ampk inhibitor bml 275  (MedChemExpress)

Journal: Redox Biology

Article Title: Pioglitazone-induced AMPK-Glutaminase-1 prevents high glucose-induced pancreatic β-cell dysfunction by glutathione antioxidant system

doi: 10.1016/j.redox.2021.102029

Figure Lengend Snippet: Effect of pioglitazone in the absence of AMPK on high glucose induced mitochondrial dysfunction. (A–C) INS-1 cells were treated with high glucose (30 mM) with Pioglitazone (10 μM) for 36 h with or without BML-275 (10 μM). The cell extracts were harvested and tested for protein levels with indicated antibodies. Actin was used as the loading control. (A) (* P < 0.001 vs control; ** P < 0.001 vs HG; *** P < 0.01 vs PIO) (B) (* P < 0.001 vs control; ** P < 0.001 vs HG; *** P < 0.001 vs PIO) (C) * P < 0.05 vs control; ** P < 0.05 vs HG; *** P < 0.05 vs PIO) (D) Co-IP of GLS1 with TRAP1 after pioglitazone treatment with BML-275 (10 μM) in high glucose conditions. (E) Measurement of relative GSH/GSSG ratios in INS-1 cells after pioglitazone treatment with BML-275 (10 μM) in high glucose conditions after 36 h (*P < 0.001 vs. control; **P < 0.005 vs. HG; ***P < 0.005 PIO). (F, G) Cellular ROS production and mitochondrial membrane potential after BML-275 treatment. (H) Cytosolic cytochrome c , cleaved casapase-3, BCL-2 and BCL-XL protein levels were quantified by immunoblotting (* P < 0.01 vs control; ** P < 0.01 vs HG; *** P < 0.01 vs PIO). Data are represented as mean ± SEM of three independent experiments.

Article Snippet: GW9662 (PPARγ antagonist) was purchased from Cayman (USA) and AMPK inhibitor (BML-275) from Enzo Life Sciences (USA).

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Redox Biology

Article Title: Pioglitazone-induced AMPK-Glutaminase-1 prevents high glucose-induced pancreatic β-cell dysfunction by glutathione antioxidant system

doi: 10.1016/j.redox.2021.102029

Figure Lengend Snippet: Pioglitazone effect on AMPK-GLS1 activation in Human pancreatic beta 1.1b4 cells. (A) Human pancreatic 1.1b4 cells were incubated with Pioglitazone (10 μM) for 48 h with high glucose (30 mM). Pioglitazone-induced AMPKα (Thr172) and ACC (Ser79), TRAP1/HSP75, and GLS1 was analyzed with western blotting (* P < 0.001 vs control; ** P < 0.005 vs HG). (B) Human pancreatic 1.1b4 cells were treated with high glucose (30 mM) with Pioglitazone (10 μM) for 48 h with or without BML-275 (10 μM). The cell extracts were harvested and tested for protein levels with indicated antibodies (* P < 0.001 vs control; ** P < 0.001 vs HG; *** P < 0.001 vs PIO). (C, D) Measurement of relative GSH/GSSG ratios and cellular ROS production were measured (*P < 0.001 vs. control; **P < 0.005 vs. HG; ***P < 0.005 PIO). (E, F) Cleaved caspase-3 and cell viability were measured using the Cell Counting Kit-8 (* P < 0.01 vs control; ** P < 0.005 vs HG; *** P < 0.005 vs PIO). (G) Pioglitazone activates AMPK, which causes a TRAP1/HSP75-GLS1 interaction, which increases the GSH/GSSG ratio and blocks mTORC1-induced maladaptive ER stress, protecting cells from high glucose toxicity.

Article Snippet: GW9662 (PPARγ antagonist) was purchased from Cayman (USA) and AMPK inhibitor (BML-275) from Enzo Life Sciences (USA).

Techniques: Activation Assay, Incubation, Western Blot, Cell Counting

Journal: Nutrients

Article Title: Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK

doi: 10.3390/nu12113326

Figure Lengend Snippet: Activation of AMPK in vitro upregulates bHLH factors, suppresses appetite and increases POMC neuropeptide expression. ( A ) Control NPCs were treated in vitro with AMPK activator (0.5, 1, 2 mM; □) or vehicle (■). NPC protein expression with representative immunoblots of pAMPK, bHLH factors (Mash1, Ngn3) and ( B ) neuropeptides (POMC, AgRP). Values are fold change of vehicle treated ( N = 5; mean ± SE). * p < 0.01 vs control. Abbreviations: AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); POMC (proopiomelanocortin); AgRP (agouti-related protein); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

Article Snippet: To further explore AMPK-mediated effects, NPCs from postnatal 1-day old (p1) Control newborn males were cultured in differentiating medium and treated in vitro with AMPK activator (AICAR; 0.25, 1, 2 mM; #A9978, Sigma-Aldrich, St. Louis, MO, USA), AMPK inhibitor (1, 5, 25 μM Dorsomorphin dihydrochloride, Compound C BML-275,#21207, Cayman Chemicals, Ann Arbor, MI, USA) or vehicle.

