human active ampk  (Sino Biological)

Journal: Cells

Article Title: Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells.

doi: 10.3390/cells14040294

Figure Lengend Snippet: Figure 1. Aspirin has a preventive effect on mice with chemically induced CRC lesions. (A) Representative images of lesions in AOM-treated mice. Lesions were examined by colonoscopy. Tumors were counted, and each tumor scored from 1 to 5 based on the burden relative to colon circumference, according to [29]. (B) Tumor number and (C) tumor score in untreated, AOM- treated, and AOM+aspirin-treated mice. Significance was evaluated with unpaired Student’s t-test. (D) immunohistochemical analysis of c-Myc in normal colonic tissue in AOM-treated (subpanel (a)) and AOM+aspirin-treated mice (subpanel (b)). The entire intestinal tract was collected using the Swiss-rolling technique (subpanel (c)), and lesions (subpanel (d)) were identified and analyzed, both in AOM-treated (subpanel (e)) and AOM+aspirin-treated mice (subpanel (f)). Colonic cells with expression of c-Myc are indicated by black arrows and cells with no expression by red arrows. Yellow arrow indicates immune cells. Original magnification was 40× for subpanels (a,b), 4× for subpanels (c,d), and 20× for subpanels (e,f). (E) Histological analysis of colorectal adenomas. Depicted are representative c-Myc stainings of HG adenomas (subpanels (a–c): c-Myc high, intermediate, and low expression, respectively). Original magnification was 40× for all subpanels. (F) Bar graph represents quantification of c-Myc-positive cells (n = 20 samples; * p < 0.05, Student’s t-test) in samples with high or low p-AMPKα T172 immunoreactivity.

Article Snippet: Recombinant full-length human protein AMPK (α1/β1/γ1), active (P47-10H-05), and GSK3β, active (G09-10G) were from SignalChem Biotech (Richmond, BC, Canada).

Techniques: Immunohistochemical staining, Expressing

Journal: Cells

Article Title: Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells.

doi: 10.3390/cells14040294

Figure Lengend Snippet: Figure 2. AMPK activation causes loss of c-Myc. (A) Exposure of HCT116 colon cancer cells to 3 mM salicylate for 1, 4, 24, or 48 h (lanes 2 through 5) elicited AMPK activation, evaluated by phosphorylation of AMPKα at T172, followed by loss of c-Myc expression. Acetyl-CoA carboxylase (ACC) phosphorylation (S79) was used as a marker of functional AMPK activation. (B) Schematic illustration of the molecular mechanisms leading to the activation or inhibition of AMPK. Modulators of AMPK used in this study, negative as well as positive, and their expected mechanism of action are represented. (C) Decreased levels of c-Myc are associated with AMPK activation. HCT116 cells were grown in complete medium for 48 h in the absence (lane 1) or presence of salicylate (lane 2), the synthetic AMPK activator A76966 (lane 3), phenformin (lane 5), or grown in glucose-free medium for 8 h (lane 6). Cells were also treated with a combination of salicylate (3 mM) and compound C (dorsomorphin 100 nM) for 48 h (lane 4). (D) HCT116 cells were cultured in glucose-free medium for up to 5 h in the absence (lanes 1 through 6) or presence of compound C (lanes 7 through 12). Representative western blots and quantification graphs of triplicate experiments are shown. Data are presented as mean ± SEM, n = 3.

Article Snippet: Recombinant full-length human protein AMPK (α1/β1/γ1), active (P47-10H-05), and GSK3β, active (G09-10G) were from SignalChem Biotech (Richmond, BC, Canada).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Marker, Functional Assay, Inhibition, Cell Culture, Western Blot

Journal: Cells

Article Title: Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells.

doi: 10.3390/cells14040294

Figure Lengend Snippet: Figure 4. Dynamic interaction between AMPK and c-Myc regulates phosphorylation of c-Myc. En- dogenous protein complexes were immunoprecipitated from HEK293 cell lysates with an AMPK1/2 antibody before and after (A) exposure to salicylate (3 mM for 3 h) or (B) glucose deprivation (20 min). Immunoprecipitated proteins were analyzed by immunoblotting, as indicated. Normal serum IgG served as a negative control. Immunoblots of the corresponding cell lysates are shown in the lower panel (input 5%). (C) Quantitation of three replicate experiments. Conditions were compared using

Article Snippet: Recombinant full-length human protein AMPK (α1/β1/γ1), active (P47-10H-05), and GSK3β, active (G09-10G) were from SignalChem Biotech (Richmond, BC, Canada).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Negative Control, Quantitation Assay

Journal: Cells

Article Title: Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells.

