Journal: Cell Death & Disease

Article Title: Therapeutic metformin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy

doi: 10.1038/cddis.2012.13

Figure Lengend Snippet: Dysregulation of AMPK/mTOR pathway in human lymphoma. Expression profile of phosphorylated form of AMPK, mTOR, as well as its downstream effectors p70S6K and 4EBP1 was assessed in human lymphoma tissue using tissue array. The melanoma tissue was served as the positive control of phosphorylated AMPK

Article Snippet: The AMPK inhibitor compound C and the autophagy inhibitor 3-methyladenine were from Merck KGaA (Darmstadt, Germany).

Techniques: Expressing, Positive Control

Journal: Cell Death & Disease

Article Title: Therapeutic metformin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy

doi: 10.1038/cddis.2012.13

Figure Lengend Snippet: The inhibitory effect of metformin on growth of human lymphoma cell lines. ( a ) IC 50 results obtained from MTT assay in a series of B- and T-lymphoma cell lines after metformin treatment. ( b and c ) Response curves ( b ) and AMPK/ACC expression detected by western blot ( c ) in malignant B and T cells treated with metformin. ( d ) Metformin inhibited growth of primary lymphoma cells. ( e ) The proliferation of CD34+ cells enriched from cord blood samples was not altered by metformin up to 120 mM

Article Snippet: The AMPK inhibitor compound C and the autophagy inhibitor 3-methyladenine were from Merck KGaA (Darmstadt, Germany).

Techniques: MTT Assay, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Therapeutic metformin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy

doi: 10.1038/cddis.2012.13

Figure Lengend Snippet: Metformin-induced mTOR inhibition in an AMPK-dependent manner. ( a ) Metformin decreased the expression of phosphorylated mTOR, as well as its effectors p70S6K and 4EBP1. ( b and c ) Specific AMPK inhibitor compound C ( b ) and AMPK siRNA ( c ) abrogated metformin-induced lymphoma cell growth inhibition and cell cycle arrest. Comparing with the negative control (Con siRNA), Jurkat cells transfected with AMPK- α siRNA (AMPK siRNA) failed to induce phosphorylated ACC and inhibit phosphorylated mTOR. * P <0.001 comparing with the metformin group. + P <0.001 comparing with the Con siRNA group. ( d ) The expression of phosphorylated AKT remained unchanged during metformin treatment in Jurkat cells. ( e ) Cell sensitivity to metformin was not affected in Jurkat cells transfected with AKT

Article Snippet: The AMPK inhibitor compound C and the autophagy inhibitor 3-methyladenine were from Merck KGaA (Darmstadt, Germany).

Techniques: Inhibition, Expressing, Negative Control, Transfection

Journal: Cell Death & Disease

Article Title: Therapeutic metformin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy

doi: 10.1038/cddis.2012.13

Figure Lengend Snippet: The effect of metformin in murine lymphoma xenograft models. ( a ) In both B- and T-cell lymphoma models, intraperitoneal treatment of metformin, both at the high-dose (IP MET H) and low-dose (IP MET L) group, significantly diminished xenograft tumor size. ( b ) As compared with the control group (Con), decreased proliferative index Ki-67 was identified, while TUNEL staining was negative in the metformin group (MET). Immunohistochemical study showed increased numbers of cells positive for phosphorylated AMPK, but decreased cells positive for phosphorylated mTOR in the metformin-treated tumors

Article Snippet: The AMPK inhibitor compound C and the autophagy inhibitor 3-methyladenine were from Merck KGaA (Darmstadt, Germany).

