Journal: The Journal of biological chemistry

Article Title: Glucose-6-phosphate dehydrogenase exerts antistress effects independently of its enzymatic activity.

doi: 10.1016/j.jbc.2022.102587

Figure Lengend Snippet: Figure 3. G6PD facilitates AMPK activity independently of its dehydrogenase activity. A, Western blotting analysis of G6PD, pACC1-Ser79, ACC1, pAMPKα-Thr172, and AMPK in HeLa/WT and HeLa/G6PD-KO cells treated with 500 μM AICAR for 8 h or 24 h. B, cell survival of HeLa/G6PD-KO cells treated with 1 μM antimycin A for 24 h, in the presence or absence of 500 μM AICAR (pretreatment for 12 h). C, Western blotting analysis of G6PD, pACC1-Ser79, ACC1, pAMPKα-Thr172, and AMPKα in HeLa cells with different states of G6PD, as indicated, treated with 50 μM or 100 μM A769662 for 8 h. D, cell survival of HeLa/G6PD-KO cells treated with or without PMS (1 μM), H2O2 (100 μM), antimycin A (1 μM) for 24 h, in the presence or absence of 100 μM A769662 (pretreatment for 16 h). E and F, Flag-AMPKα1 immunoprecipitates from HeLa/G6PD-KO cells were aliquoted to incubate with recombinant G6PD-Wt or R198P protein in the presence or absence of ATP in an in vitro kinase assay. pACC1-Ser79, ACC1, pAMPKα-The172, and AMPKα were detected using Western blot. **p < 0.01 (t test). AICAR, 5-amino-4-imidazolecarboxamide ribonucleoside; G6PD, glucose-6-phosphate dehydrogenase.

Article Snippet: A769662 was obtained from Selleck.

Techniques: Activity Assay, Western Blot, Recombinant, In Vitro, Kinase Assay

Journal: eLife

Article Title: Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis

doi: 10.7554/eLife.74903

Figure Lengend Snippet:

Article Snippet: A769662 , DMSO , Tocris , 3336.

Techniques: Solvent

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of CPT2 exacerbates cardiac dysfunction and inflammation in experimental endotoxaemia

doi: 10.1111/jcmm.15809

Figure Lengend Snippet: Changes in body temperature and blood glucose concentration 4 h after LPS administration

Article Snippet: C75 and A769662 were purchased from Tocris Bioscience and Carbosynth Ltd., respectively.

Techniques: Concentration Assay, Control, Saline

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of CPT2 exacerbates cardiac dysfunction and inflammation in experimental endotoxaemia

doi: 10.1111/jcmm.15809

Figure Lengend Snippet: LPS‐induced changes in mitochondrial energy metabolism pattern in cardiac tissues. Respiration rate (A) and flux control factors (B) in permeabilized cardiac fibres. LPS administration decreased fatty acid oxidation pathway‐dependent oxidative phosphorylation (A,B) and induced pyruvate metabolism stimulation without affecting other pathways (B). Treatment with both C75 and A769662 restored the fatty acid oxidation pathway‐linked OXPHOS coupling efficiency and subsequently decreased pyruvate metabolism (B). Alongside after aminocarnitine administration, the fatty acid oxidation pathway‐linked OXPHOS coupling efficiency remained decreased, and pyruvate metabolism was inhibited (B). Each value represents the mean ± SEM of 5 animals. *Significant difference between saline control and LPS control groups (Student's t test, P < .05). # Significantly different from the LPS control group (ANOVA followed by Dunnett's test, P < .05). FAO, fatty acid oxidation; F(N), fatty acid oxidation‐dependent pathway (FADH 2 and NADH); N, NADH‐pathway; LEAK, substrate metabolism‐dependent state; OXPHOS, oxidative phosphorylation‐dependent state; D, ADP; P, pyruvate; PC, palmitoylcarnitine; Rot, rotenone; S, succinate

Article Snippet: C75 and A769662 were purchased from Tocris Bioscience and Carbosynth Ltd., respectively.

Techniques: Control, Phospho-proteomics, Saline

Journal:

Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H + -ATPase accumulation in epididymal clear cells

doi: 10.1152/ajpcell.00004.2009

Figure Lengend Snippet: AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibits the pH-mediated V-ATPase accumulation at the apical membrane of clear cells. A and B: confocal images of double immunofluorescence labeling for V-ATPase distribution (green) and for the endocytic marker horseradish peroxidase (HRP, red) in clear cells perfused for 60 min with phosphate-buffered saline (PBS) pH 7.8 (A) or PBS pH 7.8 + AICAR (2 mM) (B). Arrows demarcate the base of the microvilli in clear cells. Treatment of cells with the AMPK activator AICAR prevented the elongation of apical microvilli observed upon exposure to pH 7.8 and prevented accumulation of the V-ATPase at the apical pole. C and D: level of V-ATPase accumulation in microvilli in the cells from A and B was quantified by measuring the area occupied by V-ATPase-labeled microvilli (enclosed in the white line), normalized for the width of the cells at the apical pole (blue line) for each cell. E: quantification of the surface occupied by V-ATPase-labeled microvilli of clear cells normalized by the apical width of each cell under the conditions shown in A and B. Data shown are means ± SE from three tissues and at least 30 cells per condition (*P < 0.05). Scale bar = 5 μm.

