levels (c) when compared to rh-sestrin2 treatment group animals. (d) Representative picture of Western blot data showed bands of the expression levels of proteins of interest with or without AMPK activator (AICAR). (e–f), Western blot data showed that silencing sestrin2 with siRNA sestrin2 reduced sestrin2 and p-AMPK expression levels in the brain compared to vehicle and scramble siRNA, while animal groups treated with siRNA sestrin2 + AMPK activator (AICAR) did not change sestrin2 expression levels when compared to sham (e) however, it significantly increased p-AMPK expression levels when compared to siRNA sestrin2 animal group (f). Statistical differences between groups were analyzed using one-way ANOVA followed by Tukey multiple-comparison post hoc analysis. * versus sham; # versus vehicle; versus scramble siRNA. n = 6/group. (Western blot samples were from those animals euthanized after short-term neurobehavioral tests.)
    Figure Legend Snippet: Representative pictures of modulation of sestrin2 on AMPK at 24 h post HIE. (a) Representative picture of Western blot data showed bands of the expression levels of proteins of interest with or without AMPK inhibitor (Dorsomorphin). (b–c) Western blot data quantification showed that animals treated with rh-sestrin2 significantly increased sestrin2 and p-AMPK expression in the brain compared to vehicle, while animal groups treated with rh-sestrin + AMPK inhibitor (Dorsomorphin) did not affect sestrin2 expression levels (b) however, it reduced p-AMPK levels (c) when compared to rh-sestrin2 treatment group animals. (d) Representative picture of Western blot data showed bands of the expression levels of proteins of interest with or without AMPK activator (AICAR). (e–f), Western blot data showed that silencing sestrin2 with siRNA sestrin2 reduced sestrin2 and p-AMPK expression levels in the brain compared to vehicle and scramble siRNA, while animal groups treated with siRNA sestrin2 + AMPK activator (AICAR) did not change sestrin2 expression levels when compared to sham (e) however, it significantly increased p-AMPK expression levels when compared to siRNA sestrin2 animal group (f). Statistical differences between groups were analyzed using one-way ANOVA followed by Tukey multiple-comparison post hoc analysis. * versus sham; # versus vehicle; versus scramble siRNA. n = 6/group. (Western blot samples were from those animals euthanized after short-term neurobehavioral tests.)

    Techniques Used: Western Blot, Expressing

    The effects of sestrin2 on mTOR signaling at 24 h post HIE. (a) Western blot data showed time course expression of p-mTOR levels, with p-mTOR peaking at 6 h and slowly decreasing by 72 h. (b–c) Significant decrease in both sestrin2 and p-mTOR expression levels after rapamycin treatment compared to vehicle at 24 h post hypoxic-ischemic injury. (d) rh-sestrin2 treatment significantly reduced p-mTOR expression levels in the brain compared to vehicle, while rh-sestrin2 + Dorsomorphin reversed that effect. AICAR significantly reduced p-mTOR expression even after sestrin2 was silenced with siRNA sestrin2. Statistical differences between groups were analyzed using one-way ANOVA followed by Tukey multiple-comparison post hoc analysis. * versus sham; # versus vehicle; versus scramble siRNA. n = 6/group.
    Figure Legend Snippet: The effects of sestrin2 on mTOR signaling at 24 h post HIE. (a) Western blot data showed time course expression of p-mTOR levels, with p-mTOR peaking at 6 h and slowly decreasing by 72 h. (b–c) Significant decrease in both sestrin2 and p-mTOR expression levels after rapamycin treatment compared to vehicle at 24 h post hypoxic-ischemic injury. (d) rh-sestrin2 treatment significantly reduced p-mTOR expression levels in the brain compared to vehicle, while rh-sestrin2 + Dorsomorphin reversed that effect. AICAR significantly reduced p-mTOR expression even after sestrin2 was silenced with siRNA sestrin2. Statistical differences between groups were analyzed using one-way ANOVA followed by Tukey multiple-comparison post hoc analysis. * versus sham; # versus vehicle; versus scramble siRNA. n = 6/group.

    Techniques Used: Western Blot, Expressing

    5) Product Images from "Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis"

    Article Title: Defects in the mitochondrial-tRNA modification enzymes MTO1 and GTPBP3 promote different metabolic reprogramming through a HIF-PPARγ-UCP2-AMPK axis

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19587-5

    Regulation of UCP2 is AMPK- and PPARγ-dependent in MTO1 fibroblasts. (A) qRT-PCR analysis of the UCP2 mRNA expression in MTO1 human fibroblasts (MTO1 HF), treated or not with 5 μM rosiglitazone (RGZ) for 1 h. Data are represented as fold change respect to WT HF values. (B) Representative immunoblots of phosphor-Thr172-AMPKα, AMPKα and UCP2 in WT HF and MTO1 HF, treated or not with 5 μM RGZ for 1 h. Porin and AMPKα were used as loading controls. Full-length western blots are included in supplementary information (Fig. S 23 ). The scatter plot shows densitometric data for UCP2 normalized to porin and phosphor-Thr172-AMPKα normalized to AMPKα, and represented as fold change relative to WT HF. (C) Representative immunoblots of phosphor-Thr172-AMPKα, AMPKα and UCP2 in WT HF and MTO1 HF, treated or not with 1 mM AICAR for 1 h. Porin was used as a loading control. Full-length western blots are included in supplementary information (Fig. S 23 ). The scatter plot shows densitometric data for UCP2 normalized to porin and phosphor-Thr172-AMPKα normalized to AMPKα, and represented as fold change relative to WT HF. All data are the mean ± SD of at least three different experiments. Differences from WT HF values were found to be statistically significant at *p
    Figure Legend Snippet: Regulation of UCP2 is AMPK- and PPARγ-dependent in MTO1 fibroblasts. (A) qRT-PCR analysis of the UCP2 mRNA expression in MTO1 human fibroblasts (MTO1 HF), treated or not with 5 μM rosiglitazone (RGZ) for 1 h. Data are represented as fold change respect to WT HF values. (B) Representative immunoblots of phosphor-Thr172-AMPKα, AMPKα and UCP2 in WT HF and MTO1 HF, treated or not with 5 μM RGZ for 1 h. Porin and AMPKα were used as loading controls. Full-length western blots are included in supplementary information (Fig. S 23 ). The scatter plot shows densitometric data for UCP2 normalized to porin and phosphor-Thr172-AMPKα normalized to AMPKα, and represented as fold change relative to WT HF. (C) Representative immunoblots of phosphor-Thr172-AMPKα, AMPKα and UCP2 in WT HF and MTO1 HF, treated or not with 1 mM AICAR for 1 h. Porin was used as a loading control. Full-length western blots are included in supplementary information (Fig. S 23 ). The scatter plot shows densitometric data for UCP2 normalized to porin and phosphor-Thr172-AMPKα normalized to AMPKα, and represented as fold change relative to WT HF. All data are the mean ± SD of at least three different experiments. Differences from WT HF values were found to be statistically significant at *p

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot

    6) Product Images from "The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells"

    Article Title: The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2018.06.014

    AMPK/p27 Kip1 Signaling Mediates Cell Survival in the MuSC Geriatric MuSCs were cultured for 48 hr treated with or without AICAR and/or overexpression of p27 Kip1 mutant-p27 Kip1(198A) . (A) Percentage of TUNEL-positive cells with respective treatment (N = 4 independent experiments). (B) Upper: representative blot of total and cleaved PARP; lower: quantification of protein expression normalized to untreated geriatric cells (N = 3 independent experiments). (C) Percentage of TUNEL-positive cells during Atg5 knockdown with concurrent overexpression of p27 Kip1 mutants p27 Kip1(198D) or p27 Kip1(198A) (N = 3 independent experiments). (D) Percentage of TUNEL-positive cells during treatment of compound C to young MuSCs with concurrent overexpression of p27 Kip1 mutant p27 Kip1(198D) (N = 3 independent experiments). (E) Geriatric MuSCs treated with or without AICAR and infected with respective p27 Kip1 mutant lentivirus. Cells were transplanted into injured young SCID muscle and quantified 4 or 28 days later. (F) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 4 days after transplantation. (G) Quantification of GFP(+) cells per muscle (N = 6 independent experiments). (H) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 28 days after transplantation. (I) Quantification of GFP(+) myofibers per muscle (N = 6 independent experiments). Scale bar, 100 μm. ∗ Signifies difference from young or untreated control cells (p
    Figure Legend Snippet: AMPK/p27 Kip1 Signaling Mediates Cell Survival in the MuSC Geriatric MuSCs were cultured for 48 hr treated with or without AICAR and/or overexpression of p27 Kip1 mutant-p27 Kip1(198A) . (A) Percentage of TUNEL-positive cells with respective treatment (N = 4 independent experiments). (B) Upper: representative blot of total and cleaved PARP; lower: quantification of protein expression normalized to untreated geriatric cells (N = 3 independent experiments). (C) Percentage of TUNEL-positive cells during Atg5 knockdown with concurrent overexpression of p27 Kip1 mutants p27 Kip1(198D) or p27 Kip1(198A) (N = 3 independent experiments). (D) Percentage of TUNEL-positive cells during treatment of compound C to young MuSCs with concurrent overexpression of p27 Kip1 mutant p27 Kip1(198D) (N = 3 independent experiments). (E) Geriatric MuSCs treated with or without AICAR and infected with respective p27 Kip1 mutant lentivirus. Cells were transplanted into injured young SCID muscle and quantified 4 or 28 days later. (F) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 4 days after transplantation. (G) Quantification of GFP(+) cells per muscle (N = 6 independent experiments). (H) Geriatric MuSCs treated with or without AICAR and overexpression of p27 Kip1 mutants analyzed 28 days after transplantation. (I) Quantification of GFP(+) myofibers per muscle (N = 6 independent experiments). Scale bar, 100 μm. ∗ Signifies difference from young or untreated control cells (p

    Techniques Used: Cell Culture, Over Expression, Mutagenesis, TUNEL Assay, Expressing, Infection, Transplantation Assay

    Restored AMPK Activation Rescues Inherent Apoptotic Susceptibility in Geriatric MuSCs (A) Phosphorylated and total protein expression of young MuSC AMPK and p27 Kip1 throughout 48 hr in culture. (B) Phosphorylated and total protein expression of AMPK and p27 Kip1 at 48 hr in culture across different ages. (C) TUNEL-positive cells in young, geriatric and geriatric MuSCs treated with AICAR (N = 3 independent experiments). (D). Left: representative blot of total and cleaved PARP; right: quantification of cleaved PARP protein expression normalized to young cells (N = 3 independent experiments). (E) Young and geriatric MuSCs infected with respective AMPK lentivirus, transplanted into injured SCID muscle and quantified 4 or 28 days later. (F and G) Upper: young GFP-labeled MuSCs overexpressing GFP control or a dominant-negative (DN) AMPK 4 days; lower: 28 days after transplantation (N = 5 independent experiments) (F). Upper: geriatric GFP-labeled MuSCs overexpressing GFP control or constitutively active (CA) AMPK 4 days; lower: 28 days after transplantation. Quantification includes counts of GFP(+) cells (4d) or myofibers (28d) per muscle (N = 5 independent experiments) (G). ∗ Signifies difference from young cells; # signifies difference from GFP control transplanted cells (p
    Figure Legend Snippet: Restored AMPK Activation Rescues Inherent Apoptotic Susceptibility in Geriatric MuSCs (A) Phosphorylated and total protein expression of young MuSC AMPK and p27 Kip1 throughout 48 hr in culture. (B) Phosphorylated and total protein expression of AMPK and p27 Kip1 at 48 hr in culture across different ages. (C) TUNEL-positive cells in young, geriatric and geriatric MuSCs treated with AICAR (N = 3 independent experiments). (D). Left: representative blot of total and cleaved PARP; right: quantification of cleaved PARP protein expression normalized to young cells (N = 3 independent experiments). (E) Young and geriatric MuSCs infected with respective AMPK lentivirus, transplanted into injured SCID muscle and quantified 4 or 28 days later. (F and G) Upper: young GFP-labeled MuSCs overexpressing GFP control or a dominant-negative (DN) AMPK 4 days; lower: 28 days after transplantation (N = 5 independent experiments) (F). Upper: geriatric GFP-labeled MuSCs overexpressing GFP control or constitutively active (CA) AMPK 4 days; lower: 28 days after transplantation. Quantification includes counts of GFP(+) cells (4d) or myofibers (28d) per muscle (N = 5 independent experiments) (G). ∗ Signifies difference from young cells; # signifies difference from GFP control transplanted cells (p

    Techniques Used: Activation Assay, Expressing, TUNEL Assay, Infection, Labeling, Dominant Negative Mutation, Transplantation Assay

    The AMPK/p27 Kip1 Pathway Regulates Cellular Senescence (A) Upper: β-gal staining in geriatric MuSCs treated with AICAR and simultaneous overexpression of p27 Kip1(198A) . Arrows represent β-gal-positive cells. Lower: percentage of β-gal-positive cells in each group (N = 3 independent experiments). (B) Upper: representative western blot of phosphorylated and total S6 kinase protein expression. Lower: quantification of phospho/total S6 ratio normalized to geriatric control cells (N = 4 independent experiments). (C) mRNA expression of p21 CIP1 and p16 INK4a in young and geriatric MuSCs treated with AICAR with or without p27 Kip1(198A) overexpression (N = 4 independent experiments). (D) Summary figure illustrating role of AMPK/p27 Kip1 signaling on autophagy, proliferation, senescence, and apoptosis. ∗ Signifies difference from untreated geriatric control cells; † signifies difference from AICAR-treated geriatric cells; # signifies difference from young cells; ˆ signifies difference from untreated geriatric control cells (p
    Figure Legend Snippet: The AMPK/p27 Kip1 Pathway Regulates Cellular Senescence (A) Upper: β-gal staining in geriatric MuSCs treated with AICAR and simultaneous overexpression of p27 Kip1(198A) . Arrows represent β-gal-positive cells. Lower: percentage of β-gal-positive cells in each group (N = 3 independent experiments). (B) Upper: representative western blot of phosphorylated and total S6 kinase protein expression. Lower: quantification of phospho/total S6 ratio normalized to geriatric control cells (N = 4 independent experiments). (C) mRNA expression of p21 CIP1 and p16 INK4a in young and geriatric MuSCs treated with AICAR with or without p27 Kip1(198A) overexpression (N = 4 independent experiments). (D) Summary figure illustrating role of AMPK/p27 Kip1 signaling on autophagy, proliferation, senescence, and apoptosis. ∗ Signifies difference from untreated geriatric control cells; † signifies difference from AICAR-treated geriatric cells; # signifies difference from young cells; ˆ signifies difference from untreated geriatric control cells (p

    Techniques Used: Staining, Over Expression, Western Blot, Expressing

    7) Product Images from "Estradiol Regulates Brown Adipose Tissue Thermogenesis via Hypothalamic AMPK"

    Article Title: Estradiol Regulates Brown Adipose Tissue Thermogenesis via Hypothalamic AMPK

    Journal: Cell Metabolism

    doi: 10.1016/j.cmet.2014.03.031

    Effect of SC E2 on Energy Balance (A–F) (A) Body weight change, (B) daily food intake, (C) in situ hybridization autoradiographic images, (D) Npy and Pomc mRNA levels in the ARC, (E) western blot autoradiographic images (left panel) and hypothalamic levels of proteins of AMPK pathway (right panel), and (F) hypothalamic malonyl-CoA levels of OVX rats and OVX rats SC treated with vehicle or E2. (G and H) (G) Hypothalamic malonyl-CoA levels and (H) food intake of OVX rats SC treated with vehicle or E2 and ICV treated with vehicle or AICAR. Error bars represent SEM; n = 8–16 animals per experimental group. 3V: third ventricle; ∗ , ∗∗ and ∗∗∗ p
    Figure Legend Snippet: Effect of SC E2 on Energy Balance (A–F) (A) Body weight change, (B) daily food intake, (C) in situ hybridization autoradiographic images, (D) Npy and Pomc mRNA levels in the ARC, (E) western blot autoradiographic images (left panel) and hypothalamic levels of proteins of AMPK pathway (right panel), and (F) hypothalamic malonyl-CoA levels of OVX rats and OVX rats SC treated with vehicle or E2. (G and H) (G) Hypothalamic malonyl-CoA levels and (H) food intake of OVX rats SC treated with vehicle or E2 and ICV treated with vehicle or AICAR. Error bars represent SEM; n = 8–16 animals per experimental group. 3V: third ventricle; ∗ , ∗∗ and ∗∗∗ p

    Techniques Used: In Situ Hybridization, Western Blot

    8) Product Images from "Metformin Inhibits Nuclear Receptor TR4-Mediated Hepatic Stearoyl-CoA Desaturase 1 Gene Expression With Altered Insulin Sensitivity"

    Article Title: Metformin Inhibits Nuclear Receptor TR4-Mediated Hepatic Stearoyl-CoA Desaturase 1 Gene Expression With Altered Insulin Sensitivity

    Journal: Diabetes

    doi: 10.2337/db10-0393

    Effect of metformin on TR4 activity. A : AMPK and phosphor-AMPK in Hepa1–6 cells treated with metformin. B : TR4 phosphorylation status by Western blot. Hepa1–6 cells were treated with AICAR and metformin, and cell lysates were harvested. Cell lysates were then treated with CIP. C : DR1 × 3-Luc reporter vector was cotransfected with TR4 in Hepa1–6 cells. After overnight recovery, the transfected cells were treated with metformin and luciferase activity was measured (* P
    Figure Legend Snippet: Effect of metformin on TR4 activity. A : AMPK and phosphor-AMPK in Hepa1–6 cells treated with metformin. B : TR4 phosphorylation status by Western blot. Hepa1–6 cells were treated with AICAR and metformin, and cell lysates were harvested. Cell lysates were then treated with CIP. C : DR1 × 3-Luc reporter vector was cotransfected with TR4 in Hepa1–6 cells. After overnight recovery, the transfected cells were treated with metformin and luciferase activity was measured (* P

    Techniques Used: Activity Assay, Western Blot, Plasmid Preparation, Transfection, Luciferase

    9) Product Images from "Vav3, a GEF for RhoA, Plays a Critical Role under High Glucose Conditions"

    Article Title: Vav3, a GEF for RhoA, Plays a Critical Role under High Glucose Conditions

    Journal: Endocrinology and Metabolism

    doi: 10.3803/EnM.2014.29.3.363

    (A) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMPK agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). Total cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-PAK and anti-PAK antibodies. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-paxillin and anti-paxillin antibodies was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence of metformin. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-PAK and anti-phospho-paxillin antibodies was performed. Blotting with anti-PAK and anti-paxillin antibodies was conducted as a protein loading control. The results shown are from three independent experiments.
    Figure Legend Snippet: (A) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMPK agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). Total cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-PAK and anti-PAK antibodies. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-paxillin and anti-paxillin antibodies was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence of metformin. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-PAK and anti-phospho-paxillin antibodies was performed. Blotting with anti-PAK and anti-paxillin antibodies was conducted as a protein loading control. The results shown are from three independent experiments.