Techniques: Control, TUNEL Assay, Staining, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: Therapeutic metformin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy

doi: 10.1038/cddis.2012.13

Figure Lengend Snippet: Metformin enhanced lymphoma cell sensitivity to the chemotherapeutic agent and stimulate autophagy. ( a ) In Daudi and Jurkat cells, metformin increased the cytotoxicity of the chemotherapeutic agent doxorubicin. ( b ) Metformin combined with doxorubicin induced less lymphoma cell growth inhibition in the AMPK siRNA group than in the Con siRNA group, * P <0.001 comparing with the Con siRNA group. ( c ) Autophagy-related LC3-II expression was significantly elevated in cells co-treated with metformin and doxorubicin. ( d ) Autophagy inhibitor 3-methyladenine abrogated lymphoma cell growth inhibition induced by co-treatment. ( e ) Combined oral metformin with doxorubicin significantly inhibited xenograft lymphoma growth, comparing with the control group or each agent alone. ( f ) The metformin/doxorubicin combination frequently induced lymphoma cell autophagy

Article Snippet: The AMPK inhibitor compound C and the autophagy inhibitor 3-methyladenine were from Merck KGaA (Darmstadt, Germany).

Techniques: Inhibition, Expressing, Control

Journal: Cell Death & Disease

Article Title: Therapeutic metformin/AMPK activation blocked lymphoma cell growth via inhibition of mTOR pathway and induction of autophagy

doi: 10.1038/cddis.2012.13

Figure Lengend Snippet: Metformin potentiated the effect of mTOR inhibitor to induce lymphoma cell autophagy. ( a ) In Daudi and Jurkat cells, metformin enhanced the growth-inhibitory effect of the mTOR inhibitor temsirolimus. ( b ) Metformin combined with temsirolimus induced less lymphoma cell growth inhibition in the AMPK siRNA group than in the Con siRNA group, * P <0.001 comparing with the Con siRNA group. ( c ) Co-treatment resulted in LC3-II upregulation. ( d ) Lymphoma cell growth inhibition was overcome by autophagy inhibitor 3-methyladenine. ( e ) Combined oral metformin with temsirolimus significantly inhibited xenograft lymphoma growth, comparing with the control group or each agent alone. ( f ) The metformin/temsirolimus combination frequently induced lymphoma cell autophagy

Article Snippet: The AMPK inhibitor compound C and the autophagy inhibitor 3-methyladenine were from Merck KGaA (Darmstadt, Germany).

Techniques: Inhibition, Control

Journal: Journal of Inflammation Research

Article Title: Irisin Ameliorates PM2.5-Induced Acute Lung Injury by Regulation of Autophagy Through AMPK/mTOR Pathway

doi: 10.2147/JIR.S390497

Figure Lengend Snippet: Irisin improved impaired autophagy flux triggered by PM2.5 through AMPK/mTOR pathway. ( A ) Transmission electron microscopy images of MH-S cells with different treatment. Green Arrows represented autophagosome. Red Arrows represented autolysosome. Scale bars=2 µm in top panel (5000× magnification) and 5 µm in bottom panel (1500×magnification). ( B ) Fluorescence images of MH-S cells expressing mCherry-GFP-LC3B. Scale bars = 5 μm (400×magnification). ( C ) p-AMPK, p-mTOR, P62, LC3II protein expression by Western blotting. ( D ) Number of autophagosomes and autolysosomes in TEM. ( E ) Number of yellow and red fluorescence puncta in MH-S cells transfected with Ad-mCherry-GFP-LC3B. ( F ) Qualification of p-AMPK, p-mTOR, P62, LC3II protein expression by Western blotting. Significant differences were presented as p < 0.05. *p < 0.05, control group vs PM2.5 group. **p < 0.05, PM2.5 group vs PM2.5 +Irisin group. ***p < 0.05, PM2.5 +Irisin group vs PM2.5 +Irisin+CC group, # p < 0.05, control group vs CC group.

Article Snippet: Irisin (20 nM) or AMPK inhibitor compound C (Glpbio Technology, Montclair, USA) (10 uM) was treated 30 mins before PM2.5 administration., Then, levels of IL-1β, IL-18, and TNF-α in culture cells were determined by commercially mouse specific kits according to the manufacturer’s instructions.