doi: 10.3390/cells14040294

Figure Lengend Snippet: Figure 5. AMPK activation with sodium salicylate mediates c-Myc ubiquitinylation and nuclear depletion. (A) Exposure of HCT116 colon cancer cells to 1 or 3 mM salicylate for 48 h (lanes 2 and 3, respectively) elicited the loss of c-Myc expression accompanied by CIP2A, but no change in the levels of S62 phosphorylated c-Myc. Cells were also treated with a combination of salicylate (1 or 3 mM) and compound C (dorsomorphin 100 nM) for 8 h (lanes 5 and 6, respectively). (B) Salicylate induced phosphorylation of PP2A Y307. Samples were analyzed with an anti-pY307-PP2Ac antibody, and the blots were reprobed with an anti-PP2Ac antibody. (C) Sodium salicylate induced AMPK-mediated ubiquitinylation of c-Myc. HEK293 cells were transiently transfected with c-Myc-HA (lanes 1 through 4) or a mock construct (HA-vector, lane 5). Cells were then either left untreated (lanes 1 and 5) or treated with compound C (lane 2), sodium salicylate (lane 3), or a combination of both (lane 4), for three hours. C-Myc-HA-tagged protein was then immunopurified and its ubiquitination levels analyzed with a ubiquitin antibody by western blot (upper panel, ubiquitin). Immunoblots of the corresponding cell lysates (ubiquitin antibody) and purified proteins (HA antibody) are shown (lower

Article Snippet: Recombinant full-length human protein AMPK (α1/β1/γ1), active (P47-10H-05), and GSK3β, active (G09-10G) were from SignalChem Biotech (Richmond, BC, Canada).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Transfection, Construct, Plasmid Preparation, Ubiquitin Proteomics, Western Blot, Purification

Journal: Cells

Article Title: Salicylate-Elicited Activation of AMP-Activated Protein Kinase Directly Triggers Degradation of C-Myc in Colorectal Cancer Cells.

doi: 10.3390/cells14040294

Figure Lengend Snippet: Figure 6. Model of potential mode of action of salicylate on c-Myc. Salicylate decreases the expression of MYC mRNA at the transcriptional level. In addition, salicylate activates AMPK both through directly binding to the ADaM site and changes in the mitochondrial membrane potential, which lead to an increased AMP:ATP ratio. Activated AMPK can directly bind to c-Myc and phosphorylate it at the bHLH-LZ region (S373 and T400), disrupting DNA binding. Additionally, phosphorylation of PP2A at Y307 and downregulation of CIP2A further compound the regulatory loop, decreasing c-Myc levels and affecting the c-Myc transcriptional program. Modulation of miR-34 defines another regulatory loop, further linking AMPK activation and c-Myc expression.

Article Snippet: Recombinant full-length human protein AMPK (α1/β1/γ1), active (P47-10H-05), and GSK3β, active (G09-10G) were from SignalChem Biotech (Richmond, BC, Canada).

Techniques: Expressing, Binding Assay, Membrane, Phospho-proteomics, Activation Assay

Journal: bioRxiv

Article Title: AMPK activation promotes transcriptional activation of TFEB through its dephosphorylation

doi: 10.1101/2024.10.22.619589

Figure Lengend Snippet: (A and B) Heatmaps of gene expression of MK-8722 and torin1-regulated genes in (A) WT and TFEB/TFE3 DKO MEF and (B) in WT and AMPKα1/α2 DKO MEF. (C-F) Venn diagrams depicting significantly altered transcripts across treatments, with (C) TFEB/TFE3-regulated genes by MK-8722 and torin1 were obtained by selecting significantly downregulated genes in TFEB/TFE3 DKO MEF cells compared to DMSO WT and across treatment conditions. Significantly upregulated genes upon MK-8722 (D) and torin1 (E) treatment across WT and AMPK DKO genotypes and (F) significantly upregulated TFEB/TFE3-genes present in torin1 upregulated genes. All significant genes were defined as having a fold change (FC) ≥ 1.2 and false discovery rate (FDR) < 0.05. (G) Heatmap of the previously reported TFEB-regulated lysosomal genes present in current MK-8722- and torin1-regulated TFEB/TFE3-dependent genes. All expression data is shown as log2 counts per million (logCPM). (H, I) Lysosomal proteolytic activity expressed as fold change in DQ-BSA fluorescence in WT and (H) AMPKα1/α2 DKO MEF or (I) TFEB/TFE3 DKO MEF treated with vehicle (0.1% DMSO), 10 μM MK-8722 or 100 nM torin1 for 2 h. Representative data from three independent experiments are shown. Two-way ANOVA with Šídák’s multiple comparison was performed (* p < 0.05 vehicle vs. treatment and # p < 0.05 KO vs. WT).