    Techniques Used: Cell Culture, Western Blot

    (A) Total RNA was cultured under high glucose culture conditions in the presence or absence of 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR) treatment, and reverse transcription-polymerase chain reaction (RT-PCR) was conducted using specific Vav3 primers. PCR products were then run on 1% agarose gels and visualized under ultraviolet light. β-Actin mRNA was employed as a positive control. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). The cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 antibody. Blotting with anti-β-actin antibody was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPK antibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. (D) C2C12 cells were cultured under high glucose conditions in the presence or absence of metformin. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPKantibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. The results shown are from three independent experiments.
    Figure Legend Snippet: (A) Total RNA was cultured under high glucose culture conditions in the presence or absence of 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR) treatment, and reverse transcription-polymerase chain reaction (RT-PCR) was conducted using specific Vav3 primers. PCR products were then run on 1% agarose gels and visualized under ultraviolet light. β-Actin mRNA was employed as a positive control. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). The cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 antibody. Blotting with anti-β-actin antibody was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPK antibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. (D) C2C12 cells were cultured under high glucose conditions in the presence or absence of metformin. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPKantibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. The results shown are from three independent experiments.

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control, Western Blot

    10) Product Images from "Genomic deletion of malic enzyme 2 confers collateral lethality in pancreatic cancer"

    Article Title: Genomic deletion of malic enzyme 2 confers collateral lethality in pancreatic cancer

    Journal: Nature

    doi: 10.1038/nature21052

    Increase in ROS activates AMPK pathway a , b , Measurement of NADPH ( a ) and total ROS ( b ) in ME2-rescued and ME3-depleted PATU8988T cells. Error bars represent s.d. of at least n = 5 replicates. c , Immunoblot of ME3, BCAT2 and AMPK expression upon depletion of ME3 followed by Trolox treatment for 24 h. Structure of Trolox (above), a synthetic vitamin E analogue that acts as a potent antioxidant. d , Expression of BCAT2 in PATU8988T and Panc1 cells upon treatment wit h AICAR for 14 h. e , Colony-formation assay showing decreased cell growth upon shRNA-mediated depletion of BCAT2 and SREBP1 using two independent shRNAs. Error bars represent s.d. of at least n = 3 replicates. f , OCR in cells depleted of BCAT2 by shRNA. g , OCR in cells upon overexpression of BCAT2. Error bars represent s.e.m of at least n = 5 replicates. P values were determined by two-tailed t -test.
    Figure Legend Snippet: Increase in ROS activates AMPK pathway a , b , Measurement of NADPH ( a ) and total ROS ( b ) in ME2-rescued and ME3-depleted PATU8988T cells. Error bars represent s.d. of at least n = 5 replicates. c , Immunoblot of ME3, BCAT2 and AMPK expression upon depletion of ME3 followed by Trolox treatment for 24 h. Structure of Trolox (above), a synthetic vitamin E analogue that acts as a potent antioxidant. d , Expression of BCAT2 in PATU8988T and Panc1 cells upon treatment wit h AICAR for 14 h. e , Colony-formation assay showing decreased cell growth upon shRNA-mediated depletion of BCAT2 and SREBP1 using two independent shRNAs. Error bars represent s.d. of at least n = 3 replicates. f , OCR in cells depleted of BCAT2 by shRNA. g , OCR in cells upon overexpression of BCAT2. Error bars represent s.e.m of at least n = 5 replicates. P values were determined by two-tailed t -test.

    Techniques Used: Expressing, Colony Assay, shRNA, Over Expression, Two Tailed Test

    11) Product Images from "Study of the AMP-activated protein kinase role in energy metabolism changes during the postmortem aging of yak longissimus lumborum"

    Article Title: Study of the AMP-activated protein kinase role in energy metabolism changes during the postmortem aging of yak longissimus lumborum

    Journal: bioRxiv

    doi: 10.1101/670851

    pH values of postmortem yak LL muscle with AICAR and STO-609 treatment. One-way ANOVA was used for statistical analyses between the control group and two treatment group at 0 h to 168 h (x, y, z P
    Figure Legend Snippet: pH values of postmortem yak LL muscle with AICAR and STO-609 treatment. One-way ANOVA was used for statistical analyses between the control group and two treatment group at 0 h to 168 h (x, y, z P

    Techniques Used:

    AMPK Activity of postmortem yak LL muscle with AICAR and STO-609 treatment. At a specific postmortem time, indicates significant difference at P
    Figure Legend Snippet: AMPK Activity of postmortem yak LL muscle with AICAR and STO-609 treatment. At a specific postmortem time, indicates significant difference at P

    Techniques Used: Activity Assay

    Lactic acid in postmortem yak LL muscle with AICAR and STO-609 treatment.
    Figure Legend Snippet: Lactic acid in postmortem yak LL muscle with AICAR and STO-609 treatment.

    Techniques Used:

    Effect of AICAR and STO-609 on AMPKα1(A), AMPKα2 (B) mRNA were treated with or without AICAR (10 mM) and STO-609 (10 mM) for the time as indicated above and total RNA was subjected to real-time RT-PCR as described in Materials and methods. The results were expressed as a relative value compared to the untreated sample as 100%. All data were represented as means ± SEM of three independent experiments. P
    Figure Legend Snippet: Effect of AICAR and STO-609 on AMPKα1(A), AMPKα2 (B) mRNA were treated with or without AICAR (10 mM) and STO-609 (10 mM) for the time as indicated above and total RNA was subjected to real-time RT-PCR as described in Materials and methods. The results were expressed as a relative value compared to the untreated sample as 100%. All data were represented as means ± SEM of three independent experiments. P

    Techniques Used: Quantitative RT-PCR

    (A) Effects of yak LL muscle injection of AICAR and STO-609 on AMPK phosphorylation (Thr 172) in postmortem yak LL muscle. Representative immunoblots of AMPK phosphorylation and b-actin, and the relative band density of phospho-AMPK after normalizing to b-actin were shown. (B) Densitometric analysis of AMPK expression of bovine muscle during postmortem aging.
    Figure Legend Snippet: (A) Effects of yak LL muscle injection of AICAR and STO-609 on AMPK phosphorylation (Thr 172) in postmortem yak LL muscle. Representative immunoblots of AMPK phosphorylation and b-actin, and the relative band density of phospho-AMPK after normalizing to b-actin were shown. (B) Densitometric analysis of AMPK expression of bovine muscle during postmortem aging.

    Techniques Used: Injection, Western Blot, Expressing

    12) Product Images from "Activation of AMP-activated protein kinase stimulates the nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in human diploid fibroblasts"

    Article Title: Activation of AMP-activated protein kinase stimulates the nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in human diploid fibroblasts

    Journal: Experimental & Molecular Medicine

    doi: 10.3858/emm.2010.42.4.025

    SFM- and AICAR-induced GAPDH translocation in AMPK siRNA-transfected HDFs. HDFs were grown in 10% FBS medium for 1 day and transfected with siRNAs of AMPKα1/α2 and a si CONTROL complete kit as a negative mock control for 2 days. Cells were
    Figure Legend Snippet: SFM- and AICAR-induced GAPDH translocation in AMPK siRNA-transfected HDFs. HDFs were grown in 10% FBS medium for 1 day and transfected with siRNAs of AMPKα1/α2 and a si CONTROL complete kit as a negative mock control for 2 days. Cells were

    Techniques Used: Translocation Assay, Transfection

    Effect of NOS inhibitors on SFM- and AICAR-induced GAPDH translocation. HDFs were cultured in 10% FBS medium, and then treated with SFM for 4 days or 2 mM AICAR in 10% FBS in the absence (-) or presence (+) of 100 µM of L-NMMA (A) or 0.5 mg/ml
    Figure Legend Snippet: Effect of NOS inhibitors on SFM- and AICAR-induced GAPDH translocation. HDFs were cultured in 10% FBS medium, and then treated with SFM for 4 days or 2 mM AICAR in 10% FBS in the absence (-) or presence (+) of 100 µM of L-NMMA (A) or 0.5 mg/ml

    Techniques Used: Translocation Assay, Cell Culture

    13) Product Images from "AICAR inhibits NFκB DNA binding independently of AMPK to attenuate LPS-triggered inflammatory responses in human macrophages"

    Article Title: AICAR inhibits NFκB DNA binding independently of AMPK to attenuate LPS-triggered inflammatory responses in human macrophages

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26102-3

    Stimulus specific regulation of transcriptional responses by AICAR. ( A ) mRNA Expression of SOCS3 in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. ( B ) mRNA Expression of CCL18 in macrophages treated for 24 h with 20 ng/ml IL-4, 1 mM AICAR and 0.5 µM ABT-702. ( C ) mRNA Expression of SLC2A1 in macrophages treated for 24 h with 1 mM DMOG, 1 mM AICAR, and 0.5 µM ABT-702. ( D .
    Figure Legend Snippet: Stimulus specific regulation of transcriptional responses by AICAR. ( A ) mRNA Expression of SOCS3 in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. ( B ) mRNA Expression of CCL18 in macrophages treated for 24 h with 20 ng/ml IL-4, 1 mM AICAR and 0.5 µM ABT-702. ( C ) mRNA Expression of SLC2A1 in macrophages treated for 24 h with 1 mM DMOG, 1 mM AICAR, and 0.5 µM ABT-702. ( D .

    Techniques Used: Expressing

    AICAR suppresses LPS transcriptional response. ( A , B ) mRNA expression of TNFα and IL-6 in macrophages treated for 3 h with 100 ng/ml LPS and indicated concentrations of AICAR ( A ) or with 100 ng/ml LPS, 0.1 mM AICAR and 0.5 µM ABT-702 ( B ). ( C ) mRNA expression of LPS-induced genes in macrophages treated for 1 h with 100 ng/ml LPS and 1 mM AICAR. ( D ) Cytokine secretion into culture medium of macrophages treated for 24 h with 100 ng/ml LPS and 1 mM AICAR. *p
    Figure Legend Snippet: AICAR suppresses LPS transcriptional response. ( A , B ) mRNA expression of TNFα and IL-6 in macrophages treated for 3 h with 100 ng/ml LPS and indicated concentrations of AICAR ( A ) or with 100 ng/ml LPS, 0.1 mM AICAR and 0.5 µM ABT-702 ( B ). ( C ) mRNA expression of LPS-induced genes in macrophages treated for 1 h with 100 ng/ml LPS and 1 mM AICAR. ( D ) Cytokine secretion into culture medium of macrophages treated for 24 h with 100 ng/ml LPS and 1 mM AICAR. *p

    Techniques Used: Expressing

    14) Product Images from "Exendin-4 improves ER stress-induced lipid accumulation and regulates lipin-1 signaling in HepG2 cells"

    Article Title: Exendin-4 improves ER stress-induced lipid accumulation and regulates lipin-1 signaling in HepG2 cells

    Journal: Cell Stress & Chaperones

    doi: 10.1007/s12192-017-0872-z

    Exendin-4 regulates lipin-1 signaling through SIRT1 and AMPK in HepG2 cells. a HepG2 cells were pretreated with 3 μg/ml tunicamycin (Tuni), followed by treatment with or without exendin-4 (Ex-4; 100 nM) for 24 h. SIRT1 and phosphorylated AMPK levels were analyzed by performing western blotting and were normalized to the lamin B1 and AMPK of each sample, respectively. b , c HepG2 cells were treated with 100 μM resveratrol (a SIRT1 agonist) or 2 mM AICAR (an AMPK agonist) for 24 h. d HepG2 cells transfected with 10 nM lipin-1 siRNA or control siRNA for 24 h. The mRNA expression levels of the genes encoding lipin-1, lipin-1α, lipin-1β, SIRT1, and AMPK were normalized to that of the β-actin gene. All values are expressed as the mean ± SE ( n = 4–6). * p
    Figure Legend Snippet: Exendin-4 regulates lipin-1 signaling through SIRT1 and AMPK in HepG2 cells. a HepG2 cells were pretreated with 3 μg/ml tunicamycin (Tuni), followed by treatment with or without exendin-4 (Ex-4; 100 nM) for 24 h. SIRT1 and phosphorylated AMPK levels were analyzed by performing western blotting and were normalized to the lamin B1 and AMPK of each sample, respectively. b , c HepG2 cells were treated with 100 μM resveratrol (a SIRT1 agonist) or 2 mM AICAR (an AMPK agonist) for 24 h. d HepG2 cells transfected with 10 nM lipin-1 siRNA or control siRNA for 24 h. The mRNA expression levels of the genes encoding lipin-1, lipin-1α, lipin-1β, SIRT1, and AMPK were normalized to that of the β-actin gene. All values are expressed as the mean ± SE ( n = 4–6). * p

    Techniques Used: Western Blot, Transfection, Expressing

    15) Product Images from "AMPK activation enhances the anti-atherogenic effects of high density lipoproteins in apoE−/− mice"

    Article Title: AMPK activation enhances the anti-atherogenic effects of high density lipoproteins in apoE−/− mice

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M073270

    AMPK activation enhances RCT in vivo and vitro. 3 H-cholesterol and acLDL-loaded J774 macrophages were injected into apoE −/− mice fed a high-fat diet (containing 1.2% cholesterol and 10% lard) and treated with vehicle, A-769662 (30 mg/kg, ip), AICAR (200 mg/kg, ip), metformin (260 mg/kg, by gavage), or IMM-H007 (200 mg/kg, by gavage) for 2 weeks, followed by radioactive counting in the serum (A), liver (B), and feces (C). D: Cholesterol efflux from cAMP pretreated J774.A1 macrophages was measured by 4 h incubation of labeled cells to AMPK activator-treated PEG-HDL. Data are mean ± SEM for n = 6 per group. * P
    Figure Legend Snippet: AMPK activation enhances RCT in vivo and vitro. 3 H-cholesterol and acLDL-loaded J774 macrophages were injected into apoE −/− mice fed a high-fat diet (containing 1.2% cholesterol and 10% lard) and treated with vehicle, A-769662 (30 mg/kg, ip), AICAR (200 mg/kg, ip), metformin (260 mg/kg, by gavage), or IMM-H007 (200 mg/kg, by gavage) for 2 weeks, followed by radioactive counting in the serum (A), liver (B), and feces (C). D: Cholesterol efflux from cAMP pretreated J774.A1 macrophages was measured by 4 h incubation of labeled cells to AMPK activator-treated PEG-HDL. Data are mean ± SEM for n = 6 per group. * P

    Techniques Used: Activation Assay, In Vivo, Injection, Mouse Assay, Incubation, Labeling

    16) Product Images from "Crystal Structure and Function of 5-Formaminoimidazole-4-carboxamide-1-?-d-ribofuranosyl 5?-Monophosphate Synthetase from Methanocaldococcus jannaschii † † , ‡"

    Article Title: Crystal Structure and Function of 5-Formaminoimidazole-4-carboxamide-1-?-d-ribofuranosyl 5?-Monophosphate Synthetase from Methanocaldococcus jannaschii † † , ‡

    Journal:

    doi: 10.1021/bi701406g

    Modeled formylphosphate intermediate for the FAICAR synthetase reaction at the Mj PurP active site. The intermediates were modeled to optimize the reaction trajectories while using the observed AICAR/FAICAR binding sites as a constraint.
    Figure Legend Snippet: Modeled formylphosphate intermediate for the FAICAR synthetase reaction at the Mj PurP active site. The intermediates were modeled to optimize the reaction trajectories while using the observed AICAR/FAICAR binding sites as a constraint.

    Techniques Used: Binding Assay

    The active site of Mj PurP. (A) Fo – Fc density contoured at 3 σ around AICAR and the chloride anion (green) of the AICAR-ATP structure. (B) The stereodiagram of the AICAR binding site for the AICAR-ATP structure. The γ-phosphate
    Figure Legend Snippet: The active site of Mj PurP. (A) Fo – Fc density contoured at 3 σ around AICAR and the chloride anion (green) of the AICAR-ATP structure. (B) The stereodiagram of the AICAR binding site for the AICAR-ATP structure. The γ-phosphate

    Techniques Used: Binding Assay

    17) Product Images from "Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1"

    Article Title: Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1

    Journal: Journal of Molecular Signaling

    doi: 10.1186/1750-2187-3-18

    Downregulation of cyclin D1 corresponds to activation of AMP-activated protein kinase . A . MCF7 cells were treated with (Met) or without (Con) metformin for 1.5 days and then western blotting was performed to detect active phospho-AMPK, cyclin D1, and β-actin. B . MCF7 cells were treated with antimycin A (AM, 1 μM), AICAR (4 mM), or metformin (8 mM) for 1.5 days. Control cells (Con) were treated with the appropriate vehicle for each reagent. Western blotting was performed to detect active phospho-AMPK, cyclin D1, and β-actin. C . MCF7 cells were pretreated with DMSO (vehicle) or the AMPK-specific inhibitor compound C (20 μM) for 1 day and then treated with (Met) or without (Con) metformin for 1.5 days. Western blotting was performed to detect cyclin D1 or the phosphorylated form of the AMPK substrate ACC. β-actin was used as a loading control.
    Figure Legend Snippet: Downregulation of cyclin D1 corresponds to activation of AMP-activated protein kinase . A . MCF7 cells were treated with (Met) or without (Con) metformin for 1.5 days and then western blotting was performed to detect active phospho-AMPK, cyclin D1, and β-actin. B . MCF7 cells were treated with antimycin A (AM, 1 μM), AICAR (4 mM), or metformin (8 mM) for 1.5 days. Control cells (Con) were treated with the appropriate vehicle for each reagent. Western blotting was performed to detect active phospho-AMPK, cyclin D1, and β-actin. C . MCF7 cells were pretreated with DMSO (vehicle) or the AMPK-specific inhibitor compound C (20 μM) for 1 day and then treated with (Met) or without (Con) metformin for 1.5 days. Western blotting was performed to detect cyclin D1 or the phosphorylated form of the AMPK substrate ACC. β-actin was used as a loading control.