Techniques: Transmission Assay, Electron Microscopy, Fluorescence, Expressing, Western Blot, Transfection, Control

Journal: Cells

Article Title: APN Expression in Serum and Corpus Luteum: Regulation of Luteal Steroidogenesis Is Possibly Dependent on the AdipoR2/AMPK Pathway in Goats

doi: 10.3390/cells12101393

Figure Lengend Snippet: APN activates the AMPK signal in goat luteal steroidogenic cells. ( A ) Luteal tissue was paraffin-embedded and processed for mIHC. P-AMPK (dark red), AMPK (olive) and DAPI staining (blue) for nucleus. NC, negative control; SLC, small luteal cells; LLC, large luteal cells. Scale bar = 20 µm. ( B ) WB for P-AMPK, AMPK and STAR of cyclic CLs. ( C ) WB for P-AMPK in mid-cycle and pregnant CLs. M, mid-cycle; P, pregnancy. ( D ) Cells were treated with 1 μg/mL APN (left) or 25 μM AdipoRon (right) for 0, 0.5, 1 and 2 h to detect P-AMPK by WB. ( E ) Cells were treated with 1 μg/mL APN or 25 μM AdipoRon for 1 h to detect whole and cytoplasmic APPL1 and LKB1 by WB. Days 0, 4, 11 and 17 represent the days in the estrous cycle, with day 0 as the onset of estrus. Data are shown as means ± SEM. A Student’s t -test was used for the comparison of two datasets. A one-way analysis of variance was followed by a Tukey’s multiple comparisons test. Statistical significance is indicated with asterisks *, ** and ***, reflecting p values of <0.05, p < 0.01 and p < 0.001, respectively.

Article Snippet: Cells were treated with APN (Sangon, Shanghai, China) [ ], AdipoRon (MedChemExpress, Monmouth Junction, NJ, USA) [ ] or AMPK antagonist (Compound C, Apexbio, Houston, TX, USA) [ , ].

Techniques: Staining, Negative Control, Comparison

Journal: Cells

Article Title: APN Expression in Serum and Corpus Luteum: Regulation of Luteal Steroidogenesis Is Possibly Dependent on the AdipoR2/AMPK Pathway in Goats

doi: 10.3390/cells12101393

Figure Lengend Snippet: APN/AdipoRon modulates steroidogenesis through the AMPK signal in goat luteal cells. ( A ) Luteal cells were pretreated with Compound C (10 μM, 1 h) prior to treatment with AdipoRon (25 μM, 1 h), and cell lysates were processed to detect P-AMPK by WB. Luteal cells were pretreated with Compound C (10 μM, 1 h) prior to treatment with AdipoRon (25 μM, 24 h), media were collected to detect the P4 concentration by RIA ( B , upper) and cell lysates were processed to detect STAR, CYP11A1 and HSD3B by WB ( C ). ( D ) Luteal cells were pretreated with SiAMPK for 48 h, then cells were treated with 1 μg/mL APN for 24 h to detect AMPK, STAR, CYP11A1 and HSD3B by WB, and media were collected to detect the P4 concentration by RIA ( B , lower). SiAMPK, SiRNA for AMPK; scrambled non-targeting RNA was transfected into cells as a negative control. Data are expressed as means ± SEM and the statistical difference was analyzed by a one-way ANOVA followed by a Tukey’s multiple comparisons test. * and ** reflect p values of <0.05 and p < 0.01, respectively.

Article Snippet: Cells were treated with APN (Sangon, Shanghai, China) [ ], AdipoRon (MedChemExpress, Monmouth Junction, NJ, USA) [ ] or AMPK antagonist (Compound C, Apexbio, Houston, TX, USA) [ , ].