Article Snippet: Recombinant human AMPK (α1β1γ1 complex, SignalChem Biotech, P47-110GH) was diluted in enzyme dilution buffer (50 mM Tris, 0.1 mM EGTA, 1 mg/ml BSA, 1 mM DTT).

Techniques: Gene Expression, Expressing, Activity Assay, Fluorescence, Comparison

Journal: bioRxiv

Article Title: AMPK activation promotes transcriptional activation of TFEB through its dephosphorylation

doi: 10.1101/2024.10.22.619589

Figure Lengend Snippet: (A) Sequence alignment of human and mouse TFEB. Sequence of immunogen used for generation of phospho-specific antibodies. Sequence of peptides used for dot blot analysis. Images of Dot blot assay for the phospho-TFEB-S466, -S467, -S466/S467 (dual) and -S469 antibodies. (B) Immunoblot images from in vitro phosphorylation of recombinant TFEB by recombinant AMPK in the presence of 1 μM MK-8722 co-incubated with 0.2 μM BAY-3827 or with 0.2 μM BAY-974. (C) In vitro AMPK activity assay in the presence of different peptides, including benchmarked peptide substrates SAMS and AMARA and peptides encompassing WT and mutated C-terminal serine residues of TFEB. (D and E) WT and AMPKα1/α2 DKO MEFs were treated with vehicle (0.1% DMSO), 10 μM MK-8722 or 100 nM torin1 for 1 hour. (D) TFEB/TFE3 DKO MEFs were used as controls. (D, E) TFEB was immunoprecipitated from protein lysates and C-terminal phospho-sites were detected by phospho-specific antibodies. pTFEB-S211 was used as a control. IgG was used as negative control for the TFEB immunoprecipitation. (F) TFEB-GFP knockin (KI) MEF were treated with vehicle (0.1% DMSO), 10 μM MK-8722 or 100 nM torin1 for 1 h. Pulldown TFEB-GFP by GFP-trap was analyzed by quantitative mass spectrometry. Phospho-peptide corresponds to TFEB (S467) quantitation based on intensity in the DMSO, MK-8722 and torin1 samples using data-dependent analysis. TFEB protein quantitation based on label-free quantitation (LFQ) in the DMSO, MK-8722 and torin1 samples using data-dependent analysis. TFEB phosphorylation in DMSO, MK-8722 and torin1 treated samples were quantified by taking ratio of the TFEB-S467 phosphorylation intensity with total TFEB quantitation.

Article Snippet: Recombinant human AMPK (α1β1γ1 complex, SignalChem Biotech, P47-110GH) was diluted in enzyme dilution buffer (50 mM Tris, 0.1 mM EGTA, 1 mg/ml BSA, 1 mM DTT).

Techniques: Sequencing, Dot Blot, Western Blot, In Vitro, Phospho-proteomics, Recombinant, Incubation, Activity Assay, Immunoprecipitation, Control, Negative Control, Knock-In, Mass Spectrometry, Quantitation Assay, Protein Quantitation

Journal: iScience

Article Title: Activation of AMP-activated protein kinase (AMPK) through inhibiting interaction with prohibitins

doi: 10.1016/j.isci.2023.106293

Figure Lengend Snippet:

Article Snippet: In brief, active recombinant human AMPK α1β1γ1 (02-113, Carna Biosciences, Kobe, Japan) or α2β1γ1 (02-114, Carna Biosciences) (0.3 nM) was incubated with indicated concentrations of RX-375 or Compound B, or 100 μM AMP in 10 μL of kinase buffer (50 mM HEPES-NaOH [pH 7.5], 1 mM EGTA, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.01% Tween 20) containing 50 nM ULight-SAMS peptide and 10 μM ATP in white OptiPlate 384-well microplates (6007290, PerkinElmer) at room temperature for 30 min. After the addition of 10 μL of detection mix (CR97–100, PerkinElmer) containing 40 mM EDTA and 2 nM Eu-conjugated antibodies to phospho-ACC (TRF0208, PerkinElmer) and incubation for an additional 1 h, phosphorylation of the peptide was determined by time-resolved fluorescence resonance energy transfer (excitation at 320 nm, emission at 615 and 665 nm).

Techniques: Produced, FLAG-tag, Control, Recombinant, Membrane, Transfection, Reverse Transcription, Silver Staining, Isolation, Cell Culture, Expressing, Plasmid Preparation, Luciferase, shRNA, Software