    Techniques Used: Activation Assay, Western Blot

    18) Product Images from "Sucrose non-fermenting related kinase enzyme is essential for cardiac metabolism"

    Article Title: Sucrose non-fermenting related kinase enzyme is essential for cardiac metabolism

    Journal: Biology Open

    doi: 10.1242/bio.20149811

    Cardiomyocyte-specific loss of SNRK results in cellular metabolic defects in vitro. The expression of ACC, pACC, AMPKα and pAMPKα in hESC-derived CMs infected with empty vector shRNA control (Control) lentivirus and SNRK shRNA lentivirus (shSNRK) was determined by western blotting (A) followed by densitometry quantitation (B,C). The CMs were treated with either DMSO, 2 mM Metformin or 1 mM AICAR. The results are the mean ± SEM of the ratio of the phosphorylated protein normalized to the total protein. pACC/ACC ratio: DMSO Control 100%, shSNRK 35.03%, p = 0.0002, n = 4; Metformin Control 100%, shSNRK 126.27%, p = 0.5, n = 4; AICAR Control 100%, shSNRK 52.41%, p = 0.017, n = 5. pAMPK/AMPK ratio: DMSO Control 100%, shSNRK 66.40%, p = 0.036, n = 4; Metformin Control 100%, shSNRK 82.29%, p = 0.261, n = 4; AICAR Control 100%, shSNRK 100.63%, p = 0.983, n = 4. (D,E) SNRK knockdown in CM results in a decrease in the oxygen consumption rate (OCR) displayed as Basal respiration, ATP production and Maximal Respiration. The CM OCR rate was assessed in basal medium containing bovine serum albumin (BSA) (Basal Respiration Control 100%, shSNRK 84.71%, p = 0.074, n = 3; ATP Production Control 100%, shSNRK 91.57%, p = 0.402, n = 3; Maximal Control 100%, shSNRK 84.69%, p = 0.103, n = 3) (D) or basal medium containing the FAO substrate (BSA-Palmitate) (Basal Control 100%, shSNRK 53.60%, p = 0.077, n = 3; ATP Production Control 100%, shSNRK 65.64%, p = 0.146, n = 3; Maximal Control 100%, shSNRK 48.30%, p = 0.073, n = 3) (E). The results are the mean ± SEM of the percentage of the OCR rate normalized to total protein. * p-value
    Figure Legend Snippet: Cardiomyocyte-specific loss of SNRK results in cellular metabolic defects in vitro. The expression of ACC, pACC, AMPKα and pAMPKα in hESC-derived CMs infected with empty vector shRNA control (Control) lentivirus and SNRK shRNA lentivirus (shSNRK) was determined by western blotting (A) followed by densitometry quantitation (B,C). The CMs were treated with either DMSO, 2 mM Metformin or 1 mM AICAR. The results are the mean ± SEM of the ratio of the phosphorylated protein normalized to the total protein. pACC/ACC ratio: DMSO Control 100%, shSNRK 35.03%, p = 0.0002, n = 4; Metformin Control 100%, shSNRK 126.27%, p = 0.5, n = 4; AICAR Control 100%, shSNRK 52.41%, p = 0.017, n = 5. pAMPK/AMPK ratio: DMSO Control 100%, shSNRK 66.40%, p = 0.036, n = 4; Metformin Control 100%, shSNRK 82.29%, p = 0.261, n = 4; AICAR Control 100%, shSNRK 100.63%, p = 0.983, n = 4. (D,E) SNRK knockdown in CM results in a decrease in the oxygen consumption rate (OCR) displayed as Basal respiration, ATP production and Maximal Respiration. The CM OCR rate was assessed in basal medium containing bovine serum albumin (BSA) (Basal Respiration Control 100%, shSNRK 84.71%, p = 0.074, n = 3; ATP Production Control 100%, shSNRK 91.57%, p = 0.402, n = 3; Maximal Control 100%, shSNRK 84.69%, p = 0.103, n = 3) (D) or basal medium containing the FAO substrate (BSA-Palmitate) (Basal Control 100%, shSNRK 53.60%, p = 0.077, n = 3; ATP Production Control 100%, shSNRK 65.64%, p = 0.146, n = 3; Maximal Control 100%, shSNRK 48.30%, p = 0.073, n = 3) (E). The results are the mean ± SEM of the percentage of the OCR rate normalized to total protein. * p-value

    Techniques Used: In Vitro, Expressing, Derivative Assay, Infection, Plasmid Preparation, shRNA, Western Blot, Quantitation Assay

    19) Product Images from "Fanconi anemia protein FANCD2 is activated by AICAR, a modulator of AMPK and cellular energy metabolism"

    Article Title: Fanconi anemia protein FANCD2 is activated by AICAR, a modulator of AMPK and cellular energy metabolism

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12185

    AMPK ‐activating AICAR treatment activates FANCD 2, a pivotal molecule of Fanconi anemia DNA damage signaling pathway. (A) AICAR treatment induces FANCD 2 monoubiquitination in transformed normal fibroblasts ( GM 00637I). GM 00637I cells were treated with 1 m m 2‐deoxyglucose, 0.25 mM AICAR , or 1 m m phenformin for 24 h. Lysates were subjected to western blotting with anti‐ FANCD 2, phospho‐ AMPK α1 (T172), and AMPK α and β‐actin. In FANCD 2 blots, the position of monoubiquitinated FANCD 2 (Ub‐ FANCD 2) is indicated by an arrow. (B) AICAR treatment induces formation of FANCD 2 nuclear foci in GM 00637I fibroblasts. Cells grown on coverslips in 12‐well plates were treated with 0.25 m m AICAR for 24 h. Cells were immunostained with FANCD 2 antibody and Alexa 488‐conjugated anti‐rabbit secondary antibody. FANCD 2 foci were visualized by confocal microscopy. Representative images are shown at the top. The number of foci per cell was counted and plotted for ≥ 20 cells (bottom panel). The values represent the mean ± SEM (Student's t ‐test, *** P
    Figure Legend Snippet: AMPK ‐activating AICAR treatment activates FANCD 2, a pivotal molecule of Fanconi anemia DNA damage signaling pathway. (A) AICAR treatment induces FANCD 2 monoubiquitination in transformed normal fibroblasts ( GM 00637I). GM 00637I cells were treated with 1 m m 2‐deoxyglucose, 0.25 mM AICAR , or 1 m m phenformin for 24 h. Lysates were subjected to western blotting with anti‐ FANCD 2, phospho‐ AMPK α1 (T172), and AMPK α and β‐actin. In FANCD 2 blots, the position of monoubiquitinated FANCD 2 (Ub‐ FANCD 2) is indicated by an arrow. (B) AICAR treatment induces formation of FANCD 2 nuclear foci in GM 00637I fibroblasts. Cells grown on coverslips in 12‐well plates were treated with 0.25 m m AICAR for 24 h. Cells were immunostained with FANCD 2 antibody and Alexa 488‐conjugated anti‐rabbit secondary antibody. FANCD 2 foci were visualized by confocal microscopy. Representative images are shown at the top. The number of foci per cell was counted and plotted for ≥ 20 cells (bottom panel). The values represent the mean ± SEM (Student's t ‐test, *** P

    Techniques Used: Transformation Assay, Western Blot, Confocal Microscopy

    20) Product Images from "Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells"

    Article Title: Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04609-7

    Impact of small-molecule combinations on targeted nucleotide substitution (TNS) efficiency in iPSCs and hESCs. Shown are TNS efficiencies in CALD1 , KATNA1 , and SLITRK1 with Cas9n and Cas9, and in HPRT and DNMT1 with Cpf1. Small molecules have an additive effect on TNS efficiency with Cas9n ( a ) but not with Cas9 ( b ) in the 409-B2 iCRISPR hiPSC lines. TNS of HPRT and DNMT1 in 409-B2 hiPSCs with recombinant Cpf1 was increased using the CRISPY mix as well ( c ). Using the CRISPY mix, TNS efficiency was also increased in SC102 A1 hiPSCs and H9 hESCs with plasmid-delivered Cas9n-2A-GFP (GFP-FACS enriched), and in chimpanzee SandraA ciPSCs with recombinant Cpf1 ( d ). Shown are TNS, TNS + indels, and indels with green, gray, or blue bars, respectively. Error bars show the SD of three technical replicates for a , b , and c , and two technical replicates for d . Concentrations used were 20 µM of NU7026, 0.01 µM of Trichostatin A, 0.5 µM MLN4924, 1 µM NSC 19630, 5 µM NSC 15520, 20 µM AICAR, and 1 µM RS-1. CRISPY mix indicates a small-molecule mix of NU7026, Trichostatin A, MLN4924, and NSC 15520. Statistical significances of TNS efficiency changes was determined using a two-way ANOVA and Tukey’s multiple comparison pooled across the three genes CALD1 , KATNA1 , and SLITRK1 . Genes and treatments were treated as random and fixed effect, respectively. P -values are adjusted for multiple comparison (** P ≤ 0.01, *** P ≤ 0.001). Overall, there was a clear treatment effect (F(12, 24) = 32.954, P ≤ 0.001)
    Figure Legend Snippet: Impact of small-molecule combinations on targeted nucleotide substitution (TNS) efficiency in iPSCs and hESCs. Shown are TNS efficiencies in CALD1 , KATNA1 , and SLITRK1 with Cas9n and Cas9, and in HPRT and DNMT1 with Cpf1. Small molecules have an additive effect on TNS efficiency with Cas9n ( a ) but not with Cas9 ( b ) in the 409-B2 iCRISPR hiPSC lines. TNS of HPRT and DNMT1 in 409-B2 hiPSCs with recombinant Cpf1 was increased using the CRISPY mix as well ( c ). Using the CRISPY mix, TNS efficiency was also increased in SC102 A1 hiPSCs and H9 hESCs with plasmid-delivered Cas9n-2A-GFP (GFP-FACS enriched), and in chimpanzee SandraA ciPSCs with recombinant Cpf1 ( d ). Shown are TNS, TNS + indels, and indels with green, gray, or blue bars, respectively. Error bars show the SD of three technical replicates for a , b , and c , and two technical replicates for d . Concentrations used were 20 µM of NU7026, 0.01 µM of Trichostatin A, 0.5 µM MLN4924, 1 µM NSC 19630, 5 µM NSC 15520, 20 µM AICAR, and 1 µM RS-1. CRISPY mix indicates a small-molecule mix of NU7026, Trichostatin A, MLN4924, and NSC 15520. Statistical significances of TNS efficiency changes was determined using a two-way ANOVA and Tukey’s multiple comparison pooled across the three genes CALD1 , KATNA1 , and SLITRK1 . Genes and treatments were treated as random and fixed effect, respectively. P -values are adjusted for multiple comparison (** P ≤ 0.01, *** P ≤ 0.001). Overall, there was a clear treatment effect (F(12, 24) = 32.954, P ≤ 0.001)

    Techniques Used: Recombinant, Plasmid Preparation, FACS

    Small molecules described or anticipated to target key proteins of NHEJ and HDR. Proteins are labeled with black text and inhibitors and enhancing small molecules are marked red and green, respectively. STL127705, NU7026, or SCR7 have been described to inhibit Ku70/80, DNA-PK, or DNA ligase IV, respectively. MLN4924, RS-1, Trichostatin A, or Resveratrol have been described to enhance CtIP, RAD51, or ATM, respectively. NSC 15520 has been described to block the association of RPA to p53 and RAD9. AICAR is an inhibitor of RAD52 and B02 is an ihibitor of RAD51. For simplicity, some proteins and protein interactions are not depicted
    Figure Legend Snippet: Small molecules described or anticipated to target key proteins of NHEJ and HDR. Proteins are labeled with black text and inhibitors and enhancing small molecules are marked red and green, respectively. STL127705, NU7026, or SCR7 have been described to inhibit Ku70/80, DNA-PK, or DNA ligase IV, respectively. MLN4924, RS-1, Trichostatin A, or Resveratrol have been described to enhance CtIP, RAD51, or ATM, respectively. NSC 15520 has been described to block the association of RPA to p53 and RAD9. AICAR is an inhibitor of RAD52 and B02 is an ihibitor of RAD51. For simplicity, some proteins and protein interactions are not depicted

    Techniques Used: Non-Homologous End Joining, Labeling, Blocking Assay, Recombinase Polymerase Amplification

    21) Product Images from "Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation"

    Article Title: Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation

    Journal: Journal of Clinical Investigation

    doi: 10.1172/JCI25256

    Effect of PUFAs and AICAR on AMPK activation and ChREBP localization. In vitro studies: After plating, hepatocytes were cultured for 24 hours in the presence of 5 mM glucose and 100 nM insulin. ( A ) Hepatocytes were then incubated with 25 mM glucose and 100 nM insulin in the presence or not of 0.3 mM of albumin-bound linoleate or 500 μM AICAR. After 1, 2, 6, and 10 hours, cytosolic form of phospho-AMPK and nuclear forms of ChREBP were measured. ( B ) After a 6-hour incubation period with 25 mM glucose, 100 nM insulin, and 0.3 mM of albumin-bound stearate (C18), oleate [C18:1 (n-9)], linoleate [C18:2 (n-6)], EPA [C20:5 (n-3)], DHA [C22:6 (n-3)], or 500 μM AICAR, the cytosolic form of P-AMPK protein was measured. Representative Western blots of 4 independent cultures are shown. In vivo studies: ( C ) The phosphorylation status of AMPK was measured in liver extracts from 24 hour–fasted mice refed for 3 hours on a HCHO or a HCHO-PUFA diet. n = 4/group. ( D ) Cytosolic and nuclear ChREBP content from livers of 24 hour–fasted control and AMPKα1 –/– or AMPKα2 –/– (α1 –/– and α2 –/– ) mice refed 18 hours upon HCHO diet supplemented or not with PUFAs. A representative Western blot is shown; n = 3/group. ( E ) RTQ-PCR analysis of L-PK and FAS genes from livers of 24 hour–fasted mice and mice refed 18 hours on a HCHO or HCHO-PUFA diet were performed in control and AMPKα1 –/– or AMPKα2 –/– mice. Results are the mean ± SEM; n = 3/group. *Significantly different from mice refed a HCHO diet for 18 hours ( P
    Figure Legend Snippet: Effect of PUFAs and AICAR on AMPK activation and ChREBP localization. In vitro studies: After plating, hepatocytes were cultured for 24 hours in the presence of 5 mM glucose and 100 nM insulin. ( A ) Hepatocytes were then incubated with 25 mM glucose and 100 nM insulin in the presence or not of 0.3 mM of albumin-bound linoleate or 500 μM AICAR. After 1, 2, 6, and 10 hours, cytosolic form of phospho-AMPK and nuclear forms of ChREBP were measured. ( B ) After a 6-hour incubation period with 25 mM glucose, 100 nM insulin, and 0.3 mM of albumin-bound stearate (C18), oleate [C18:1 (n-9)], linoleate [C18:2 (n-6)], EPA [C20:5 (n-3)], DHA [C22:6 (n-3)], or 500 μM AICAR, the cytosolic form of P-AMPK protein was measured. Representative Western blots of 4 independent cultures are shown. In vivo studies: ( C ) The phosphorylation status of AMPK was measured in liver extracts from 24 hour–fasted mice refed for 3 hours on a HCHO or a HCHO-PUFA diet. n = 4/group. ( D ) Cytosolic and nuclear ChREBP content from livers of 24 hour–fasted control and AMPKα1 –/– or AMPKα2 –/– (α1 –/– and α2 –/– ) mice refed 18 hours upon HCHO diet supplemented or not with PUFAs. A representative Western blot is shown; n = 3/group. ( E ) RTQ-PCR analysis of L-PK and FAS genes from livers of 24 hour–fasted mice and mice refed 18 hours on a HCHO or HCHO-PUFA diet were performed in control and AMPKα1 –/– or AMPKα2 –/– mice. Results are the mean ± SEM; n = 3/group. *Significantly different from mice refed a HCHO diet for 18 hours ( P

    Techniques Used: Activation Assay, In Vitro, Cell Culture, Incubation, Western Blot, In Vivo, Mouse Assay, Polymerase Chain Reaction

    22) Product Images from "The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells"

    Article Title: The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells

    Journal: Nature

    doi: 10.1038/s41586-018-0282-0

    P2RX7 ablation leads to aberrant metabolism and depressed AMPK activation in CD8 + T cells (a–d) In vitro activated WT and P2rx7 −/− P14 were activated then polarized with IL-2 or IL-15 (cells pooled from 2 mice/experiment). (a) Cell viability during IL-2 or IL-15 cultures (p≥0.2 for all IL-2 polarized cells and IL-15 polarized at 24h). (b,c) OCR (b) and calculated SRC (c) for IL-2 or IL-15-polarized cells. (d) electron microscopy of mitochondria (arrows) in IL-15-polarized WT or P2rx7 −/− P14 (black bars =500nm). (e–k) mouse (e,i–k) or human (f–h) CD8 + T cells were stimulated in vitro in the presence of A-438079 (e–h) , BzATP (i) , Probenecid (j,k) , or vehicle controls. Mouse cells activated as in ( a ), human cells assayed 72h post-stimulation. OCR (e,f,i,j) and SRC (k) were measured and human cells assayed for proliferation (Ki67) (g) and Granzyme B/IFN-γ (h). (l) pACC in IL-15-polarized WT and P2rx7 −/− P14 (representative flow cytometric histograms and median average values (right). In (m) , cells were incubated (6h) with/without AICAR prior to OCR measurement. (n–o) Mice receiving co-transferred WT and P2rx7 −/− P14 were LCMV-infected primed and treated with metformin (1–7 dpi; n) or rapamycin (4–8 dpi; o) . P2rx7 −/− /WT ratios for splenic memory subsets (n) and P14 T CM numbers were determined ( o ). (a–o) Three independent experiments, n=3–6 ( a ), 8–9 ( b–c ), 6 ( d,g–h,l–m ), 4–5 ( e,i ), 4 ( f ), 5–6 ( j–k ), 10–11 ( n ), 12–17 ( o ) total. (a–c,e–o) Mean ± SEM; (a,c,g–h,l,n) , Two-tailed Student’s t-test; (k,o) One-way ANOVA + Tukey post-test, *P≤0.05, **P≤0.01, ***P≤0.001.
    Figure Legend Snippet: P2RX7 ablation leads to aberrant metabolism and depressed AMPK activation in CD8 + T cells (a–d) In vitro activated WT and P2rx7 −/− P14 were activated then polarized with IL-2 or IL-15 (cells pooled from 2 mice/experiment). (a) Cell viability during IL-2 or IL-15 cultures (p≥0.2 for all IL-2 polarized cells and IL-15 polarized at 24h). (b,c) OCR (b) and calculated SRC (c) for IL-2 or IL-15-polarized cells. (d) electron microscopy of mitochondria (arrows) in IL-15-polarized WT or P2rx7 −/− P14 (black bars =500nm). (e–k) mouse (e,i–k) or human (f–h) CD8 + T cells were stimulated in vitro in the presence of A-438079 (e–h) , BzATP (i) , Probenecid (j,k) , or vehicle controls. Mouse cells activated as in ( a ), human cells assayed 72h post-stimulation. OCR (e,f,i,j) and SRC (k) were measured and human cells assayed for proliferation (Ki67) (g) and Granzyme B/IFN-γ (h). (l) pACC in IL-15-polarized WT and P2rx7 −/− P14 (representative flow cytometric histograms and median average values (right). In (m) , cells were incubated (6h) with/without AICAR prior to OCR measurement. (n–o) Mice receiving co-transferred WT and P2rx7 −/− P14 were LCMV-infected primed and treated with metformin (1–7 dpi; n) or rapamycin (4–8 dpi; o) . P2rx7 −/− /WT ratios for splenic memory subsets (n) and P14 T CM numbers were determined ( o ). (a–o) Three independent experiments, n=3–6 ( a ), 8–9 ( b–c ), 6 ( d,g–h,l–m ), 4–5 ( e,i ), 4 ( f ), 5–6 ( j–k ), 10–11 ( n ), 12–17 ( o ) total. (a–c,e–o) Mean ± SEM; (a,c,g–h,l,n) , Two-tailed Student’s t-test; (k,o) One-way ANOVA + Tukey post-test, *P≤0.05, **P≤0.01, ***P≤0.001.