Techniques: Concentration Assay, Transfection, Negative Control

Journal: eLife

Article Title: Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis

doi: 10.7554/eLife.87674

Figure Lengend Snippet: ( A ) Experimental design used to conduct real-time ATP analysis of HSCs treated with 5-FU or PBS. PLFA medium containing mitochondrial substrates (pyruvate, lactate, fatty acids, and amino acids) but no glucose, was used for experiments with 2-DG; Ba-M containing neither mitochondrial substrates nor glucose was used for experiments with oligomycin, PFKFB3 inhibitor, or AMPK inhibitor. ( B–E ) Results of real-time ATP analysis of PBS- (red) or 5-FU-treated (blue) HSCs after treatment with 2-DG ( B, D ), oligomycin ( C, E ). ( F ) Normalized mRNA counts of PFKFB isozymes based on the RNA sequencing of HSCs. ( G-J ) Results of real-time ATP analysis of PBS- (red) or 5-FU-treated (blue) HSCs after treatment with PFKFB3 inhibitor ( G, I ), or AMPK inhibitor ( H, J ). Bar graphs show corrected ATP concentrations for the last 2 min ( D ) of ( B ), 6–7 min ( E ) of ( C ), or the last 1 min ( I, J ) of ( G, H ) for PFKFB3 and AMPK inhibitors, respectively. Each group represents at least 60 cells. Data are representative results of pooled samples from three biological replicates. (see ‘Time-course analysis of FRET values’ in ‘Materials and methods’ for details of the correction method used to calculate ATP concentration.) Data are presented as mean ± SD. * p≤0.05, ** p≤0.01, *** p≤0.001 as determined by Student’s t -test (D, E, I, and J) or a one-way ANOVA followed by Tukey’s test ( F ). See also – . Figure 4—source data 1. Raw data for .

Article Snippet: In analysis using the AMPK inhibitor dorsomorphin (Cayman Chemical, Cat# 21207), CD150-APC (BioLegend, Cat# 115910) and CD48-Alexa Fluor700 (BioLegend, Cat# 103426) were used to stain LSK-SLAM to eliminate effects of dorsomorphin fluorescence on cell staining.

Techniques: RNA Sequencing, Concentration Assay

Journal: eLife

Article Title: Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis

doi: 10.7554/eLife.87674

Figure Lengend Snippet: ( A ) Experimental design of real-time ATP analysis using GO-ATeam2 knock-in BMMNCs. Ba-M was used in experiments with oligomycin. For other experiments, PLFA medium was used. ( B–C ) Evaluation of factors affecting ATP concentration in HSCs ( B ) and GMPs ( C ) based on the GO-ATeam2 system. GO-ATeam2 knock-in BMMNCs were incubated with glucose, oligomycin, 2-DG, or glucose plus oligomycin, and the FRET/EGFP ratio was calculated. ( D ) ATP concentration in indicated stem/progenitor fractions in PLFA medium (red bars) alone or PLFA medium plus 2-DG (blue bars). ATP concentration for the last 2 min of the analysis time is shown. Data is summarized from and . Each group represents at least 110 cells. Data are representative results of pooled samples from three biological replicates. ( E ) ATP concentration in indicated stem/progenitor fractions in Ba-M plus glucose (dark blue bars) or Ba-M plus glucose and oligomycin (orange bars). ATP concentration for the last 1 min of the analysis period is shown. Data is summarized from and . Each group represents at least 43 cells. Data are representative results of pooled samples from three biological replicates. ( F–I ) Effects of PFKFB3 or AMPK inhibitors (PFKFB3i or AMPKi, respectively) on ATP concentration in HSCs from GO-ATeam2 mice in Ba-M plus glucose only ( F ) or Ba-M plus glucose and oligomycin ( G ). ATP concentrations for the last 1 min of the analysis period are shown in ( H ) and ( I ) for glucose only and glucose with oligomycin groups, respectively. Each group represents at least 90 cells. Data are representative results of pooled samples from three biological replicates. ( J ) Experimental schema for cell cycle assay and real-time ATP concentration analysis after overexpression of Pfkfb3 . ( K ) Cell cycle status of Pfkfb3 -overexpressing ( Pfkfb3 OE) and mock -transduced HSCs. ( L–M ) Effects of inhibitors on ATP concentration in Pfkfb3 -overexpressing GO-ATeam2 + HSCs. Cells were exposed to vehicle or 2-DG ( L ), oligomycin in the presence or absence of glucose 12.5 mg/dL ( M ), and ATP concentrations for the last 2 min ( L ) or 1 min ( M ) of the analysis period were calculated. Data are representative results of pooled samples from three biological replicates. Data are presented as mean ± SD. * p≤0.05, ** p≤0.01, *** p≤0.001 as determined by Student’s t -test (D, E, and K) or one-way ANOVA followed by Tukey’s test (H, I, L, and M). See also . Figure 5—source data 1. Raw data for .