Article Snippet: To further confirm that these findings resulted from AMPK activation in this epididymis/VD in vivo perfusion system, we repeated the above experiments using a second direct and specific AMPK activator, Abbott compound A-769662.

Techniques: Membrane, Immunofluorescence, Labeling, Marker, Saline

Journal:

Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H + -ATPase accumulation in epididymal clear cells

doi: 10.1152/ajpcell.00004.2009

Figure Lengend Snippet: The AMPK activator A-769662 inhibits the pH-mediated V-ATPase accumulation at the apical membrane of clear cells. A and B: confocal images of double immunofluroscence labeling of the V-ATPase distribution (green) and the endocytic marker HRP (red) in clear cells perfused for 60 min with PBS pH 7.8 (A) or PBS pH 7.8 containing AMPK activator Abbott compound A-769662 (200 μM) (B). Arrows demarcate the base of the microvilli in clear cells. Treatment of cells A-769662 prevented the elongation of apical microvilli observed upon exposure to pH 7.8 and prevented accumulation of the V-ATPase at the apical pole. C: quantification of the surface occupied by V-ATPase-labeled microvilli of clear cells normalized by the apical width of each cell under the conditions shown in A and B. Data shown are means ± SE from three tissues and at least 30 cells per condition (*P < 0.05). Scale bar = 5 μm.

Article Snippet: To further confirm that these findings resulted from AMPK activation in this epididymis/VD in vivo perfusion system, we repeated the above experiments using a second direct and specific AMPK activator, Abbott compound A-769662.

Techniques: Membrane, Labeling, Marker

Journal:

Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H + -ATPase accumulation in epididymal clear cells

doi: 10.1152/ajpcell.00004.2009

Figure Lengend Snippet: AMPK activator AICAR inhibits the PKA-mediated V-ATPase accumulation at the apical membrane of clear cells. A, B, and C: confocal images of the distribution of V-ATPase (green) and of the endocytic marker HRP (red) by immunofluorescence labeling in clear cells perfused with PBS pH 6.5 for 75 min (A); PBS pH 6.5 for 45 min, followed by PBS pH 6.5 containing PKA activator N6-monobutyryl-cAMP (6-MB-cAMP, 100 μM) for an additional 30 min (B); or PBS (pH 6.5) containing AICAR (2 mM) for 45 min, followed by PBS (pH 6.5) plus AICAR plus 6-MB-cAMP for an additional 30 min (C). Arrows demarcate the bases of the microvilli. Treatment of cells with AMPK activator AICAR prevented the elongation of apical microvilli observed upon exposure to a specific PKA activator 6-MB-cAMP and prevented accumulation of the V-ATPase at the apical pole. D: quantification of V-ATPase accumulation in apical microvilli for at least three independent perfusions and at least 30 cells per condition. Data shown are the means ± SE (*P < 0.05, relative to PBS, pH 6.5; #P < 0.05, relative to PBS, pH 6.5; **P < 0.05, relative to + 6-MB-cAMP). Scale bar = 7.5 μm.

Article Snippet: To further confirm that these findings resulted from AMPK activation in this epididymis/VD in vivo perfusion system, we repeated the above experiments using a second direct and specific AMPK activator, Abbott compound A-769662.

Techniques: Membrane, Marker, Immunofluorescence, Labeling

Journal:

Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H + -ATPase accumulation in epididymal clear cells