    Techniques Used: Activation Assay, In Vitro, Mouse Assay, Electron Microscopy, Flow Cytometry, Incubation, Infection, Two Tailed Test

    P2RX7-mediated eATP sensing is crucial for optimal CD8 + T cell immunometabolism via AMPK/mTOR pathway regulation (a–g) , WT and P2rx7 −/− P14 cells were in vitro activated and polarized with IL-15 (as in Fig. 3b ). Data from three independent experiments, samples pooled from n=6 mice per experiment; n=3–12 total samples. (a–c) show the numbers of P14 cells (left) and viability (right) in cultures supplemented with (a) the eATP hydrolytic enzyme Apyrase, (b) the inhibitor oATP or (c) the eATP analog BzATP, during cell culture. In (d,e,g) IL-15-polarized WT or P2rx7 −/− P14 cells were assayed for OCR 1h after addition of apyrase (d) or oATP (e) , or 6h after addition of A-438079 (g) . In (f) , IL-15-polarized cells or ex vivo WT P14 T CM (isolated 4w after LCMV infection) were incubated with either DAPI (left) or Indo-1 (right) and stimulated with the indicated concentrations of Bz-ATP during kinetic flow cytometric analysis. The percentage of cells showing DAPI uptake (left) or Ca 2+ influx (right) over 30m are shown. (f) Data from two independent experiments, samples pooled from n=5 mice total; n=2–5 samples. In (h) , in vitro -activated (72h) WT and P2rx7 −/− P14 cells were assayed for intracellular ATP concentrations. Data from three independent experiments, n=9 total. In (i), In vitro -activated, IL-15 polarized (24h post-polarization) WT or P2rx7 −/− P14 cells were assayed for extracellular ATP concentration, following culture without or with the Panx1 inhibitor 10 Panx. Data from two independent experiments, n=3–4 total samples (pooled from 6 mice). (j) , WT and P2rx7 −/− P14 cells were co-adoptively transferred and assayed 4w post LCMV infection (as in Fig. 1a ) and the ex vivo frequency of pS6-expressing cells determined by flow cytometry. Data are from two independent experiments (n=6 total). (k) Representative histograms showing expression of pACC in IL-15-polarized WT (black) and P2rx7 −/− (red) P14 cells (relative to Fig. 3l ; representative from three independent experiments, n=6 total). (l) In vitro activated and IL-15-polarized WT and P2rx7 −/− P14 cells were cultured for 6h with the indicated concentrations of BzATP then stained for pACC (left) and pS6 (center) and the pACC/pS6 ratio was determined (right). Data from three independent experiments, n=6–8 total. (m) In vitro -activated WT and P2rx7 −/− P14 cells were IL-15 polarized in presence or absence of the AMPK activator AICAR as in Fig. 3l . The percentage of viable cells at the indicated times following initiation of IL-15 +/− AICAR culture is indicated. Data are from three independent experiments (n=3–6 total; samples pooled from n=6 mice total). (n–p) WT and P2rx7 −/− P14 CD8 + T cells were mixed 1:1, co-adoptively transferred into B6.SJL mice that were subsequently infected with LCMV, and donor cells identified as in Fig. 1 . The animals were treated with Metformin or PBS control during the first week of LCMV infection, and the cells analyzed at day 30. Data are compiled from three independent experiments (n=11–12 total, n=4 for FRT samples). Panel (n) relates to Fig. 3m and shows P2rx7 −/− /WT P14 ratio in indicated non-lymphoid tissues (n=9 except female reproductive tract – FRT – where n=4). (o,p) shows measurements of mitochondrial mass (measured by MTG) (o) and mitochondrial membrane potential (measured by TMRE staining, normalized to MTG staining) (p) for indicated splenocyte subsets (n=3–6 total samples). (q) WT and P2rx7 −/− P14 CD8 + T cells were mixed 1:1, co-adoptively transferred into B6.SJL mice that were subsequently infected with LCMV, and donor cells identified as in Fig. 1 . The animals were treated with Rapamycin or PBS control between days 4–8 post-LCMV infection, and the cells analyzed at day 30. The numbers of WT or P2rx7 −/− P14 cells are shown (log-transformed values). Data are compiled from three independent experiments (n=15 total). (a–j,l–q) , mean ± SEM is shown; (a–c,h–j,m–p) Two-tailed Student’s t-test; (l,q) One-way ANOVA with Tukey’s post-test; *P≤0.05, **P≤0.01, ***P≤0.001.
    Figure Legend Snippet: P2RX7-mediated eATP sensing is crucial for optimal CD8 + T cell immunometabolism via AMPK/mTOR pathway regulation (a–g) , WT and P2rx7 −/− P14 cells were in vitro activated and polarized with IL-15 (as in Fig. 3b ). Data from three independent experiments, samples pooled from n=6 mice per experiment; n=3–12 total samples. (a–c) show the numbers of P14 cells (left) and viability (right) in cultures supplemented with (a) the eATP hydrolytic enzyme Apyrase, (b) the inhibitor oATP or (c) the eATP analog BzATP, during cell culture. In (d,e,g) IL-15-polarized WT or P2rx7 −/− P14 cells were assayed for OCR 1h after addition of apyrase (d) or oATP (e) , or 6h after addition of A-438079 (g) . In (f) , IL-15-polarized cells or ex vivo WT P14 T CM (isolated 4w after LCMV infection) were incubated with either DAPI (left) or Indo-1 (right) and stimulated with the indicated concentrations of Bz-ATP during kinetic flow cytometric analysis. The percentage of cells showing DAPI uptake (left) or Ca 2+ influx (right) over 30m are shown. (f) Data from two independent experiments, samples pooled from n=5 mice total; n=2–5 samples. In (h) , in vitro -activated (72h) WT and P2rx7 −/− P14 cells were assayed for intracellular ATP concentrations. Data from three independent experiments, n=9 total. In (i), In vitro -activated, IL-15 polarized (24h post-polarization) WT or P2rx7 −/− P14 cells were assayed for extracellular ATP concentration, following culture without or with the Panx1 inhibitor 10 Panx. Data from two independent experiments, n=3–4 total samples (pooled from 6 mice). (j) , WT and P2rx7 −/− P14 cells were co-adoptively transferred and assayed 4w post LCMV infection (as in Fig. 1a ) and the ex vivo frequency of pS6-expressing cells determined by flow cytometry. Data are from two independent experiments (n=6 total). (k) Representative histograms showing expression of pACC in IL-15-polarized WT (black) and P2rx7 −/− (red) P14 cells (relative to Fig. 3l ; representative from three independent experiments, n=6 total). (l) In vitro activated and IL-15-polarized WT and P2rx7 −/− P14 cells were cultured for 6h with the indicated concentrations of BzATP then stained for pACC (left) and pS6 (center) and the pACC/pS6 ratio was determined (right). Data from three independent experiments, n=6–8 total. (m) In vitro -activated WT and P2rx7 −/− P14 cells were IL-15 polarized in presence or absence of the AMPK activator AICAR as in Fig. 3l . The percentage of viable cells at the indicated times following initiation of IL-15 +/− AICAR culture is indicated. Data are from three independent experiments (n=3–6 total; samples pooled from n=6 mice total). (n–p) WT and P2rx7 −/− P14 CD8 + T cells were mixed 1:1, co-adoptively transferred into B6.SJL mice that were subsequently infected with LCMV, and donor cells identified as in Fig. 1 . The animals were treated with Metformin or PBS control during the first week of LCMV infection, and the cells analyzed at day 30. Data are compiled from three independent experiments (n=11–12 total, n=4 for FRT samples). Panel (n) relates to Fig. 3m and shows P2rx7 −/− /WT P14 ratio in indicated non-lymphoid tissues (n=9 except female reproductive tract – FRT – where n=4). (o,p) shows measurements of mitochondrial mass (measured by MTG) (o) and mitochondrial membrane potential (measured by TMRE staining, normalized to MTG staining) (p) for indicated splenocyte subsets (n=3–6 total samples). (q) WT and P2rx7 −/− P14 CD8 + T cells were mixed 1:1, co-adoptively transferred into B6.SJL mice that were subsequently infected with LCMV, and donor cells identified as in Fig. 1 . The animals were treated with Rapamycin or PBS control between days 4–8 post-LCMV infection, and the cells analyzed at day 30. The numbers of WT or P2rx7 −/− P14 cells are shown (log-transformed values). Data are compiled from three independent experiments (n=15 total). (a–j,l–q) , mean ± SEM is shown; (a–c,h–j,m–p) Two-tailed Student’s t-test; (l,q) One-way ANOVA with Tukey’s post-test; *P≤0.05, **P≤0.01, ***P≤0.001.

    Techniques Used: In Vitro, Mouse Assay, Cell Culture, Ex Vivo, Isolation, Infection, Incubation, Flow Cytometry, Concentration Assay, Expressing, Cytometry, Staining, Transformation Assay, Two Tailed Test

    23) Product Images from "LKB1 loss promotes endometrial cancer progression via CCL2-dependent macrophage recruitment"

    Article Title: LKB1 loss promotes endometrial cancer progression via CCL2-dependent macrophage recruitment

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI82152

    LKB1 suppresses CCL2 production by human EM cells via an AMPK-dependent mechanism. ( A ) Human CCL2 ELISA of conditioned media harvested 24 hours after plating of EM cells previously transduced with lentivirus. LKB1 knockdown led to a significant increase of CCL2 in the media ( n = 3 biological replicates per experiment). ( B ) Human CCL2 ELISA on conditioned media containing AICAR (0.5 mM), metformin (5 mM), or vehicle (PBS) only. AICAR or metformin significantly reduced CCL2 secretion 24 hours after addition of drug ( n = 3 biological replicates). ( C ) Representative Western blot of lysates from cells shown in panel B revealing partial restoration of pAMPK levels. ( D ) Human CCL2 ELISA on conditioned media harvested 24 hours after plating cells transduced with control or AMPKα1/2-shRNA lentivirus. AMPK knockdown led to a significant increase in CCL2 ( n = 3 biological replicates per experiment). ( E ) Representative Western blot of lysates from cells shown in D showing knockdown of AMPK and undetectable pAMPK levels. * P
    Figure Legend Snippet: LKB1 suppresses CCL2 production by human EM cells via an AMPK-dependent mechanism. ( A ) Human CCL2 ELISA of conditioned media harvested 24 hours after plating of EM cells previously transduced with lentivirus. LKB1 knockdown led to a significant increase of CCL2 in the media ( n = 3 biological replicates per experiment). ( B ) Human CCL2 ELISA on conditioned media containing AICAR (0.5 mM), metformin (5 mM), or vehicle (PBS) only. AICAR or metformin significantly reduced CCL2 secretion 24 hours after addition of drug ( n = 3 biological replicates). ( C ) Representative Western blot of lysates from cells shown in panel B revealing partial restoration of pAMPK levels. ( D ) Human CCL2 ELISA on conditioned media harvested 24 hours after plating cells transduced with control or AMPKα1/2-shRNA lentivirus. AMPK knockdown led to a significant increase in CCL2 ( n = 3 biological replicates per experiment). ( E ) Representative Western blot of lysates from cells shown in D showing knockdown of AMPK and undetectable pAMPK levels. * P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transduction, Western Blot, shRNA

    24) Product Images from "Protein Kinase C Epsilon Promotes Cerebral Ischemic Tolerance Via Modulation of Mitochondrial Sirt5"

    Article Title: Protein Kinase C Epsilon Promotes Cerebral Ischemic Tolerance Via Modulation of Mitochondrial Sirt5

    Journal: Scientific Reports

    doi: 10.1038/srep29790

    Nampt and Sirtuin Activity are Downstream of PKCε-Mediated Neuroprotection. Bar graphs representing neuronal cell death measured by LDH release at 48 hours of reperfusion after lethal OGD from rat neuronal-astrocyte cultures. ( a ) IPC, resveratrol (Resv) (25 μM), and the AMPK activator AICAR (0.5 mM)–mediated neuroprotection is blocked upon exposure to εV1–2 (100 nM), an inhibitor of PKCε activity. ( b ) ΨεRACK treatment provided significant protection against OGD-induced neuronal death which was blocked with the Nampt inhibitor FK866 (25 nM) or pan-sirtuin inhibitor sirtinol (10 μM). **p
    Figure Legend Snippet: Nampt and Sirtuin Activity are Downstream of PKCε-Mediated Neuroprotection. Bar graphs representing neuronal cell death measured by LDH release at 48 hours of reperfusion after lethal OGD from rat neuronal-astrocyte cultures. ( a ) IPC, resveratrol (Resv) (25 μM), and the AMPK activator AICAR (0.5 mM)–mediated neuroprotection is blocked upon exposure to εV1–2 (100 nM), an inhibitor of PKCε activity. ( b ) ΨεRACK treatment provided significant protection against OGD-induced neuronal death which was blocked with the Nampt inhibitor FK866 (25 nM) or pan-sirtuin inhibitor sirtinol (10 μM). **p

    Techniques Used: Activity Assay

    25) Product Images from "Use of Cells Expressing ? Subunit Variants to Identify Diverse Mechanisms of AMPK Activation"

    Article Title: Use of Cells Expressing ? Subunit Variants to Identify Diverse Mechanisms of AMPK Activation

    Journal: Cell Metabolism

    doi: 10.1016/j.cmet.2010.04.001

    Phosphorylation of Thr-172 on AMPK-α in WT or RG Cells in Response to Various Agents Cells were incubated in duplicate for 1 hr (except metformin, 16 hr) with the indicated agent at the following concentrations: oligomycin (1 μM), DNP (100 μM), 2-deoxyglucose (30 mM), AICAR (3 mM), hydrogen peroxide (1 mM), sorbitol (1.5 M), A23187 (3 μM), A769662 (300 μM), metformin (3 mM), phenformin (3 mM), galegine (100 μM), troglitazone (100 μM), phenobarbital (3 mM), quercetin (100 μM), resveratrol (300 μM), and berberine (100 μM).
    Figure Legend Snippet: Phosphorylation of Thr-172 on AMPK-α in WT or RG Cells in Response to Various Agents Cells were incubated in duplicate for 1 hr (except metformin, 16 hr) with the indicated agent at the following concentrations: oligomycin (1 μM), DNP (100 μM), 2-deoxyglucose (30 mM), AICAR (3 mM), hydrogen peroxide (1 mM), sorbitol (1.5 M), A23187 (3 μM), A769662 (300 μM), metformin (3 mM), phenformin (3 mM), galegine (100 μM), troglitazone (100 μM), phenobarbital (3 mM), quercetin (100 μM), resveratrol (300 μM), and berberine (100 μM).

    Techniques Used: Incubation

    26) Product Images from "Structural and Biochemical Characterization of Human Adenylosuccinate Lyase (ADSL) and the R303C ADSL Deficiency Associated Mutation"

    Article Title: Structural and Biochemical Characterization of Human Adenylosuccinate Lyase (ADSL) and the R303C ADSL Deficiency Associated Mutation

    Journal: Biochemistry

    doi: 10.1021/bi300796y

    Proposed mechanism for SAICAR to AICAR plus fumarate and for SAMP to AMP plus fumarate.
    Figure Legend Snippet: Proposed mechanism for SAICAR to AICAR plus fumarate and for SAMP to AMP plus fumarate.