Article Snippet: In analysis using the AMPK inhibitor dorsomorphin (Cayman Chemical, Cat# 21207), CD150-APC (BioLegend, Cat# 115910) and CD48-Alexa Fluor700 (BioLegend, Cat# 103426) were used to stain LSK-SLAM to eliminate effects of dorsomorphin fluorescence on cell staining.

Techniques: Knock-In, Concentration Assay, Incubation, Cell Cycle Assay, Over Expression

Journal: eLife

Article Title: Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis

doi: 10.7554/eLife.87674

Figure Lengend Snippet: ( A ) Normalized Pfkfb3 mRNA counts based on RNA sequencing of PBS-treated (red) or 5-FU-treated (blue) HSCs. Data are representative results of pooled samples from three biological replicates. Data were extracted from the same pooled samples as in and . ( B ) Quantification of mean fluorescent intensity (MFI) of PFKFB3 protein in PBS- or 5-FU-treated HSCs. The lower part of the graph shows representative images of immunocytochemistry of PFKFB3 in each group. n=26–27 single HSCs for each group. The data are representative results from two independent experiments. ( C ) Quantification of MFI of phosphorylated-PFKFB3 (p-PFKFB3) protein in PBS- or 5-FU-treated HSCs. The lower part of the graph shows representative images of immunocytochemistry of p-PFKFB3 in each group. n=27 single HSCs for each group. The data are representative results from two independent experiments. ( D ) Quantification of mean fluorescence intensity (MFI) of p-PFKFB3 in HSCs treated with glucose (200 mg/dL); glucose plus oligomycin (1 µM); and glucose, oligomycin, and dorsomorphin (100 µM) for 5 min. The lower part of the graph shows representative images of immunocytochemistry of p-PFKFB3 in each group. n=32–36 for each group. The data are representative results from two independent experiments. ( E ) Normalized Prmt1 mRNA counts based on RNA sequencing of PBS-treated (red) or 5-FU-treated (blue) HSCs. Data are representative results of pooled samples from three biological replicates. ( F ) MFI quantification of methylated-PFKFB3 (m-PFKFB3) in PBS- or 5-FU-treated HSCs. The lower part of the graph shows representative images of immunocytochemistry of m-PFKFB3 in each group. n=23–41 for each group. The data are representative results from three independent experiments. ( G ) Quantification of MFI of m-PFKFB3 in PBS- or 5-FU-treated HSCs or 5-FU-treated HSCs after 15 min treatment with a PRMT1 inhibitor (90 μg/mL GSK3368715); n=25–35 single HSCs for each group. The lower part of the graph shows representative images showing immunocytochemistry of m-PFKFB3. Data represent a single experiment. ( H ) Quantitation of m-PFKFB3 in NBDG-positive or -negative HSCs in mice treated with PBS or 5-FU. The lower part of the graph shows representative images of immunocytochemistry of m-PFKFB3 in each group. n=28–41 for each group. The data are representative results from two independent experiments. ( I ) Corrected ATP levels in PBS- (red) or 5-FU-treated (blue) HSCs 15 min after treatment with vehicle or a PRMT1 inhibitor (90 µg/mL GSK3368715). Each group represents at least 101 cells. Data are representative results of pooled samples of two biological replicates. (see ‘Time-course analysis of FRET values’ in ‘Materials and methods’ for details of the correction method used to calculate ATP concentration.) ( J ) ATP concentration in mock-transduced (Ctl) or Pfkfb3 -overexpressed (OE) HSCs after treatment with the PRMT1 inhibitor (90 µg/mL GSK3368715). ATP concentration for the last 1 min of the analysis period is shown. Data are presented as mean ± SD. * p≤0.05, ** p≤0.01, *** p≤0.001 as determined by Student’s t -test (A-C, E-F, and I-J) or one-way ANOVA followed by Tukey’s test (D, G, and H). See also . Figure 6—source data 1. Raw data for .