doi: 10.1152/ajpcell.00004.2009

Figure Lengend Snippet: PKA- and AMPK-dependent in vivo phosphorylation of the V-ATPase A subunit in HEK-293 cells. FLAG-tagged A subunit was transfected into HEK-293 cells expressing an irrelevant mammalian shRNA (Fig. 7, “CON” cells) or in AMPK KD cells expressing an shRNA for AMPK-α1. Cells were then incubated with [32P]orthophosphate for 2 h in the presence of a PKA activator (6-MB-cAMP, last 20 min of labeling period) or in the presence of PKA inhibitor myristoylated protein kinase inhibitor (mPKI) (for entire labeling period), followed by lysis of the cells, immunoprecipitation using an anti-FLAG antibody, SDS-PAGE, and immunoblotting using an anti-FLAG antibody. A: typical phospho-screen image (top) revealing the signal of phosphorylated A subunit under the indicated conditions in CON or AMPK KD cells. The Western blot (bottom) confirms similar protein expression and loading of the gel for the different conditions. B: quantification of V-ATPase A subunit phosphorylation signal normalized for protein expression in vivo (*P = 1.6 × 10−5; **P = 1.3 × 10−4; #P = 3.7 × 10−4; ##P = 3.2 × 10−6; §P = 4.9 × 10−6, and §§P = 0.016) indicates that the differences between the indicated conditions were statistically significant by analysis of variance; (n = 4 experiments). C: relative PKA-dependent A subunit phosphorylation in CON and AMPK KD cells as calculated by the difference in phosphorylation signal between 6-MB-cAMP- and mPKI-treated cells over the total phosphorylation signal with 6-MB-cAMP treatment (comparing lanes 2 and 3 for CON and lanes 5 and 6 for AMPK KD). *P < 0.05, unpaired t-test.

Article Snippet: To further confirm that these findings resulted from AMPK activation in this epididymis/VD in vivo perfusion system, we repeated the above experiments using a second direct and specific AMPK activator, Abbott compound A-769662.

Techniques: In Vivo, Phospho-proteomics, Transfection, Expressing, shRNA, Incubation, Labeling, Lysis, Immunoprecipitation, SDS Page, Western Blot

Journal: Redox Biology

Article Title: AMPK leads to phosphorylation of the transcription factor Nrf2, tuning transactivation of selected target genes

doi: 10.1016/j.redox.2019.101393

Figure Lengend Snippet: Nrf2 is AMPK-dependently phosphorylated at serine 374, 408 and 433, interacts with AMPK and is an AMPK substrate . ( A) AMPK wt or AMPKα1 −/− MEF were transiently transfected with EGFP-WT-Nrf2 and treated with AMPK activator A769662 (50 μM, 4 h) and proteasome inhibitor (MG132, 20 μM). Accumulated Nrf2 was pulled down from cell lysates and after tryptic/chymotryptic digestion subjected to LC-MS analysis and screened for AMPK-dependent phosphosites. For S 374, 408 and 433 the relative amount (area by LFQ) of phosphosite specific peptides normalized to the total amount of Nrf2 is shown for WT and AMPKα1 −/−MEF (S 215 depicted as presumably AMPK independent site). Dots show the obtained area in single biological replicates, bars indicate the mean. (B) HEK cells were transfected with expression plasmids for EGFP-WT-Nrf2, PRKAA1 (AMPK) or EGFP as indicated. After stabilization of Nrf2 (via proteasome inhibition), lysis and pulldown via GFP-Trap®, eluates as well as an aliquot (20 μg protein) of the unprecipitated lysate (input control) were immunoblotted for GFP (= EGFP-WT-Nrf2 or EGFP) or AMPKα1. Representative blots and compiled densitometric analyses (AMPK signal/GFP signal) are depicted (n = 3, unpaired Student's t-test, two tailed, α = 0.05, *P < 0.05). (C) Recombinant AMPK and Nrf2 were subjected to an in vitro kinase assay with varying ratios of ATP to AMP (as indicated, fixed ATP concentration of 200 μM). After termination of the reaction, Nrf2 was prepared for and subjected to MS analysis for phosphosite identification and quantification. The obtained area of the most abundant peptide of Nrf2 for each indicated phosphosite was normalized as follows: [area P peptide/(area P peptide + area same peptide without phosphorylation)], which gives the degree of phosphorylation. A value of 1 would indicate that 100% of the detected peptide was phosphorylated at that position and no unphosphorylated peptide was found. (n = 3, mean +SD, unpaired Student's t-test, two tailed, α = 0.05, *P < 0.05, ns: not significant).

Article Snippet: Sulforaphane (Sfn) and cycloheximide (CHX) were purchased from Sigma –Aldrich (Vienna, Austria), A769662 and MG132 came from ApexBio (Houston, TX, USA), dorsomorphin (= Compound C) was obtained from Abcam (Cambridge, UK).