    Techniques Used:

    27) Product Images from "AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells"

    Article Title: AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-020-1565-6

    Effects of AICAR, NAM, and AICAR+NAM on the intracellular signaling pathways of AMPK, mTORC1, and autophagy. a MSCs at passage 5 were treated with AICAR, NAM, and AICAR+NAM for further five passages. Immunofluorescence staining of the study groups at P10 using anti-phospho-AMPK (AMPK-α1-pThr183 and AMPK-α2-pThr172) antibody, anti-phospho-p70 S6 Kinase (Thr389) antibody, and anti-LC3B antibody. The nuclei were visualized using DAPI (scale bar = 100 μm). b Representative data of phospho-AMPK, Phospho-p70 S6 kinase (Thr389), and LC3B immunofluorescence, calculated as the number of positive cells per total number of cells. Each bar indicates mean ± SD (* p
    Figure Legend Snippet: Effects of AICAR, NAM, and AICAR+NAM on the intracellular signaling pathways of AMPK, mTORC1, and autophagy. a MSCs at passage 5 were treated with AICAR, NAM, and AICAR+NAM for further five passages. Immunofluorescence staining of the study groups at P10 using anti-phospho-AMPK (AMPK-α1-pThr183 and AMPK-α2-pThr172) antibody, anti-phospho-p70 S6 Kinase (Thr389) antibody, and anti-LC3B antibody. The nuclei were visualized using DAPI (scale bar = 100 μm). b Representative data of phospho-AMPK, Phospho-p70 S6 kinase (Thr389), and LC3B immunofluorescence, calculated as the number of positive cells per total number of cells. Each bar indicates mean ± SD (* p

    Techniques Used: Immunofluorescence, Staining

    The schematic mechanism of action of AICAR and NAM. a AMP binds to the gamma subunit of AMPK, allosterically increases the exposure of T172 to the activating upstream kinases like LKB1 and CaMKK-beta, and hinders the access of upstream phosphatases like PP2A to this residue. b AMPK may undergo a series of phosphorylations by inhibitory upstream kinases like GSK3, AKT, and ERK; GSK3 phosphorylates the ST loop of the alpha subunit and primes the subsequent phosphorylations for others. Later on, the alpha subunit changes its conformation, and the T172 gets exposed to the inhibitory phosphatases. c Acting like AMP, AICAR increases T172 phosphorylation of the alpha subunit by facilitating the phosphorylation and hindering the dephosphorylation. Following that, AMPK activates autophagy in general. Additionally, AMPK increases the biogenesis of new mitochondria and the degradation of dysfunctional and damaged ones. Moreover, NAM increases SIRT1 activity. In turn, SIRT1 increases the LKB1 activity and indirectly increases the AMPK activity. Furthermore, it increases the functionality of mitochondria and decreases ROS production by damaged mitochondria. These sequence of events prevents the MSCs to age and to show senescence-associated changes like dysfunctional autophagosome accumulation, and enlarged and flattened morphology
    Figure Legend Snippet: The schematic mechanism of action of AICAR and NAM. a AMP binds to the gamma subunit of AMPK, allosterically increases the exposure of T172 to the activating upstream kinases like LKB1 and CaMKK-beta, and hinders the access of upstream phosphatases like PP2A to this residue. b AMPK may undergo a series of phosphorylations by inhibitory upstream kinases like GSK3, AKT, and ERK; GSK3 phosphorylates the ST loop of the alpha subunit and primes the subsequent phosphorylations for others. Later on, the alpha subunit changes its conformation, and the T172 gets exposed to the inhibitory phosphatases. c Acting like AMP, AICAR increases T172 phosphorylation of the alpha subunit by facilitating the phosphorylation and hindering the dephosphorylation. Following that, AMPK activates autophagy in general. Additionally, AMPK increases the biogenesis of new mitochondria and the degradation of dysfunctional and damaged ones. Moreover, NAM increases SIRT1 activity. In turn, SIRT1 increases the LKB1 activity and indirectly increases the AMPK activity. Furthermore, it increases the functionality of mitochondria and decreases ROS production by damaged mitochondria. These sequence of events prevents the MSCs to age and to show senescence-associated changes like dysfunctional autophagosome accumulation, and enlarged and flattened morphology

    Techniques Used: De-Phosphorylation Assay, Activity Assay, Sequencing

    Effect of AICAR and NAM on doubling time and MTT assay of the aging MSCs. a AICAR, NAM, and combination of AICAR+NAM reduce the doubling time of MSCs compared to the control group, from P5 to P10. b Proliferative capacity of MSCs at P10 was determined by MTT assay at the third and seventh day of culture ( n = 3 independent experiments). Each bar indicates mean ± SD (* p
    Figure Legend Snippet: Effect of AICAR and NAM on doubling time and MTT assay of the aging MSCs. a AICAR, NAM, and combination of AICAR+NAM reduce the doubling time of MSCs compared to the control group, from P5 to P10. b Proliferative capacity of MSCs at P10 was determined by MTT assay at the third and seventh day of culture ( n = 3 independent experiments). Each bar indicates mean ± SD (* p

    Techniques Used: MTT Assay

    Distinct effects of AICAR, NAM, and concomitant AICAR+NAM treatment on senescence-associated changes of MSCs and total cellular reactive oxygen species (ROS). MSCs at passage 5 were treated with AICAR, NAM, and AICAR+NAM for further five passages. a Phase-contrast images of MSCs (P10) (scale bar = 500 μm), SA-β-gal expression, visualized using light microscopy (scale bar = 100 μm), and fluorescent micrograph (scale bar = 50 μm) of the Acridine Orange stained MSCs at P10 of the four groups. b Left panel: the surface area of the MSCs (P10), calculated using ImageJ software, indicates that cells treated with AICAR alone or AICAR+NAM displayed a significantly lower cross-sectional surface area compared to the NAM-treated cells and the untreated group. Middle panel: prevalence of the SA-β-gal-positive cells, calculated as the number of blue cells per the total number of cells counted. Our data show that treatment with AICAR and NAM reduces the expression of SA-β-gal. Right panel: prevalence of senescent cells determined by the number of green fluorescence-emitting cells per the total number of cells counted. Untreated cells displayed the highest frequency of cells emitting green fluorescence and the least frequency of red fluorescence-emitting cells, compared to the treatment groups. Each bar indicates mean ± SD. c Total cellular ROS was measured at P5 and P10 by staining with DCFDA, followed by flow cytometry analysis ( n = 3 independent experiments). d Comparison between total cellular ROS of the study groups at P10 ( n = 3 independent experiments). Each bar indicates mean ± SD (* p
    Figure Legend Snippet: Distinct effects of AICAR, NAM, and concomitant AICAR+NAM treatment on senescence-associated changes of MSCs and total cellular reactive oxygen species (ROS). MSCs at passage 5 were treated with AICAR, NAM, and AICAR+NAM for further five passages. a Phase-contrast images of MSCs (P10) (scale bar = 500 μm), SA-β-gal expression, visualized using light microscopy (scale bar = 100 μm), and fluorescent micrograph (scale bar = 50 μm) of the Acridine Orange stained MSCs at P10 of the four groups. b Left panel: the surface area of the MSCs (P10), calculated using ImageJ software, indicates that cells treated with AICAR alone or AICAR+NAM displayed a significantly lower cross-sectional surface area compared to the NAM-treated cells and the untreated group. Middle panel: prevalence of the SA-β-gal-positive cells, calculated as the number of blue cells per the total number of cells counted. Our data show that treatment with AICAR and NAM reduces the expression of SA-β-gal. Right panel: prevalence of senescent cells determined by the number of green fluorescence-emitting cells per the total number of cells counted. Untreated cells displayed the highest frequency of cells emitting green fluorescence and the least frequency of red fluorescence-emitting cells, compared to the treatment groups. Each bar indicates mean ± SD. c Total cellular ROS was measured at P5 and P10 by staining with DCFDA, followed by flow cytometry analysis ( n = 3 independent experiments). d Comparison between total cellular ROS of the study groups at P10 ( n = 3 independent experiments). Each bar indicates mean ± SD (* p

    Techniques Used: Expressing, Light Microscopy, Staining, Software, Fluorescence, Flow Cytometry

    Effect of AICAR and NAM treatment on differentiation potential of MSCs after long-term in vitro culture. MSCs at P8 were cultured in osteogenic or adipogenic induction media in the presence of AICAR (1 mM), NAM (5 mM), or AICAR+NAM (1 mM and 5 mM, respectively) or in the absence of AICAR and NAM (control group). a Cells were stained with Alizarin Red S, and the accumulation of calcium deposits was visualized using light microscopy (scale bar = 100 μm) and quantified by spectrophotometry ( n = 3 independent experiments). Each bar indicates mean ± SD. b Expression of markers of osteogenesis—Runx-2, osteopontin, and ALP—was determined by qRT-PCR analysis ( n = 3 independent experiments). Each bar indicates mean ± SD. c For the evaluation of adipogenesis, cells were stained with Oil Red O, and lipid accumulation was visualized using light microscopy (scale bar = 100 μm) and quantified by spectrophotometry ( n = 3 independent experiments). Each bar indicates mean ± SD. d mRNA expression of markers of adipogenesis—LPL and PPAR-γ—was determined by qRT-PCR analysis ( n = 3 independent experiments). Each bar indicates mean ± SD (* p
    Figure Legend Snippet: Effect of AICAR and NAM treatment on differentiation potential of MSCs after long-term in vitro culture. MSCs at P8 were cultured in osteogenic or adipogenic induction media in the presence of AICAR (1 mM), NAM (5 mM), or AICAR+NAM (1 mM and 5 mM, respectively) or in the absence of AICAR and NAM (control group). a Cells were stained with Alizarin Red S, and the accumulation of calcium deposits was visualized using light microscopy (scale bar = 100 μm) and quantified by spectrophotometry ( n = 3 independent experiments). Each bar indicates mean ± SD. b Expression of markers of osteogenesis—Runx-2, osteopontin, and ALP—was determined by qRT-PCR analysis ( n = 3 independent experiments). Each bar indicates mean ± SD. c For the evaluation of adipogenesis, cells were stained with Oil Red O, and lipid accumulation was visualized using light microscopy (scale bar = 100 μm) and quantified by spectrophotometry ( n = 3 independent experiments). Each bar indicates mean ± SD. d mRNA expression of markers of adipogenesis—LPL and PPAR-γ—was determined by qRT-PCR analysis ( n = 3 independent experiments). Each bar indicates mean ± SD (* p

    Techniques Used: In Vitro, Cell Culture, Staining, Light Microscopy, Spectrophotometry, Expressing, Quantitative RT-PCR

    Effects of AICAR, NAM, and AICAR+NAM treatment on markers of apoptosis and cellular apoptosis. a Immunofluorescence staining of the study groups at P10, for Bcl-2, Bax, and Caspase-3. The nuclei were visualized using DAPI (blue fluorescence). AICAR and NAM treatments decrease pro-apoptotic proteins, Bax, and Caspase-3 and increase the anti-apoptotic protein, Bcl-2 (scale bar = 100 μm). b Representative data of Bcl-2, Bax, and Caspase-3 immunofluorescence, calculated as the number of positive cells per the total number of cells. Each bar indicates mean ± SD. c Flow cytometry analysis of Annexin V assay (* p
    Figure Legend Snippet: Effects of AICAR, NAM, and AICAR+NAM treatment on markers of apoptosis and cellular apoptosis. a Immunofluorescence staining of the study groups at P10, for Bcl-2, Bax, and Caspase-3. The nuclei were visualized using DAPI (blue fluorescence). AICAR and NAM treatments decrease pro-apoptotic proteins, Bax, and Caspase-3 and increase the anti-apoptotic protein, Bcl-2 (scale bar = 100 μm). b Representative data of Bcl-2, Bax, and Caspase-3 immunofluorescence, calculated as the number of positive cells per the total number of cells. Each bar indicates mean ± SD. c Flow cytometry analysis of Annexin V assay (* p

    Techniques Used: Immunofluorescence, Staining, Fluorescence, Flow Cytometry, Annexin V Assay

    28) Product Images from "The cross talk of two family members of β-TrCP in the regulation of cell autophagy and growth"

    Article Title: The cross talk of two family members of β-TrCP in the regulation of cell autophagy and growth

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-019-0402-x

    Activated AMPK triggers β-TrCP1 degradation upon glucose deprivation. a Cells were cultured in high-glucose or glucose-free medium for the indicated time periods and then harvested for IB with anti-β-TrCP1, p-ACC, t-ACC, p-RSK, t-RSK, p-AMPK, t-AMPK, p-AKT, t-AKT, p-ERK1/2, t-ERK1/2, and Actin Abs. b Cells transferred to glucose-free medium were treated with or without 10 μM Compound C for 1 h, treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, p-ACC, and Actin Abs. c Cells were pretreated with DMSO or 0.5 mM AICAR for 24 h, lysed under denaturing conditions and then subjected to pull-down by Ni-NTA beads. Pull-downs (top) and whole-cell extracts (bottom) were subject to IB with anti-HA and Actin Abs. SK-BR3 cells were transfected with siRNA targeting AMPKα or scrambled control siRNA for 48 h ( d ); AMPK WT or DKO MEFs were shifted to glucose-free medium for 2 h ( e ), treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, p-AMPK, t-AMPK, p-ACC, t-ACC, and Actin Abs. f SK-BR3 cells were transfected with siRNA targeting LKB1 or scrambled control siRNA. After 48 h, cells were shifted to glucose-free medium for 4 h, treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, LKB1, p-AMPK, t-AMPK, and Actin Abs. g Human β-TrCP1 has a putative AMPK motif (75-SLRQTYNSCARL-86) that is highly evolutionarily conserved. Hyd: bulky hydrophobic residues (like L, I, M, F, and V); X * : one of these sites is a basic residue. h H1299 cells were transfected with FLAG-β-TrCP1 WT or S82A mutant plasmid. After 48 h, cells were shifted to glucose-free medium for 4 h, treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-FLAG and Actin. Densitometry quantification was performed with ImageJ, and the decay curves are shown (mean ± S.E.M., n = 3, * p
    Figure Legend Snippet: Activated AMPK triggers β-TrCP1 degradation upon glucose deprivation. a Cells were cultured in high-glucose or glucose-free medium for the indicated time periods and then harvested for IB with anti-β-TrCP1, p-ACC, t-ACC, p-RSK, t-RSK, p-AMPK, t-AMPK, p-AKT, t-AKT, p-ERK1/2, t-ERK1/2, and Actin Abs. b Cells transferred to glucose-free medium were treated with or without 10 μM Compound C for 1 h, treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, p-ACC, and Actin Abs. c Cells were pretreated with DMSO or 0.5 mM AICAR for 24 h, lysed under denaturing conditions and then subjected to pull-down by Ni-NTA beads. Pull-downs (top) and whole-cell extracts (bottom) were subject to IB with anti-HA and Actin Abs. SK-BR3 cells were transfected with siRNA targeting AMPKα or scrambled control siRNA for 48 h ( d ); AMPK WT or DKO MEFs were shifted to glucose-free medium for 2 h ( e ), treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, p-AMPK, t-AMPK, p-ACC, t-ACC, and Actin Abs. f SK-BR3 cells were transfected with siRNA targeting LKB1 or scrambled control siRNA. After 48 h, cells were shifted to glucose-free medium for 4 h, treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-β-TrCP1, LKB1, p-AMPK, t-AMPK, and Actin Abs. g Human β-TrCP1 has a putative AMPK motif (75-SLRQTYNSCARL-86) that is highly evolutionarily conserved. Hyd: bulky hydrophobic residues (like L, I, M, F, and V); X * : one of these sites is a basic residue. h H1299 cells were transfected with FLAG-β-TrCP1 WT or S82A mutant plasmid. After 48 h, cells were shifted to glucose-free medium for 4 h, treated with 100 μg/ml CHX for the indicated time periods and then subjected to IB with anti-FLAG and Actin. Densitometry quantification was performed with ImageJ, and the decay curves are shown (mean ± S.E.M., n = 3, * p

    Techniques Used: Cell Culture, Transfection, Mutagenesis, Plasmid Preparation

    29) Product Images from "The Adipocyte-Expressed Forkhead Transcription Factor Foxc2 Regulates Metabolism Through Altered Mitochondrial Function"

    Article Title: The Adipocyte-Expressed Forkhead Transcription Factor Foxc2 Regulates Metabolism Through Altered Mitochondrial Function

    Journal: Diabetes

    doi: 10.2337/db10-0409

    Cells overexpressing Foxc2 have increased aerobic capacity and enhanced levels of palmitate oxidation. A : Oxygen consumption rates in MEF-derived adipocytes from WT and Foxc2 overexpressing mice. Basal rates were determined in the absence of uncoupler (black bars), whereas maximal rates were determined after the addition of uncoupler (1 μmol/L FCCP; gray bars; n = 4 multiwell plates/genotype). * P = 0.05; *** P = 0.001. B : Extracellular acidification rates, a surrogate measure of glycolytic activity (see research design and methods ), in MEF-derived adipocytes from WT and Foxc2 overexpressing mice. Basal rates were determined in the absence of uncoupler (black bars), whereas maximal rates were determined after the addition of uncoupler (1 μmol/L FCCP; gray bars; n = 4 multiwell plates/genotype). * P = 0.05; ** P = 0.01. C : Palmitate oxidation was measured in MEF-derived adipocytes from WT (black bars) and FOXC2 overexpressing (gray bars) mice under basal conditions and in the presence of 100 nmol/L insulin (inhibitor of fatty acid oxidation) or 1 mmol/L AICAR (activator of AMP-activated protein kinase [AMPK]). Values are calculated as fold differences compared with palmitate oxidation under basal conditions in MEF-derived adipocytes from WT mice ( n = six multiwell plates/genotype). ** P = 0.01; *** P = 0.001. As expected, insulin decreased, whereas AICAR stimulated fatty acid oxidation in a similar manner both in the adipocytes derived from WT and FOXC2 overexpressing mice. Oxidation of palmitate was higher in adipocytes with increased expression of FOXC2 compared with the WT adipocytes under basal conditions and after treatment with insulin and AMPK activator AICAR.
    Figure Legend Snippet: Cells overexpressing Foxc2 have increased aerobic capacity and enhanced levels of palmitate oxidation. A : Oxygen consumption rates in MEF-derived adipocytes from WT and Foxc2 overexpressing mice. Basal rates were determined in the absence of uncoupler (black bars), whereas maximal rates were determined after the addition of uncoupler (1 μmol/L FCCP; gray bars; n = 4 multiwell plates/genotype). * P = 0.05; *** P = 0.001. B : Extracellular acidification rates, a surrogate measure of glycolytic activity (see research design and methods ), in MEF-derived adipocytes from WT and Foxc2 overexpressing mice. Basal rates were determined in the absence of uncoupler (black bars), whereas maximal rates were determined after the addition of uncoupler (1 μmol/L FCCP; gray bars; n = 4 multiwell plates/genotype). * P = 0.05; ** P = 0.01. C : Palmitate oxidation was measured in MEF-derived adipocytes from WT (black bars) and FOXC2 overexpressing (gray bars) mice under basal conditions and in the presence of 100 nmol/L insulin (inhibitor of fatty acid oxidation) or 1 mmol/L AICAR (activator of AMP-activated protein kinase [AMPK]). Values are calculated as fold differences compared with palmitate oxidation under basal conditions in MEF-derived adipocytes from WT mice ( n = six multiwell plates/genotype). ** P = 0.01; *** P = 0.001. As expected, insulin decreased, whereas AICAR stimulated fatty acid oxidation in a similar manner both in the adipocytes derived from WT and FOXC2 overexpressing mice. Oxidation of palmitate was higher in adipocytes with increased expression of FOXC2 compared with the WT adipocytes under basal conditions and after treatment with insulin and AMPK activator AICAR.

    Techniques Used: Derivative Assay, Mouse Assay, Activity Assay, Expressing

    30) Product Images from "Role of AMP-Activated Protein Kinase on Steroid Hormone Biosynthesis in Adrenal NCI-H295R Cells"

    Article Title: Role of AMP-Activated Protein Kinase on Steroid Hormone Biosynthesis in Adrenal NCI-H295R Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030956

    Studies of the effect of AICAR and compound C on promoter activities of genes involved in androgen production. NCI-H295R cells were either transfected with an empty vector (pGL3, Δluc) or promoter luciferase reporter constructs ( -3.7 CYP17, -1.05 HSD3B2, -1.3 b5, -325 POR, -1.08 SULT2A1) and subsequently treated with 1 mM AICAR or 20 µM compound C for 6 h (A) or 48 h (B). Following transfection and treatment, promoter activities were assessed by Dual luciferase assay readout (Promega). Quantification data represent two independent experiments performed in duplicates. Results are expressed as a percentage of control, mean ±S.E.M. *, P
    Figure Legend Snippet: Studies of the effect of AICAR and compound C on promoter activities of genes involved in androgen production. NCI-H295R cells were either transfected with an empty vector (pGL3, Δluc) or promoter luciferase reporter constructs ( -3.7 CYP17, -1.05 HSD3B2, -1.3 b5, -325 POR, -1.08 SULT2A1) and subsequently treated with 1 mM AICAR or 20 µM compound C for 6 h (A) or 48 h (B). Following transfection and treatment, promoter activities were assessed by Dual luciferase assay readout (Promega). Quantification data represent two independent experiments performed in duplicates. Results are expressed as a percentage of control, mean ±S.E.M. *, P

    Techniques Used: Transfection, Plasmid Preparation, Luciferase, Construct

    Cell proliferation of NCI-H295R cells. Cells were treated with 1 mM AICAR (A) or 0–20 µM compound C (B) in serum-free medium for 0–48 hours. A commercially available cell proliferation assay was performed (Promega). Results are expressed as OD490 nm which corresponds directly to cell viability in this assay. Data are the mean of two independent experiments ± S.E.M.
    Figure Legend Snippet: Cell proliferation of NCI-H295R cells. Cells were treated with 1 mM AICAR (A) or 0–20 µM compound C (B) in serum-free medium for 0–48 hours. A commercially available cell proliferation assay was performed (Promega). Results are expressed as OD490 nm which corresponds directly to cell viability in this assay. Data are the mean of two independent experiments ± S.E.M.