Article Snippet: In analysis using the AMPK inhibitor dorsomorphin (Cayman Chemical, Cat# 21207), CD150-APC (BioLegend, Cat# 115910) and CD48-Alexa Fluor700 (BioLegend, Cat# 103426) were used to stain LSK-SLAM to eliminate effects of dorsomorphin fluorescence on cell staining.

Techniques: RNA Sequencing, Immunocytochemistry, Fluorescence, Methylation, Quantitation Assay, Concentration Assay

Journal: eLife

Article Title: Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis

doi: 10.7554/eLife.87674

Figure Lengend Snippet:

Article Snippet: In analysis using the AMPK inhibitor dorsomorphin (Cayman Chemical, Cat# 21207), CD150-APC (BioLegend, Cat# 115910) and CD48-Alexa Fluor700 (BioLegend, Cat# 103426) were used to stain LSK-SLAM to eliminate effects of dorsomorphin fluorescence on cell staining.

Techniques: Transplantation Assay, Generated, Cell Cycle Assay, Recombinant, Plasmid Preparation, Over Expression, Knock-Out, Flow Cytometry, ATP Assay, Sequencing, Software

Journal: PLoS ONE

Article Title: Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms

doi: 10.1371/journal.pone.0198016

Figure Lengend Snippet: INS-1E cells were cultured in the presence of 11 mM glucose and test substances as indicated and described under Materials and methods. (A,D,F) Changes of TXNIP mRNA levels (ΔΔCt) vs control set to 100% in A and D; vs max effect in F. (B,E,G) Representative western blots of 172 Thr-P-AMPKα and AMPKα1; Tubulin is used as loading control. (C) Quantitative analysis of western blots of three independent experiments of B. Results are given as mean ± SEM of n = 3–4 independent experiments. *p<0.05, **p<0.01, *** p<0.001 denotes significance to control, i.e. 11 mM Glc at 1h; ## p<0.01 to 11 mM Glc at 24 h. $ $p<0.01 significant effect of palmitate in the presence of MS. §§p<0.01 significant effect of palmitate in the presence of MS and BML. Abbreviations: Glc, glucose; Pal, palmitate; MS, MS-275 (HDAC1/2/3 inhibitor); BML, BML-275 (AMPK inhibitor); AICAR, AMPK activator.

Article Snippet: The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO.

Techniques: Cell Culture, Western Blot, Tandem Mass Spectroscopy

Journal: PLoS ONE

Article Title: Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms

doi: 10.1371/journal.pone.0198016

Figure Lengend Snippet: (A-C) INS-1E cells were cultured for 24 h in the presence of 11 mM glucose and test substances as indicated and described under Materials and methods. (A) Percentage of TUNEL positive cells, *p<0.05 significant vs control culture, (B) correlation between TUNEL positive nuclei and relative TXNIP mRNA levels (∆Ct), (C) representative western blot for cleaved caspase 3, GAPDH was used as loading control; (D) representative pictures of INS-1E cells stained with the ROS sensor CellROXGreen. INS-1E cells were cultured for 2 h in the presence of different glucose concentrations, palmitate (600 μM) and menadione (100 μM; ROS inducer) as indicated. CellROXGreen (5 μM) was applied for 1 h in culture. Note the increased green fluorescence (increased ROS levels) in the cells exposed to 30 mM Glc or menadione vs 2.8 mM glucose and 30 mM Glc+palmitate. Abbreviations: Pal, P600, palmitate; BML, BML-275 (AMPK inhibitor); CC-3, cleaved caspase-3.

Article Snippet: The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO.