Techniques: Transfection, Liquid Chromatography with Mass Spectroscopy, Expressing, Inhibition, Lysis, Two Tailed Test, Recombinant, In Vitro, Kinase Assay, Concentration Assay

Journal: Redox Biology

Article Title: AMPK leads to phosphorylation of the transcription factor Nrf2, tuning transactivation of selected target genes

doi: 10.1016/j.redox.2019.101393

Figure Lengend Snippet: The identified AMPK-dependent phosphosites in Nrf2 do not markedly influence half-life of Nrf2 in wt cells. (A) HEK293 were transfected with EGFP-tagged WT or TM-Nrf2 expression plasmids and treated with MG132 (20 μM) for 2.5 h (t = −2.5 to 0) to accumulate Nrf2. After washout of MG132 the translation inhibitor cycloheximide (100 μM) was added (time point 0) for another incubation period of 1–5 h as indicated. Cell lysates were subjected to immunoblot analysis for EGFP-Nrf2 (via α-GFP antibody) or actin. Representative blot and compiled densitometric evaluations (below) for EGFP-Nrf2/actin (related to signal at t = 0) are depicted. (n ≥ 3, mean +SD, unpaired Student's t-test, two tailed, α = 0.05, *P < 0.05, ns: not significant). (B) Wt MEF were transfected with EGFP-tagged WT or TM-Nrf2 expression plasmids and treated with MG132 (10 μM) for 1 h to accumulate Nrf2. After washout, cycloheximide (30 μM) was added (time point 0) for the indicated periods of time. Cell lysates were subjected to immunoblot analysis for EGFP-Nrf2 (via α-GFP antibody) or actin. Representative blots and compiled densitometric evaluations (below) for EGFP-Nrf2/actin (related to signal at t = 0) are depicted. (n = 3, mean +SD, unpaired Student's t-test, two tailed, α = 0.05, *P < 0.05, ns: not significant). (C) Wt MEF were transfected with EGFP-tagged WT- or TM-Nrf2 expression plasmids and treated with MG132 (10 μM) and DMSO or A769662 (50 μM) for 1 h. After washout of MG132 the translation inhibitor cycloheximid (30 μM) was added (time point 0) in the presence of A769662 (50 μM) (+) or DMSO (−) for the indicated period of time. Cell lysates were subjected to immunoblot analysis for EGFP, pACC or actin. Representative blots and compiled densitometric evaluations for DMSO and A769662-treated cells (EGFP-WT- or EGFP-TM-Nrf2/actin, related to signal at t = 0) are depicted (n = 3, mean +SD).

Article Snippet: Sulforaphane (Sfn) and cycloheximide (CHX) were purchased from Sigma –Aldrich (Vienna, Austria), A769662 and MG132 came from ApexBio (Houston, TX, USA), dorsomorphin (= Compound C) was obtained from Abcam (Cambridge, UK).

Techniques: Transfection, Expressing, Incubation, Western Blot, Two Tailed Test

Journal: Redox Biology

Article Title: AMPK leads to phosphorylation of the transcription factor Nrf2, tuning transactivation of selected target genes

doi: 10.1016/j.redox.2019.101393

Figure Lengend Snippet: Mutation of the AMPK-dependent phosphosites to alanine stabilizes Nrf2 in Keap1−/− cells and impedes interaction with β-TrCP. (A) Keap1−/− MEF were transfected with EGFP-tagged WT- or TM-Nrf2 expression plasmids, pretreated with A76996 (50 μM, 1h) as indicated and then exposed to cycloheximid (30 μM) for different periods of time. Cell lysates were subjected to immunoblot analysis for EGFP or actin. Representative blots and compiled densitometric evaluations (EGFP-WT/TM-Nrf2/actin, related to signal at t = 0) are depicted (n = 3, mean +SD, unpaired Student's t-test, two tailed, α = 0.05, different superscript letters indicate differences with p < 0.05). (B) HEK cells were transfected with an expression plasmid for myc-tagged β-TrCP together with constructs encoding EGFP-WT-Nrf2, the triple mutated version EGFP-TM-Nrf2, or EGFP as indicated. Cells were treated with vehicle (DMSO) or A769662 (50 μM) 1 h prior to addition of MG132 (20 μM) for further 3 h to stabilize Nrf2, which was pulled down using GFP-Trap®. (Co) precipitated proteins as well as input controls were immunoblotted for myc (=β-TrCP) or GFP (=EGFP-WT-/EGFP-TM-Nrf2 or EGFP) as indicated. Representative blot pictures and densitometric evaluations (relative myc (β-TrCP)/GFP (Nrf2) signal) are depicted. (n = 6, mean +SD; *P < 0.05, Welch's t -test, two tailed, α = 0.05).

Article Snippet: Sulforaphane (Sfn) and cycloheximide (CHX) were purchased from Sigma –Aldrich (Vienna, Austria), A769662 and MG132 came from ApexBio (Houston, TX, USA), dorsomorphin (= Compound C) was obtained from Abcam (Cambridge, UK).

Techniques: Mutagenesis, Transfection, Expressing, Western Blot, Two Tailed Test, Plasmid Preparation, Construct