    Techniques Used: Proliferation Assay

    Effect of AMPK activator AICAR and inhibitor compound C on steroidogenesis in NCI-H295R cells. NCI-H295R cells were either stimulated with 1 mM AICAR or inhibited with 20 µM compound C (Comp C) for 48 h. HSD3B2 activity was blocked by 1 µM trilostane to specifically study the CYP17-OHase and CYP17-17,20 lyase activities. HSD3B2 activity was studied as a conversion of DHEA into androstenedione. Steroidogenesis was either labeled using 100,000 cpm/35-mm well of ( 3 H) pregnenolone (Preg), ( 3 H) 17OH pregnenolone (17OH-Preg) or ( 3 H) dehydroepiandrosterone (DHEA) as substrates for 90 min. Steroids were extracted from medium and resolved on TLC plates. A–C, representative TLCs (upper panel) and quantifications (lower panel) of CYP17-OHase (A), CYP17-17,20 lyase (B) and HSD3B2 (C) activities. Quantification was performed on four independent experiments. Results are expressed as a percentage of control, error bars are ±S.D. Δ4A, androstenedione. **, P
    Figure Legend Snippet: Effect of AMPK activator AICAR and inhibitor compound C on steroidogenesis in NCI-H295R cells. NCI-H295R cells were either stimulated with 1 mM AICAR or inhibited with 20 µM compound C (Comp C) for 48 h. HSD3B2 activity was blocked by 1 µM trilostane to specifically study the CYP17-OHase and CYP17-17,20 lyase activities. HSD3B2 activity was studied as a conversion of DHEA into androstenedione. Steroidogenesis was either labeled using 100,000 cpm/35-mm well of ( 3 H) pregnenolone (Preg), ( 3 H) 17OH pregnenolone (17OH-Preg) or ( 3 H) dehydroepiandrosterone (DHEA) as substrates for 90 min. Steroids were extracted from medium and resolved on TLC plates. A–C, representative TLCs (upper panel) and quantifications (lower panel) of CYP17-OHase (A), CYP17-17,20 lyase (B) and HSD3B2 (C) activities. Quantification was performed on four independent experiments. Results are expressed as a percentage of control, error bars are ±S.D. Δ4A, androstenedione. **, P

    Techniques Used: Activity Assay, Labeling, Thin Layer Chromatography

    Direct effects of AICAR and compound C on enzyme activities of CYP17, HSD3B2 and CYP21A2 and effect on overall steroidogenesis in NCI-H295R cells. A–F yeast microsomes either co-expressing human CYP17 or CYP21 with human P450 oxidoreductase or expressing human HSD3B2 were incubated with 15 µM or 150 µM ( 14 C) progesterone (Prog), 50 µM ( 3 H) pregnenolone (Preg) or 50 µM ( 3 H) 17α-hydroxypregnenolone (17OH-Preg). At the same time, microsomes were incubated with either various concentrations of compound C or 1 mM AICAR to assess their effect on the activities of CYP17-OHase (A, D), CYP17-17,20 lyase (E), HSD3B2 (B, F) and CYP21A2 (C). G, effect of AICAR and compound C on overall steroidogenesis in NCI-H295R cells. Experiments were repeated independently two to three times. Error bars represent ±SEM. *, P
    Figure Legend Snippet: Direct effects of AICAR and compound C on enzyme activities of CYP17, HSD3B2 and CYP21A2 and effect on overall steroidogenesis in NCI-H295R cells. A–F yeast microsomes either co-expressing human CYP17 or CYP21 with human P450 oxidoreductase or expressing human HSD3B2 were incubated with 15 µM or 150 µM ( 14 C) progesterone (Prog), 50 µM ( 3 H) pregnenolone (Preg) or 50 µM ( 3 H) 17α-hydroxypregnenolone (17OH-Preg). At the same time, microsomes were incubated with either various concentrations of compound C or 1 mM AICAR to assess their effect on the activities of CYP17-OHase (A, D), CYP17-17,20 lyase (E), HSD3B2 (B, F) and CYP21A2 (C). G, effect of AICAR and compound C on overall steroidogenesis in NCI-H295R cells. Experiments were repeated independently two to three times. Error bars represent ±SEM. *, P

    Techniques Used: Expressing, Incubation

    Effect of AICAR and compound C on phosphorylation of AMPK and on CYP17 expression in NCI-H295R cells. A–D, cells were treated and protein extracts were prepared and analyzed by Western blot. A–B, NCI-H295R cells were stimulated with 1 mM AICAR for 3–48 h. An increase in AMPK phosphorylation was noted after 3 hours (A) while CYP17 expression remained unchanged (B). C–D, NCI-H295R cells were treated with 20 nM–20 µM compound C (Comp C), 1 mM AICAR or with both for 48 h. Note that compound C attenuated AICAR stimulated AMPK phosphorylation (C) and increased CYP17 expression at higher concentrations (D). Representative Western blots of two to three independent experiments are shown. β-actin served as loading control. Quantitative results are given as mean ± S.E.M.
    Figure Legend Snippet: Effect of AICAR and compound C on phosphorylation of AMPK and on CYP17 expression in NCI-H295R cells. A–D, cells were treated and protein extracts were prepared and analyzed by Western blot. A–B, NCI-H295R cells were stimulated with 1 mM AICAR for 3–48 h. An increase in AMPK phosphorylation was noted after 3 hours (A) while CYP17 expression remained unchanged (B). C–D, NCI-H295R cells were treated with 20 nM–20 µM compound C (Comp C), 1 mM AICAR or with both for 48 h. Note that compound C attenuated AICAR stimulated AMPK phosphorylation (C) and increased CYP17 expression at higher concentrations (D). Representative Western blots of two to three independent experiments are shown. β-actin served as loading control. Quantitative results are given as mean ± S.E.M.

    Techniques Used: Expressing, Western Blot

    31) Product Images from "Nuclear respiratory factor 1 and endurance exercise promote human telomere transcription"

    Article Title: Nuclear respiratory factor 1 and endurance exercise promote human telomere transcription

    Journal: Science Advances

    doi: 10.1126/sciadv.1600031

    AMPK activation in human myotubes induces NRF1-dependent increase in TERRA levels. ( A ) TERRA-FISH (green) combined with telomeric DNA FISH (red) in myotubes. Blue, DAPI. Scale bar, 5 μm. ( B ) Quantification of (A) on 25 nuclei. ( C ) ACC phosphorylation in myotubes treated with either AICAR, metformin, or phenformin. ( D ) qRT-PCR analysis of TERRA levels in treated myotubes normalized to β 2M cDNA and to untreated cells. Error bars indicate SD ( n = 4). ( E ) Western blot analysis of NRF1 knockdown in myotubes. ( F ) qRT-PCR analysis of TERRA in siNRF1-treated myotubes and upon phenformin treatment. Values were normalized to β 2M cDNA and to siLuci-treated cells without phenformin. Error bars indicate SD ( n = 3). ( G ) Unified theory of aging ( 34 ) revisited with data from this study (green). See text for details.
    Figure Legend Snippet: AMPK activation in human myotubes induces NRF1-dependent increase in TERRA levels. ( A ) TERRA-FISH (green) combined with telomeric DNA FISH (red) in myotubes. Blue, DAPI. Scale bar, 5 μm. ( B ) Quantification of (A) on 25 nuclei. ( C ) ACC phosphorylation in myotubes treated with either AICAR, metformin, or phenformin. ( D ) qRT-PCR analysis of TERRA levels in treated myotubes normalized to β 2M cDNA and to untreated cells. Error bars indicate SD ( n = 4). ( E ) Western blot analysis of NRF1 knockdown in myotubes. ( F ) qRT-PCR analysis of TERRA in siNRF1-treated myotubes and upon phenformin treatment. Values were normalized to β 2M cDNA and to siLuci-treated cells without phenformin. Error bars indicate SD ( n = 3). ( G ) Unified theory of aging ( 34 ) revisited with data from this study (green). See text for details.

    Techniques Used: Activation Assay, Fluorescence In Situ Hybridization, Quantitative RT-PCR, Western Blot

    32) Product Images from "Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways"

    Article Title: Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways

    Journal:

    doi: 10.1152/ajpcell.00040.2008

    Effect of U0126 on azide- or AICAR-induced stimulation of glucose transport, p-AMPK, p-p38, and p-ERK1/2. A : glucose transport. Confluent Clone 9 cells in triplicate 60-mm dishes were pretreated with diluent or 10 μM U0126 for 20 min before being
    Figure Legend Snippet: Effect of U0126 on azide- or AICAR-induced stimulation of glucose transport, p-AMPK, p-p38, and p-ERK1/2. A : glucose transport. Confluent Clone 9 cells in triplicate 60-mm dishes were pretreated with diluent or 10 μM U0126 for 20 min before being

    Techniques Used:

    Effect of azide and 5-aminoimidazole-4-carboxamide-1-β- d -ribofuranoside (AICAR) on glucose transport and on the abundance of phosphorylated AMP-activated protein kinase (p-AMPK) in wild-type (WT) and AMPK knockout (KO) fibroblasts. A : glucose
    Figure Legend Snippet: Effect of azide and 5-aminoimidazole-4-carboxamide-1-β- d -ribofuranoside (AICAR) on glucose transport and on the abundance of phosphorylated AMP-activated protein kinase (p-AMPK) in wild-type (WT) and AMPK knockout (KO) fibroblasts. A : glucose

    Techniques Used: Knock-Out

    33) Product Images from "Meformin action in human hepatocytes. Co-activation of atypical protein kinase C alters 5?-AMP-activated protein kinase effects on lipogenic and gluconeogenic enzyme expression"

    Article Title: Meformin action in human hepatocytes. Co-activation of atypical protein kinase C alters 5?-AMP-activated protein kinase effects on lipogenic and gluconeogenic enzyme expression

    Journal: Diabetologia

    doi: 10.1007/s00125-013-3010-1

    Effects of ICAP versus AICAR versus Metformin on Expression of Lipogenic and Gluconeogenic Factors in Basal and Insulin-stimulated Hepatocytes of Non-diabetic and Type 2 Diabetic (T2DM) Humans. Hepatocytes of non-diabetic and T2DM humans were treated for 24 hours without (O) or with 100nmol/l ICAP (I), 100nmol/l AICAR (A) or 1 or 3 mmol/l metformin (1M and 3M), in the presence (solid bar) and absence (open bar) of 1μmol/l insulin, and then examined for mRNA levels of SREBP-1c, FAS, PEPCK and G6Pase. Relative values are Mean ± SEM of 5 determinations. Symbols indicate: *, P
    Figure Legend Snippet: Effects of ICAP versus AICAR versus Metformin on Expression of Lipogenic and Gluconeogenic Factors in Basal and Insulin-stimulated Hepatocytes of Non-diabetic and Type 2 Diabetic (T2DM) Humans. Hepatocytes of non-diabetic and T2DM humans were treated for 24 hours without (O) or with 100nmol/l ICAP (I), 100nmol/l AICAR (A) or 1 or 3 mmol/l metformin (1M and 3M), in the presence (solid bar) and absence (open bar) of 1μmol/l insulin, and then examined for mRNA levels of SREBP-1c, FAS, PEPCK and G6Pase. Relative values are Mean ± SEM of 5 determinations. Symbols indicate: *, P

    Techniques Used: Expressing

    Dose-dependent effects of ICAP, AICAR and Metformin on AMPK Activity in Human Hepatocytes. Hepatocytes of non-diabetic humans were treated for 24 hours with indicated concentrations of ICAP, AICAR or metformin, and then examined for immunoprecipitable AMPK activity. Values are Mean ± SEM of 5 determinations.
    Figure Legend Snippet: Dose-dependent effects of ICAP, AICAR and Metformin on AMPK Activity in Human Hepatocytes. Hepatocytes of non-diabetic humans were treated for 24 hours with indicated concentrations of ICAP, AICAR or metformin, and then examined for immunoprecipitable AMPK activity. Values are Mean ± SEM of 5 determinations.

    Techniques Used: Activity Assay

    Effects of ICAP, AICAR and Metformin on aPKC Activity in Human Hepatocytes. Hepatocytes of 5 non-diabetic humans were treated for 24 hours with indicated concentrations of ICAP, AICAR or metformin, in the presence and absence of 1μmol/l insulin, and then examined for immunoprecipitable aPKC activity. Values are Mean ± SEM of 5 determinations. Asterisks indicate: *, P
    Figure Legend Snippet: Effects of ICAP, AICAR and Metformin on aPKC Activity in Human Hepatocytes. Hepatocytes of 5 non-diabetic humans were treated for 24 hours with indicated concentrations of ICAP, AICAR or metformin, in the presence and absence of 1μmol/l insulin, and then examined for immunoprecipitable aPKC activity. Values are Mean ± SEM of 5 determinations. Asterisks indicate: *, P

    Techniques Used: Activity Assay

    AICAR and Metformin Increase Expression of Lipogenic Factors by an aPKC-dependent Mechanism in Human Hepatocytes. Hepatocytes of 4 non-diabetic humans were treated for 24 hours without or with 1μmol/l ACPD, 100nmol/l AICAR (A), 1 mmol/l metformin and 1μmol/l insulin, and then examined for mRNA levels of SREBP-1c and FAS, and activities of aPKC and AMPK. Values are Mean ± SEM of 4 determinations. Asterisks indicate: *, P
    Figure Legend Snippet: AICAR and Metformin Increase Expression of Lipogenic Factors by an aPKC-dependent Mechanism in Human Hepatocytes. Hepatocytes of 4 non-diabetic humans were treated for 24 hours without or with 1μmol/l ACPD, 100nmol/l AICAR (A), 1 mmol/l metformin and 1μmol/l insulin, and then examined for mRNA levels of SREBP-1c and FAS, and activities of aPKC and AMPK. Values are Mean ± SEM of 4 determinations. Asterisks indicate: *, P

    Techniques Used: Expressing

    Effects of ICAP, AICAR and Metformin on Phosphorylation of AMPK, ACC and aPKC in Human Hepatocytes. Hepatocytes of non-diabetic humans were treated for 24 hours with 100nmol/l ICAP, 100nmol/l AICAR or 1mmol/l metformin, and then examined for relative levels of phosho-threonine-172-AMPK, phospho-serine-79-ACC and phospho-threonine-555/560-PKC-ι/ζ. Representative immunoblots are shown above bargrams. Bargram relative values are Mean ± SEM of 5 determinations. Asterisks indicate: *, P
    Figure Legend Snippet: Effects of ICAP, AICAR and Metformin on Phosphorylation of AMPK, ACC and aPKC in Human Hepatocytes. Hepatocytes of non-diabetic humans were treated for 24 hours with 100nmol/l ICAP, 100nmol/l AICAR or 1mmol/l metformin, and then examined for relative levels of phosho-threonine-172-AMPK, phospho-serine-79-ACC and phospho-threonine-555/560-PKC-ι/ζ. Representative immunoblots are shown above bargrams. Bargram relative values are Mean ± SEM of 5 determinations. Asterisks indicate: *, P

    Techniques Used: Western Blot

    34) Product Images from "Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer"

    Article Title: Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer

    Journal: Oncotarget

    doi:

    Inhibition of the AMPK/mTOR pathway attenuates the upregulation of CD133 by CD90 in cell lines and fresh liver cancer specimen A. The phosphorylation of mTOR and AMPK in the transfectants was determined by western blotting. The quantitative data represent mean ± SD ( n = 3). CD133 mRNA B. and protein expression C. in the transfectants were determined by quantitative RT-PCR and western blotting respectively after rapamycin or AICAR treatment for 24 hours. D. The transfectants were plated in soft agar and treated with the indicated inhibitors. Colonies were monitored for 14 days and quantified using Image-Pro Plus software. Data represent mean ± SEM ( n = 3). E. The CD90 transfectant was subcutaneously injected to NOD/SCID mice. The mice were intraperitoneally injected with indicated sorafenib or OSU-CG5 after 14 days and the drug was administrated to mice every other day. P value was calculated using two-way anova analysis and * indicated P
    Figure Legend Snippet: Inhibition of the AMPK/mTOR pathway attenuates the upregulation of CD133 by CD90 in cell lines and fresh liver cancer specimen A. The phosphorylation of mTOR and AMPK in the transfectants was determined by western blotting. The quantitative data represent mean ± SD ( n = 3). CD133 mRNA B. and protein expression C. in the transfectants were determined by quantitative RT-PCR and western blotting respectively after rapamycin or AICAR treatment for 24 hours. D. The transfectants were plated in soft agar and treated with the indicated inhibitors. Colonies were monitored for 14 days and quantified using Image-Pro Plus software. Data represent mean ± SEM ( n = 3). E. The CD90 transfectant was subcutaneously injected to NOD/SCID mice. The mice were intraperitoneally injected with indicated sorafenib or OSU-CG5 after 14 days and the drug was administrated to mice every other day. P value was calculated using two-way anova analysis and * indicated P

    Techniques Used: Inhibition, Western Blot, Expressing, Quantitative RT-PCR, Software, Transfection, Injection, Mouse Assay

    35) Product Images from "Discovery of TBC1D1 as an Insulin-, AICAR-, and Contraction-stimulated Signaling Nexus in Mouse Skeletal Muscle *"

    Article Title: Discovery of TBC1D1 as an Insulin-, AICAR-, and Contraction-stimulated Signaling Nexus in Mouse Skeletal Muscle *

