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: PLoS ONE

Article Title: FOXO3 Is a Glucocorticoid Receptor Target and Regulates LKB1 and Its Own Expression Based on Cellular AMP Levels via a Positive Autoregulatory Loop

doi: 10.1371/journal.pone.0042166

Figure Lengend Snippet: ( A ) MCF-10A cells were either treated with vehicle (Ctrl) and 1 µM dexamethasone (DEX) or in combination with either 1 mM AICAR (left panel) or 50 mM 2-deoxyglucose (right panel) in the presence or absence of 20 µM of the specific AMPK inhibitor “Compound C” for 18 h. Relative mRNA levels of LKB1, FOXO3 and GAPDH as internal control were analysed by semi-quantitative RT-PCR in a 1% agarose gel after ethidium bromide staining. The data shown is representative of three independent experiments. ( B ) Illustration of metabolic regulation of FOXO3’s transcriptional activity and subsequent effects on FOXO3 and LKB1 expression. Activating events are indicated in green colour, inhibiting events in red. High glucocorticoid levels immediately activate FOXO3 and SGK-1. Following stimulation of FOXO3 transcription, the protein is phosphorylated at threonine 32 (Thr 32), leading to its inactivation. Treatment with AMPK activating stimuli (metabolic stress) triggers phosphorylation of FOXO3 by AMPK at position 413 of the serine residue (Ser 413), thereby counteracting inactivation of FOXO3 by SGK-1. This results in a temporally delayed further enhancement of FOXO3 transcription since the FOXO3 protein can bind and activate its own gene promoter via a positive autoregulatory feedback loop in the presence of glucocorticoids. Functional FOXO3 in turn induces the master upstream kinase that further actives AMPK by threonine phosphorylation at position 172 (Thr 172). Restored ATP levels or inhibition of AMPK by compound C interrupts this circuit.

Article Snippet: For drug treatment, MCF-10A cells were transferred to a 1∶1 mixture of DMEM and Ham’s F12 supplemented with 2% of charcoal stripped FBS (Invitrogen, Cat. No.: 12676011) and subjected to 96 h of steroid hormone withdrawal, followed by treatment with either vehicle (0.1% ethanol), 1 µM dexamethasone (Sigma), 1 µM RU-486 (Cayman Chemicals), 1 mM AICAR (Sigma), 50 mM 2-Deoxyglucose (Sigma), 2 µM Oligomycin (Cell Signalling), 20 µM of AMPK inhibitor Compound C (Merck) or concomitant treatment with the indicated combinations for 18 h, unless otherwise indicated.

Techniques: Quantitative RT-PCR, Agarose Gel Electrophoresis, Staining, Activity Assay, Expressing, Functional Assay, Inhibition

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