Techniques: Cell Culture, TUNEL Assay, Western Blot, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms

doi: 10.1371/journal.pone.0198016

Figure Lengend Snippet: Transcription of TXNIP is under the control of a protein complex consisting of ChREBP (carbohydrate-responsive element-binding protein) and MondoA as well as the histone acetyltransferase P300. ChREBP is negatively regulated by AMPK. Consequently, stimulation of AMPK by AICAR inhibits ChREBP and TXNIP expression, while inhibition of AMPK by glucose and BML-275 activates ChREBP and increases TXNIP mRNA levels. Histone deacetylase 1 (HDAC1), counteracts P300-mediated histone acetylation, and is involved in insulin-mediated downregulation of TXNIP expression. Thus, inhibition of PI3K, AKT or HDAC1/3 increases TXNIP mRNA levels. Fatty acids-mediated stimulation of insulin secretion occurs via FFAR1/GPR40, a signalling pathway not involved in regulation of TXNIP expression. Fatty acids activate AMPK and have an additional effect on TXNIP mRNA levels. Fatty acids counteract glucose-induced TXNIP expression and ROS elevation, events which do not impede the ER strees-associated lipotoxic effect.

Article Snippet: The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO.

Techniques: Binding Assay, Expressing, Inhibition, Histone Deacetylase Assay

Journal: American Journal of Translational Research

Article Title: The roles of AMPK-mediated autophagy and mitochondrial autophagy in a mouse model of imiquimod-induced psoriasis

doi:

Figure Lengend Snippet: Effects of AMPK modulation on the mRNA expression of autophagy and mitochondrially associated proteins. The sequences of the forward and reverse primers for Atg7, P62, PINK1, and Parkin (A). The mRNA expression levels of Atg7 (B), P62 (C), PINK1 (D), and Parkin (E) in the skin after back injury in the psoriasis mouse model. Statistical analysis was conducted with one-way ANOVA, then with a Student-Newman-Keuls post hoc test. There were n=6 animals per group. Data are expressed as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001, compared with the control group. #P<0.05, ##P<0.01, ###P<0.001, compared with the IMQ + vehicle group.

Article Snippet: In the IMQ + CC group, the AMPK inhibitor CC (Cayman, USA, 10 mg/kg) was intraperitoneally injected once per day for 8 consecutive days.

Techniques: Expressing, Control

Journal: American Journal of Translational Research

Article Title: The roles of AMPK-mediated autophagy and mitochondrial autophagy in a mouse model of imiquimod-induced psoriasis

doi:

Figure Lengend Snippet: Expression levels of autophagy-associated proteins in mice after AMPK modulation. The protein-expression levels of AMPK and p-AMPK (A, B), ULK1 and p-ULK1 (C, D), Atg7 (E, F), and P62 (G, H) were assessed to determine the effects of AMPK modulation on back skin injury in a psoriasis mouse model. Statistical analysis was conducted with one-way ANOVA, then with a Student-Newman-Keuls post hoc test. There were n=6 animals per group. Data are expressed as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001, compared with the control group. #P<0.05, ###P<0.001, compared with the IMQ + vehicle group.

Article Snippet: In the IMQ + CC group, the AMPK inhibitor CC (Cayman, USA, 10 mg/kg) was intraperitoneally injected once per day for 8 consecutive days.

Techniques: Expressing, Control

Journal: American Journal of Translational Research

Article Title: The roles of AMPK-mediated autophagy and mitochondrial autophagy in a mouse model of imiquimod-induced psoriasis

doi:

Figure Lengend Snippet: Expression levels of mitophagy-associated proteins in mice after AMPK modulation. The protein-expression levels of PINK1 (A, B), Parkin (C, D), and LC3 (E, F) were evaluated to determine the effect of AMPK modulation on autophagy in a psoriasis mouse model. Statistical analysis was conducted with one-way ANOVA, then with a Student-Newman-Keuls post hoc test. There were n=6 animals per group. Data are expressed as the mean ± SD. **P<0.01, ***P<0.001, compared with the control group. ##P<0.01, ###P<0.001, compared with the IMQ + vehicle group.

Article Snippet: In the IMQ + CC group, the AMPK inhibitor CC (Cayman, USA, 10 mg/kg) was intraperitoneally injected once per day for 8 consecutive days.

Techniques: Expressing, Control