    Journal:

    doi: 10.1074/jbc.M708839200

    AICAR-stimulated PAS-160 phosphorylation is greatest in TA muscle. The time courses of AICAR-stimulated Akt substrate phosphorylation at a molecular weight of 160 (PAS-160) and AMPK Thr-172 phosphorylation ( P-AMPK ) in vivo were determined by injecting
    Figure Legend Snippet: AICAR-stimulated PAS-160 phosphorylation is greatest in TA muscle. The time courses of AICAR-stimulated Akt substrate phosphorylation at a molecular weight of 160 (PAS-160) and AMPK Thr-172 phosphorylation ( P-AMPK ) in vivo were determined by injecting

    Techniques Used: Molecular Weight, In Vivo

    36) Product Images from "The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells"

    Article Title: The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells

    Journal: Nature

    doi: 10.1038/s41586-018-0282-0

    P2RX7 ablation leads to aberrant metabolism and depressed AMPK activation in CD8 + T cells (a–d) In vitro activated WT and P2rx7 −/− P14 were activated then polarized with IL-2 or IL-15 (cells pooled from 2 mice/experiment). (a) Cell viability during IL-2 or IL-15 cultures (p≥0.2 for all IL-2 polarized cells and IL-15 polarized at 24h). (b,c) OCR (b) and calculated SRC (c) for IL-2 or IL-15-polarized cells. (d) electron microscopy of mitochondria (arrows) in IL-15-polarized WT or P2rx7 −/− P14 (black bars =500nm). (e–k) mouse (e,i–k) or human (f–h) CD8 + T cells were stimulated in vitro in the presence of A-438079 (e–h) , BzATP (i) , Probenecid (j,k) , or vehicle controls. Mouse cells activated as in ( a ), human cells assayed 72h post-stimulation. OCR (e,f,i,j) and SRC (k) were measured and human cells assayed for proliferation (Ki67) (g) and Granzyme B/IFN-γ (h). (l) pACC in IL-15-polarized WT and P2rx7 −/− P14 (representative flow cytometric histograms and median average values (right). In (m) , cells were incubated (6h) with/without AICAR prior to OCR measurement. (n–o) Mice receiving co-transferred WT and P2rx7 −/− P14 were LCMV-infected primed and treated with metformin (1–7 dpi; n) or rapamycin (4–8 dpi; o) . P2rx7 −/− /WT ratios for splenic memory subsets (n) and P14 T CM numbers were determined ( o ). (a–o) Three independent experiments, n=3–6 ( a ), 8–9 ( b–c ), 6 ( d,g–h,l–m ), 4–5 ( e,i ), 4 ( f ), 5–6 ( j–k ), 10–11 ( n ), 12–17 ( o ) total. (a–c,e–o) Mean ± SEM; (a,c,g–h,l,n) , Two-tailed Student’s t-test; (k,o) One-way ANOVA + Tukey post-test, *P≤0.05, **P≤0.01, ***P≤0.001.
    Figure Legend Snippet: P2RX7 ablation leads to aberrant metabolism and depressed AMPK activation in CD8 + T cells (a–d) In vitro activated WT and P2rx7 −/− P14 were activated then polarized with IL-2 or IL-15 (cells pooled from 2 mice/experiment). (a) Cell viability during IL-2 or IL-15 cultures (p≥0.2 for all IL-2 polarized cells and IL-15 polarized at 24h). (b,c) OCR (b) and calculated SRC (c) for IL-2 or IL-15-polarized cells. (d) electron microscopy of mitochondria (arrows) in IL-15-polarized WT or P2rx7 −/− P14 (black bars =500nm). (e–k) mouse (e,i–k) or human (f–h) CD8 + T cells were stimulated in vitro in the presence of A-438079 (e–h) , BzATP (i) , Probenecid (j,k) , or vehicle controls. Mouse cells activated as in ( a ), human cells assayed 72h post-stimulation. OCR (e,f,i,j) and SRC (k) were measured and human cells assayed for proliferation (Ki67) (g) and Granzyme B/IFN-γ (h). (l) pACC in IL-15-polarized WT and P2rx7 −/− P14 (representative flow cytometric histograms and median average values (right). In (m) , cells were incubated (6h) with/without AICAR prior to OCR measurement. (n–o) Mice receiving co-transferred WT and P2rx7 −/− P14 were LCMV-infected primed and treated with metformin (1–7 dpi; n) or rapamycin (4–8 dpi; o) . P2rx7 −/− /WT ratios for splenic memory subsets (n) and P14 T CM numbers were determined ( o ). (a–o) Three independent experiments, n=3–6 ( a ), 8–9 ( b–c ), 6 ( d,g–h,l–m ), 4–5 ( e,i ), 4 ( f ), 5–6 ( j–k ), 10–11 ( n ), 12–17 ( o ) total. (a–c,e–o) Mean ± SEM; (a,c,g–h,l,n) , Two-tailed Student’s t-test; (k,o) One-way ANOVA + Tukey post-test, *P≤0.05, **P≤0.01, ***P≤0.001.

    Techniques Used: Activation Assay, In Vitro, Mouse Assay, Electron Microscopy, Flow Cytometry, Incubation, Infection, Two Tailed Test

    P2RX7-mediated eATP sensing is crucial for optimal CD8 + T cell immunometabolism via AMPK/mTOR pathway regulation (a–g) , WT and P2rx7 −/− P14 cells were in vitro activated and polarized with IL-15 (as in ). Data from three independent experiments, samples pooled from n=6 mice per experiment; n=3–12 total samples. (a–c) show the numbers of P14 cells (left) and viability (right) in cultures supplemented with (a) the eATP hydrolytic enzyme Apyrase, (b) the inhibitor oATP or (c) the eATP analog BzATP, during cell culture. In (d,e,g) IL-15-polarized WT or P2rx7 −/− P14 cells were assayed for OCR 1h after addition of apyrase (d) or oATP (e) , or 6h after addition of A-438079 (g) . In (f) , IL-15-polarized cells or ex vivo WT P14 T CM (isolated 4w after LCMV infection) were incubated with either DAPI (left) or Indo-1 (right) and stimulated with the indicated concentrations of Bz-ATP during kinetic flow cytometric analysis. The percentage of cells showing DAPI uptake (left) or Ca 2+ influx (right) over 30m are shown. (f) Data from two independent experiments, samples pooled from n=5 mice total; n=2–5 samples. In (h) , in vitro -activated (72h) WT and P2rx7 −/− P14 cells were assayed for intracellular ATP concentrations. Data from three independent experiments, n=9 total. In (i), In vitro -activated, IL-15 polarized (24h post-polarization) WT or P2rx7 −/− P14 cells were assayed for extracellular ATP concentration, following culture without or with the Panx1 inhibitor 10 Panx. Data from two independent experiments, n=3–4 total samples (pooled from 6 mice). (j) , WT and P2rx7 −/− ) and the ex vivo frequency of pS6-expressing cells determined by flow cytometry. Data are from two independent experiments (n=6 total). (k) Representative histograms showing expression of pACC in IL-15-polarized WT (black) and P2rx7 −/− ; representative from three independent experiments, n=6 total). (l) In vitro activated and IL-15-polarized WT and P2rx7 −/− P14 cells were cultured for 6h with the indicated concentrations of BzATP then stained for pACC (left) and pS6 (center) and the pACC/pS6 ratio was determined (right). Data from three independent experiments, n=6–8 total. (m) In vitro -activated WT and P2rx7 −/− . The percentage of viable cells at the indicated times following initiation of IL-15 +/− AICAR culture is indicated. Data are from three independent experiments (n=3–6 total; samples pooled from n=6 mice total). (n–p) WT and P2rx7 −/− P14 CD8 + . The animals were treated with Metformin or PBS control during the first week of LCMV infection, and the cells analyzed at day 30. Data are compiled from three independent experiments (n=11–12 total, n=4 for FRT samples). Panel (n) and shows P2rx7 −/− /WT P14 ratio in indicated non-lymphoid tissues (n=9 except female reproductive tract – FRT – where n=4). (o,p) shows measurements of mitochondrial mass (measured by MTG) (o) and mitochondrial membrane potential (measured by TMRE staining, normalized to MTG staining) (p) for indicated splenocyte subsets (n=3–6 total samples). (q) WT and P2rx7 −/− P14 CD8 + . The animals were treated with Rapamycin or PBS control between days 4–8 post-LCMV infection, and the cells analyzed at day 30. The numbers of WT or P2rx7 −/− P14 cells are shown (log-transformed values). Data are compiled from three independent experiments (n=15 total). (a–j,l–q) , mean ± SEM is shown; (a–c,h–j,m–p) Two-tailed Student’s t-test; (l,q) One-way ANOVA with Tukey’s post-test; *P≤0.05, **P≤0.01, ***P≤0.001. Fig. 3b
    Figure Legend Snippet: P2RX7-mediated eATP sensing is crucial for optimal CD8 + T cell immunometabolism via AMPK/mTOR pathway regulation (a–g) , WT and P2rx7 −/− P14 cells were in vitro activated and polarized with IL-15 (as in ). Data from three independent experiments, samples pooled from n=6 mice per experiment; n=3–12 total samples. (a–c) show the numbers of P14 cells (left) and viability (right) in cultures supplemented with (a) the eATP hydrolytic enzyme Apyrase, (b) the inhibitor oATP or (c) the eATP analog BzATP, during cell culture. In (d,e,g) IL-15-polarized WT or P2rx7 −/− P14 cells were assayed for OCR 1h after addition of apyrase (d) or oATP (e) , or 6h after addition of A-438079 (g) . In (f) , IL-15-polarized cells or ex vivo WT P14 T CM (isolated 4w after LCMV infection) were incubated with either DAPI (left) or Indo-1 (right) and stimulated with the indicated concentrations of Bz-ATP during kinetic flow cytometric analysis. The percentage of cells showing DAPI uptake (left) or Ca 2+ influx (right) over 30m are shown. (f) Data from two independent experiments, samples pooled from n=5 mice total; n=2–5 samples. In (h) , in vitro -activated (72h) WT and P2rx7 −/− P14 cells were assayed for intracellular ATP concentrations. Data from three independent experiments, n=9 total. In (i), In vitro -activated, IL-15 polarized (24h post-polarization) WT or P2rx7 −/− P14 cells were assayed for extracellular ATP concentration, following culture without or with the Panx1 inhibitor 10 Panx. Data from two independent experiments, n=3–4 total samples (pooled from 6 mice). (j) , WT and P2rx7 −/− ) and the ex vivo frequency of pS6-expressing cells determined by flow cytometry. Data are from two independent experiments (n=6 total). (k) Representative histograms showing expression of pACC in IL-15-polarized WT (black) and P2rx7 −/− ; representative from three independent experiments, n=6 total). (l) In vitro activated and IL-15-polarized WT and P2rx7 −/− P14 cells were cultured for 6h with the indicated concentrations of BzATP then stained for pACC (left) and pS6 (center) and the pACC/pS6 ratio was determined (right). Data from three independent experiments, n=6–8 total. (m) In vitro -activated WT and P2rx7 −/− . The percentage of viable cells at the indicated times following initiation of IL-15 +/− AICAR culture is indicated. Data are from three independent experiments (n=3–6 total; samples pooled from n=6 mice total). (n–p) WT and P2rx7 −/− P14 CD8 + . The animals were treated with Metformin or PBS control during the first week of LCMV infection, and the cells analyzed at day 30. Data are compiled from three independent experiments (n=11–12 total, n=4 for FRT samples). Panel (n) and shows P2rx7 −/− /WT P14 ratio in indicated non-lymphoid tissues (n=9 except female reproductive tract – FRT – where n=4). (o,p) shows measurements of mitochondrial mass (measured by MTG) (o) and mitochondrial membrane potential (measured by TMRE staining, normalized to MTG staining) (p) for indicated splenocyte subsets (n=3–6 total samples). (q) WT and P2rx7 −/− P14 CD8 + . The animals were treated with Rapamycin or PBS control between days 4–8 post-LCMV infection, and the cells analyzed at day 30. The numbers of WT or P2rx7 −/− P14 cells are shown (log-transformed values). Data are compiled from three independent experiments (n=15 total). (a–j,l–q) , mean ± SEM is shown; (a–c,h–j,m–p) Two-tailed Student’s t-test; (l,q) One-way ANOVA with Tukey’s post-test; *P≤0.05, **P≤0.01, ***P≤0.001. Fig. 3b

    Techniques Used: In Vitro, Mouse Assay, Cell Culture, Ex Vivo, Isolation, Infection, Incubation, Flow Cytometry, Concentration Assay, Expressing, Cytometry, Staining, Transformation Assay, Two Tailed Test

    37) Product Images from "AMP Kinase Promotes Bcl6 Expression in both Mouse and Human T cells"

    Article Title: AMP Kinase Promotes Bcl6 Expression in both Mouse and Human T cells

    Journal: Molecular immunology

    doi: 10.1016/j.molimm.2016.11.020

    AICAR enhances BCL6 and T FH -dependent antibody responses in vivo (A) C57BL/6 wild-type (WT) or Bcl6 cKO mice were immunized i.p. with NP-KLH or NP-KLH plus AICAR. Sera were harvested on day 14 post-immunization for NP-KLH-specific IgG titer analysis. The X-axis shows the dilution factors. Graph shows mean ± SEM, n = 3. Data are representative of two independent experiments with similar results. (B-C) C57BL/6 mice were immunized i.p. with NP-KLH or NP-KLH plus AICAR and day 3 post immunization stained for percent T FH cells (B) and Bcl6 MFI of splenic T FH cells on (C). T FH cells were gated as % Foxp3- PD-1 high CXCR5+ cells within CD4+ population. (B) n=8, mean ± SEM, from two combined experiments. (C) n=5, mean ± SEM. (D) Germinal center B cells were analyzed day 7 post immunization of C57BL/6 mice immunized i.p. with NP-KLH or NP-KLH plus AICAR. GCB cells are defined as B220 + CD38 lo GL-7 hi cells; n=4, mean ± SEM. *p
    Figure Legend Snippet: AICAR enhances BCL6 and T FH -dependent antibody responses in vivo (A) C57BL/6 wild-type (WT) or Bcl6 cKO mice were immunized i.p. with NP-KLH or NP-KLH plus AICAR. Sera were harvested on day 14 post-immunization for NP-KLH-specific IgG titer analysis. The X-axis shows the dilution factors. Graph shows mean ± SEM, n = 3. Data are representative of two independent experiments with similar results. (B-C) C57BL/6 mice were immunized i.p. with NP-KLH or NP-KLH plus AICAR and day 3 post immunization stained for percent T FH cells (B) and Bcl6 MFI of splenic T FH cells on (C). T FH cells were gated as % Foxp3- PD-1 high CXCR5+ cells within CD4+ population. (B) n=8, mean ± SEM, from two combined experiments. (C) n=5, mean ± SEM. (D) Germinal center B cells were analyzed day 7 post immunization of C57BL/6 mice immunized i.p. with NP-KLH or NP-KLH plus AICAR. GCB cells are defined as B220 + CD38 lo GL-7 hi cells; n=4, mean ± SEM. *p

    Techniques Used: In Vivo, Mouse Assay, Staining

    38) Product Images from "Regulation of PGC-1α mediated by acetylation and phosphorylation in MPP+ induced cell model of Parkinson’s disease"

    Article Title: Regulation of PGC-1α mediated by acetylation and phosphorylation in MPP+ induced cell model of Parkinson’s disease

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.103219

    Evaluation of compounds on cell viability ( A ) cell viability after MPP + treatment; ( B ) cell viability after MB-3 treatment; ( C ) cell viability after SRC-3 treatment; ( D ) cell viability after SB203580 treatment; ( E ) cell viability after isoproterenol treatment; ( F ) cell viability after Compound C treatment; ( G ) cell viability after AICAR treatment. * P
    Figure Legend Snippet: Evaluation of compounds on cell viability ( A ) cell viability after MPP + treatment; ( B ) cell viability after MB-3 treatment; ( C ) cell viability after SRC-3 treatment; ( D ) cell viability after SB203580 treatment; ( E ) cell viability after isoproterenol treatment; ( F ) cell viability after Compound C treatment; ( G ) cell viability after AICAR treatment. * P

    Techniques Used:

    39) Product Images from "Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin"

    Article Title: Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-112

    AMPK attenuated resistin-increased SK-Hep1 cell adhesions to HUVECs through activating the NF-κB. (A) SK-Hep1 cells were kept as the control or treated with resistin (50 ng/mL) for 1, 2 and 4 h. (B) SK-Hep1 cells were kept as the control pretreated with DMSO or NF-κB inhibitors, PDTC or SN50, to inhibit activity. They were then treated with resistin (50 ng/mL) for 4 h. (C) SK-Hep1 cells were kept as the control or pretreated with AICAR at different concentrations (i.e. 0, 0.1, 0.5 and 1 ng/ml) for 1 h and then kept as the control or treated with resistin (50 ng/mL) for 4 h. (D) SK-Hep1 cells were treated with resistin (50 ng/mL) or AICAR at different concentrations (i.e. 0, 0.1, 0.5 and 1 ng/ml) for 4 h. *, P
    Figure Legend Snippet: AMPK attenuated resistin-increased SK-Hep1 cell adhesions to HUVECs through activating the NF-κB. (A) SK-Hep1 cells were kept as the control or treated with resistin (50 ng/mL) for 1, 2 and 4 h. (B) SK-Hep1 cells were kept as the control pretreated with DMSO or NF-κB inhibitors, PDTC or SN50, to inhibit activity. They were then treated with resistin (50 ng/mL) for 4 h. (C) SK-Hep1 cells were kept as the control or pretreated with AICAR at different concentrations (i.e. 0, 0.1, 0.5 and 1 ng/ml) for 1 h and then kept as the control or treated with resistin (50 ng/mL) for 4 h. (D) SK-Hep1 cells were treated with resistin (50 ng/mL) or AICAR at different concentrations (i.e. 0, 0.1, 0.5 and 1 ng/ml) for 4 h. *, P

    Techniques Used: Activity Assay

    40) Product Images from "The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer"

    Article Title: The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer

    Journal: Oncotarget

    doi:

    Inhibition of FASN by TCS causes cytotoxicity in PCa cells (A) Proliferation as a function of cell confluence. LNCaP cells were treated with control (DMSO) or the indicated concentrations of inhibitors of de novo FA synthesis, and confluence was measured every 2 h for 96 h on an IncuCyte FLR system (left panel). IC50 values for treatment of LNCaP cells for 48 h were calculated by non-linear regression analysis (n=3 ±SD, middle panel). The structures of the lipogenic inhibitors are shown (right panel). (B) Representative images of A after 24 h of treatment with control (DMSO) or inhibitors (TCS 7.5 μM, C75 20 μM, orlistat 30 μM and TOFA 20 μM). (C) IC50 values for the indicated time points were calculated from the data in A (n=3 ±SD). (D) Western blot analysis of key lipogenic and energy sensing enzymes. LNCaP cells were treated for 24 h with control (DMSO) or the indicated inhibitors (TCS 10 μM, C75 20 μM, orlistat 20 μM and TOFA 10 μM), and protein lysates were probed with antibodies directed against the indicated proteins. As controls, LNCaP cells were treated with the AMPK activators metformin (2 mM) and AICAR (0.5 mM). For quantification, total protein levels were normalized relative to loading control (eIF2α). The level of protein phosphorylation was calculated relative to the normalized total amount of the respective protein. For better clarity, irrelevant lanes were removed from the image as indicated by the gaps. (E) LNCaP cells were treated with control (DMSO), 10 μM TCS or 20 μM C75 for the indicated times, and FASN expression was analyzed as in D. (F) Cytotoxicity of TCS and orlistat is mediated by FA starvation. LNCaP cells were treated with control (DMSO), 7.5 μM TCS, 40 μM C75, 10 μM orlistat, 5 μM palmitate (PA), or a combination of FASN inhibitor with PA (TCS+PA, C75+PA and orlistat+PA), and proliferation was measured for 96 h as described in A. (G) Representative images of F after 24 h of incubation.
    Figure Legend Snippet: Inhibition of FASN by TCS causes cytotoxicity in PCa cells (A) Proliferation as a function of cell confluence. LNCaP cells were treated with control (DMSO) or the indicated concentrations of inhibitors of de novo FA synthesis, and confluence was measured every 2 h for 96 h on an IncuCyte FLR system (left panel). IC50 values for treatment of LNCaP cells for 48 h were calculated by non-linear regression analysis (n=3 ±SD, middle panel). The structures of the lipogenic inhibitors are shown (right panel). (B) Representative images of A after 24 h of treatment with control (DMSO) or inhibitors (TCS 7.5 μM, C75 20 μM, orlistat 30 μM and TOFA 20 μM). (C) IC50 values for the indicated time points were calculated from the data in A (n=3 ±SD). (D) Western blot analysis of key lipogenic and energy sensing enzymes. LNCaP cells were treated for 24 h with control (DMSO) or the indicated inhibitors (TCS 10 μM, C75 20 μM, orlistat 20 μM and TOFA 10 μM), and protein lysates were probed with antibodies directed against the indicated proteins. As controls, LNCaP cells were treated with the AMPK activators metformin (2 mM) and AICAR (0.5 mM). For quantification, total protein levels were normalized relative to loading control (eIF2α). The level of protein phosphorylation was calculated relative to the normalized total amount of the respective protein. For better clarity, irrelevant lanes were removed from the image as indicated by the gaps. (E) LNCaP cells were treated with control (DMSO), 10 μM TCS or 20 μM C75 for the indicated times, and FASN expression was analyzed as in D. (F) Cytotoxicity of TCS and orlistat is mediated by FA starvation. LNCaP cells were treated with control (DMSO), 7.5 μM TCS, 40 μM C75, 10 μM orlistat, 5 μM palmitate (PA), or a combination of FASN inhibitor with PA (TCS+PA, C75+PA and orlistat+PA), and proliferation was measured for 96 h as described in A. (G) Representative images of F after 24 h of incubation.

    Techniques Used: Inhibition, Western Blot, Expressing, Incubation

    Related Articles

    Transfection:

    Article Title: Convergence of IPMK and LKB1-AMPK Signaling Pathways on Metformin Action
    Article Snippet: .. Thirty hours after transfection, cells were treated with metformin (1mM–8mM) or AICAR (0.1mM–4mM) for 2 hours (Sigma). .. For Cre recombinase experiments, IPMK MEFs (flox/flox) cells were transfected with either Myc or Myc-Cre targeting IPMK with PolyFect reagent, and 30 hours after transfection, cells were treated with metformin or AICAR.

    other:

    Article Title: AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function
    Article Snippet: Chemicals and reagents AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), rapamycin, diclofenac, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and collagenase were purchased from Sigma Aldrich.

    Article Title: Hypothalamic AMP-Activated Protein Kinase Regulates Glucose Production
    Article Snippet: Treatments included 5% DMSO, 50 μmol/l AMPK inhibitor compound C (dissolved in 5% DMSO; Calbiochem), 2 mmol/l glucose or 5 mmol/l lactate alone, 25 mmol/l AMPK activator AICAR (dissolved in saline; Sigma) plus 2 mmol/l glucose, 25 mmol/l AICAR plus 5 mmol/l lactate, and vehicle (either saline or 25 mmol/l AICAR).

    Injection:

    Article Title: Sestrin2, as a negative feedback regulator of mTOR, provides neuroprotection by activation AMPK phosphorylation in neonatal hypoxic-ischemic encephalopathy in rat pups
    Article Snippet: .. A total of 1 μl of the siRNA was injected per pup over 5 min. AICAR (30 mg/kg, Sigma-Aldrich, USA), dorsomorphin (10 mg/kg, Sigma-Aldrich, USA) and rapamycin (3 mg/kg, Cayman Chemical) were administered intraperitoneally at 1 h, 0.5 h, and 0.5 h after HIE respectively. ..

    Cell Culture:

    Article Title: AMP-activated protein kinase (AMPK) activation inhibits nuclear translocation of Smad4 in mesangial cells and diabetic kidneys
    Article Snippet: .. Cell culture reagents were purchased from GIBCO/Invitrogen (Carlsbad, CA), AICAR and lactacystin were obtained from Sigma-Aldrich (St. Louis, MO). pAMPKT172 and AMPKα antibodies were purchased from Cell Signaling Technology (Danvers, MA). .. Smad4, Smad2/3, pSmad2, pSmad3, histone H3, and β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

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    Stimulus specific regulation of transcriptional responses by <t>AICAR.</t> ( A ) mRNA Expression of SOCS3 in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM <t>ABT-702.</t> ( B ) mRNA Expression of CCL18 in macrophages treated for 24 h with 20 ng/ml IL-4, 1 mM AICAR and 0.5 µM ABT-702. ( C ) mRNA Expression of SLC2A1 in macrophages treated for 24 h with 1 mM DMOG, 1 mM AICAR, and 0.5 µM ABT-702. ( D ) Western blot analysis of STAT3 phosphorylation in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. *p
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    Stimulus specific regulation of transcriptional responses by AICAR. ( A ) mRNA Expression of SOCS3 in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. ( B ) mRNA Expression of CCL18 in macrophages treated for 24 h with 20 ng/ml IL-4, 1 mM AICAR and 0.5 µM ABT-702. ( C ) mRNA Expression of SLC2A1 in macrophages treated for 24 h with 1 mM DMOG, 1 mM AICAR, and 0.5 µM ABT-702. ( D ) Western blot analysis of STAT3 phosphorylation in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. *p

    Journal: Scientific Reports

    Article Title: AICAR inhibits NFκB DNA binding independently of AMPK to attenuate LPS-triggered inflammatory responses in human macrophages

    doi: 10.1038/s41598-018-26102-3

    Figure Lengend Snippet: Stimulus specific regulation of transcriptional responses by AICAR. ( A ) mRNA Expression of SOCS3 in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. ( B ) mRNA Expression of CCL18 in macrophages treated for 24 h with 20 ng/ml IL-4, 1 mM AICAR and 0.5 µM ABT-702. ( C ) mRNA Expression of SLC2A1 in macrophages treated for 24 h with 1 mM DMOG, 1 mM AICAR, and 0.5 µM ABT-702. ( D ) Western blot analysis of STAT3 phosphorylation in macrophages treated for 1 h with 20 ng/ml IL-6 or IL-10, 1 mM AICAR and 0.5 µM ABT-702. *p

    Article Snippet: Cells were treated with the following reagents: 1 mM AICAR (EMD Biosciences), 0.5 µM ABT-702 (Tocris), 1 mM DMOG (Cayman), 100 ng/ml lipopolysaccharide (Sigma-Aldrich), 20 ng/ml IL-6, IL-10 or IL-4 (all Immunotools).

    Techniques: Expressing, Western Blot

    AICAR suppresses LPS transcriptional response. ( A , B ) mRNA expression of TNFα and IL-6 in macrophages treated for 3 h with 100 ng/ml LPS and indicated concentrations of AICAR ( A ) or with 100 ng/ml LPS, 0.1 mM AICAR and 0.5 µM ABT-702 ( B ). ( C ) mRNA expression of LPS-induced genes in macrophages treated for 1 h with 100 ng/ml LPS and 1 mM AICAR. ( D ) Cytokine secretion into culture medium of macrophages treated for 24 h with 100 ng/ml LPS and 1 mM AICAR. *p

    Journal: Scientific Reports

    Article Title: AICAR inhibits NFκB DNA binding independently of AMPK to attenuate LPS-triggered inflammatory responses in human macrophages

    doi: 10.1038/s41598-018-26102-3

    Figure Lengend Snippet: AICAR suppresses LPS transcriptional response. ( A , B ) mRNA expression of TNFα and IL-6 in macrophages treated for 3 h with 100 ng/ml LPS and indicated concentrations of AICAR ( A ) or with 100 ng/ml LPS, 0.1 mM AICAR and 0.5 µM ABT-702 ( B ). ( C ) mRNA expression of LPS-induced genes in macrophages treated for 1 h with 100 ng/ml LPS and 1 mM AICAR. ( D ) Cytokine secretion into culture medium of macrophages treated for 24 h with 100 ng/ml LPS and 1 mM AICAR. *p

    Article Snippet: Cells were treated with the following reagents: 1 mM AICAR (EMD Biosciences), 0.5 µM ABT-702 (Tocris), 1 mM DMOG (Cayman), 100 ng/ml lipopolysaccharide (Sigma-Aldrich), 20 ng/ml IL-6, IL-10 or IL-4 (all Immunotools).

    Techniques: Expressing

    Effect of metformin on TR4 activity. A : AMPK and phosphor-AMPK in Hepa1–6 cells treated with metformin. B : TR4 phosphorylation status by Western blot. Hepa1–6 cells were treated with AICAR and metformin, and cell lysates were harvested. Cell lysates were then treated with CIP. C : DR1 × 3-Luc reporter vector was cotransfected with TR4 in Hepa1–6 cells. After overnight recovery, the transfected cells were treated with metformin and luciferase activity was measured (* P

    Journal: Diabetes

    Article Title: Metformin Inhibits Nuclear Receptor TR4-Mediated Hepatic Stearoyl-CoA Desaturase 1 Gene Expression With Altered Insulin Sensitivity

    doi: 10.2337/db10-0393

    Figure Lengend Snippet: Effect of metformin on TR4 activity. A : AMPK and phosphor-AMPK in Hepa1–6 cells treated with metformin. B : TR4 phosphorylation status by Western blot. Hepa1–6 cells were treated with AICAR and metformin, and cell lysates were harvested. Cell lysates were then treated with CIP. C : DR1 × 3-Luc reporter vector was cotransfected with TR4 in Hepa1–6 cells. After overnight recovery, the transfected cells were treated with metformin and luciferase activity was measured (* P

    Article Snippet: We used 500 μmol/L metformin (Sigma-Aldrich), 1 mmol/L AICAR (Calbiochem, San Diego, CA), and 10 μmol/L CpdC (Dorsomorphin; Calbiochem).

    Techniques: Activity Assay, Western Blot, Plasmid Preparation, Transfection, Luciferase

    (A) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMPK agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). Total cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-PAK and anti-PAK antibodies. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-paxillin and anti-paxillin antibodies was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence of metformin. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-PAK and anti-phospho-paxillin antibodies was performed. Blotting with anti-PAK and anti-paxillin antibodies was conducted as a protein loading control. The results shown are from three independent experiments.

    Journal: Endocrinology and Metabolism

    Article Title: Vav3, a GEF for RhoA, Plays a Critical Role under High Glucose Conditions

    doi: 10.3803/EnM.2014.29.3.363

    Figure Lengend Snippet: (A) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMPK agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). Total cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-PAK and anti-PAK antibodies. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-paxillin and anti-paxillin antibodies was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence of metformin. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-PAK and anti-phospho-paxillin antibodies was performed. Blotting with anti-PAK and anti-paxillin antibodies was conducted as a protein loading control. The results shown are from three independent experiments.

    Article Snippet: Metformin and AICAR were obtained from Calbiochem (San Diego, CA, USA).

    Techniques: Cell Culture, Western Blot

    (A) Total RNA was cultured under high glucose culture conditions in the presence or absence of 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR) treatment, and reverse transcription-polymerase chain reaction (RT-PCR) was conducted using specific Vav3 primers. PCR products were then run on 1% agarose gels and visualized under ultraviolet light. β-Actin mRNA was employed as a positive control. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). The cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 antibody. Blotting with anti-β-actin antibody was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPK antibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. (D) C2C12 cells were cultured under high glucose conditions in the presence or absence of metformin. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPKantibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. The results shown are from three independent experiments.

    Journal: Endocrinology and Metabolism

    Article Title: Vav3, a GEF for RhoA, Plays a Critical Role under High Glucose Conditions

    doi: 10.3803/EnM.2014.29.3.363

    Figure Lengend Snippet: (A) Total RNA was cultured under high glucose culture conditions in the presence or absence of 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR) treatment, and reverse transcription-polymerase chain reaction (RT-PCR) was conducted using specific Vav3 primers. PCR products were then run on 1% agarose gels and visualized under ultraviolet light. β-Actin mRNA was employed as a positive control. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). The cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 antibody. Blotting with anti-β-actin antibody was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPK antibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. (D) C2C12 cells were cultured under high glucose conditions in the presence or absence of metformin. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPKantibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. The results shown are from three independent experiments.

    Article Snippet: Metformin and AICAR were obtained from Calbiochem (San Diego, CA, USA).

    Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control, Western Blot

    SFM- and AICAR-induced GAPDH translocation in AMPK siRNA-transfected HDFs. HDFs were grown in 10% FBS medium for 1 day and transfected with siRNAs of AMPKα1/α2 and a si CONTROL complete kit as a negative mock control for 2 days. Cells were

    Journal: Experimental & Molecular Medicine

    Article Title: Activation of AMP-activated protein kinase stimulates the nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in human diploid fibroblasts

    doi: 10.3858/emm.2010.42.4.025

    Figure Lengend Snippet: SFM- and AICAR-induced GAPDH translocation in AMPK siRNA-transfected HDFs. HDFs were grown in 10% FBS medium for 1 day and transfected with siRNAs of AMPKα1/α2 and a si CONTROL complete kit as a negative mock control for 2 days. Cells were

    Article Snippet: The followings are the reagents used in this study and their sources: PDGF-BB, LPA, LMB, FITC-conjugated goat antimouse secondary antibody, and mouse anti-β-actin monoclonal antibody from Sigma-Aldrich (St. Louis, MO); DMEM, FBS, penicillin, and streptomycin from Gibco/BRL Life Technologies, Inc (Carlsbad, CA); PI3K inhibitor LY 294002, anti-rabbit monoclonal antibodies against phospho-AMPKα (Thr172 ) and GSK-3β (Ser9 ), anti-mouse monoclonal antibodies against phospho-Bad (Ser136 ) and phospho-Akt (Ser473 ), and anti-rabbit polyclonal antibodies against AMPK-α, Akt, and GSK-3β from Cell Signaling Technology (Denver, MA); compound C, AICAR, SH-5, L-NAME, and L-NMMA from Calbiochem (San Diego, CA); mouse anti-GAPDH monoclonal antibody from Chemicon (Bedford, MA); anti-rabbit monoclonal antibodies against ACC1 and phospho-ACC1 (Ser79 ) from Upstate (Waltham, MA); horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies from Vector Laboratories (Burlingame, CA); Lipofectamine™ RNAiMAX and mounting solution including DAPI from Invitrogen Life Technologies (Carlsbad, CA); protein assay kit from Bio-Rad Laboratories (Hercules, CA); enhanced chemiluminescence (ECL) system and Amersham hyperfilm™ ECL from GE Healthcare (Buckinghamshire, UK); sense and complement strands of human AMPK-α1 and α2 siRNAs, si CONTROL complete kit, and 5 × siRNA buffer from Dharmacon Inc. (Lafayette, CO); 5'-ITC from Biomol Research Labs (Plymouth Meeting, PA).

    Techniques: Translocation Assay, Transfection

    Effect of NOS inhibitors on SFM- and AICAR-induced GAPDH translocation. HDFs were cultured in 10% FBS medium, and then treated with SFM for 4 days or 2 mM AICAR in 10% FBS in the absence (-) or presence (+) of 100 µM of L-NMMA (A) or 0.5 mg/ml

    Journal: Experimental & Molecular Medicine

    Article Title: Activation of AMP-activated protein kinase stimulates the nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in human diploid fibroblasts

    doi: 10.3858/emm.2010.42.4.025

    Figure Lengend Snippet: Effect of NOS inhibitors on SFM- and AICAR-induced GAPDH translocation. HDFs were cultured in 10% FBS medium, and then treated with SFM for 4 days or 2 mM AICAR in 10% FBS in the absence (-) or presence (+) of 100 µM of L-NMMA (A) or 0.5 mg/ml

    Article Snippet: The followings are the reagents used in this study and their sources: PDGF-BB, LPA, LMB, FITC-conjugated goat antimouse secondary antibody, and mouse anti-β-actin monoclonal antibody from Sigma-Aldrich (St. Louis, MO); DMEM, FBS, penicillin, and streptomycin from Gibco/BRL Life Technologies, Inc (Carlsbad, CA); PI3K inhibitor LY 294002, anti-rabbit monoclonal antibodies against phospho-AMPKα (Thr172 ) and GSK-3β (Ser9 ), anti-mouse monoclonal antibodies against phospho-Bad (Ser136 ) and phospho-Akt (Ser473 ), and anti-rabbit polyclonal antibodies against AMPK-α, Akt, and GSK-3β from Cell Signaling Technology (Denver, MA); compound C, AICAR, SH-5, L-NAME, and L-NMMA from Calbiochem (San Diego, CA); mouse anti-GAPDH monoclonal antibody from Chemicon (Bedford, MA); anti-rabbit monoclonal antibodies against ACC1 and phospho-ACC1 (Ser79 ) from Upstate (Waltham, MA); horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies from Vector Laboratories (Burlingame, CA); Lipofectamine™ RNAiMAX and mounting solution including DAPI from Invitrogen Life Technologies (Carlsbad, CA); protein assay kit from Bio-Rad Laboratories (Hercules, CA); enhanced chemiluminescence (ECL) system and Amersham hyperfilm™ ECL from GE Healthcare (Buckinghamshire, UK); sense and complement strands of human AMPK-α1 and α2 siRNAs, si CONTROL complete kit, and 5 × siRNA buffer from Dharmacon Inc. (Lafayette, CO); 5'-ITC from Biomol Research Labs (Plymouth Meeting, PA).

    Techniques: Translocation Assay, Cell Culture