Techniques: Activation Assay, In Vitro, Expressing, Control, Western Blot

Journal: Nutrients

Article Title: Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK

doi: 10.3390/nu12113326

Figure Lengend Snippet: Suppression of AMPK in vitro downregulates bHLH factors and increases appetite and reduces satiety neuropeptide expression. ( A ) Control NPCs were treated in vitro with AMPK inhibitor (Compound C 1, 5, 25 µm; □) or vehicle (■). NPC protein expression with representative immunoblots of pAMPK, bHLH factors (Mash1, Ngn3) and ( B ) neuropeptides (POMC, AgRP). Values are fold change of vehicle treated ( N = 5; mean ± SE). * p < 0.001 vs control. Abbreviations: Compound C (Dorsomorphin dihydrochloride); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); POMC (proopiomelanocortin); AgRP (agouti-related protein); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

Article Snippet: To further explore AMPK-mediated effects, NPCs from postnatal 1-day old (p1) Control newborn males were cultured in differentiating medium and treated in vitro with AMPK activator (AICAR; 0.25, 1, 2 mM; #A9978, Sigma-Aldrich, St. Louis, MO, USA), AMPK inhibitor (1, 5, 25 μM Dorsomorphin dihydrochloride, Compound C BML-275,#21207, Cayman Chemicals, Ann Arbor, MI, USA) or vehicle.

Techniques: In Vitro, Expressing, Control, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Zyflamend induces apoptosis in pancreatic cancer cells via modulation of the JNK pathway

doi: 10.1186/s12964-020-00609-7

Figure Lengend Snippet: Zyflamend Induces Inflammatory, ER Stress, and Autophagy Responses in β-TC6 Cells. a-b Total cell lysates from control and Zyflamend treated cells for 3, 6, 12, 24, 36 and 48 h were immunoblotted for ER stress markers: pPERK, pEIF2α, pIRE1α, their respective unphosphorylated proteins, sXBP1, CHOP, and β-actin as a loading control. Representative immunoblots are shown. b Bar graphs represent pPERK/PERK, pEIF2α/EIF2α, pIRE1/IRE1, sXBP1/β-actin, and CHOP/β-actin as means + SEM. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. c-d Markers of autophagy were examined in the same lysates using antibodies against Beclin 1, LC3, I&II, ATG5, ATG7, and β-actin as a loading control. d Bar graphs represent Beclin 1/β-actin, LC3/β-actin, ATG5/β-actin, and ATG7/β-actin as means + SEM. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. e-f Immunoblots of key proteins in autophagy, AMPK, ER stress, and apoptosis signaling in β-TC6 cells treated with 200 μg/ml Zyflamend with and without the pan-caspase inhibitor Z-VAD.fmk. Representative immunoblots are shown. f Bar graphs represent pAMPK/AMPK, pPERK/PERK, pEIF2α/EIF2α, pIRE1α/IRE1α, sXBP1/β-actin, CHOP/β-actin, pJNK/JNK, Beclin 1/β-actin, LC3I&II/β-actin, and cleaved caspase3/β-actin as means + SEM. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. † p < 0.05, †† p < 0.01 indicate a significant difference between cells treated with Z-VAD.fmk and non-treated cells

Article Snippet: Finally, AMPK inhibitor (BML-275; a.k.a. compound C) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Zyflamend induces apoptosis in pancreatic cancer cells via modulation of the JNK pathway

doi: 10.1186/s12964-020-00609-7

Figure Lengend Snippet: Zyflamend-Induced Cell Death is Mediated through the ER Stress/Autophagy Pathways. Total cell lysates from control cells treated with Zyflamend and non-treated cells with or without AMPK inhibitor (Compound C: CC), ( a - b ), autophagy inhibitor (3-MA) ( c - d ), or ER stress inhibitor (4-PBA; e - h ) were immunoblotted for markers of inflammation, ER stress, autophagy, and cell death. Cells were pre-treated with the indicated inhibitors 12 h prior to adding Zyflamend or DMSO (as a vehicle control) for an additional 24 h. In b , d , and f Bar graphs represent) Bar graphs represent pAMPK/AMPK, pPERK/PERK, pEIF2α/EIF2α, pIRE1α /IRE1α, sXBP1/β-actin, CHOP/β-actin, pJNK/JNK, Beclin 1/β-actin, LC3II&II/β-actin, and cleaved caspase3/β-actin as means + SEM. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. † p < 0.05, †† p < 0.01 indicate a significant difference between cells treated with the indicated inhibitor and non-treated cells (CC, 3-MA, or 4-PBA). g Cells were treated with 200 μg/ml of Zyflamend for the indicated time with or without 4-PBA. Bar graphs represent the intensity of SRB staining reflective of the cell number and presented as means + SEM from at least three independent experiments. * p < 0.05, ** p < 0.01 indicate a significant difference between cells treated with Zyflamend and non-treated cells. † p < 0.05, †† p < 0.01 indicate a significant difference between non-treated and cells treated with 4-PBA. h-i Colony formation assay. i Bar graphs represent the relative number of colonies in each condition determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSO treated cells (Ctrl.) and expressed as a percentage. j-k Chromatin Condensation in cells treated with 200 μg/ml of Zyflamend alone or in combination with 4-PBA for 24 h. Representative images are shown. Scale bar: 50 μm. i Bar graphs represent the number of apoptotic cells (Hoechst positive) as means + SEM from at least three independent experiments. In i and k , ** p < 0.01 indicates a significant difference between cells treated with Zyflamend and non-treated cells. †† p < 0.01 indicates a significant difference between cells treated with 4-PBA and non-treated cells

Article Snippet: Finally, AMPK inhibitor (BML-275; a.k.a. compound C) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Staining, Colony Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Zyflamend induces apoptosis in pancreatic cancer cells via modulation of the JNK pathway

doi: 10.1186/s12964-020-00609-7

Figure Lengend Snippet: Overview of the Pro-Apoptotic Effects of Zyflamend in Pancreatic Cancer Cells. Zyflamend treatment induced ER stress, autophagy, MAP kinases and apoptosis in pancreatic β-TC6 Cells. Previous studies have shown that activation of AMPK in pancreatic cancer cells by chemotherapeutic agens induces apoptosis and autophagy via the AMPK/mTOR signaling pathway (dotted lines) . Similarly, activation of the JNK/SAPK pathway by stressors and chemotherapeutic agents can lead to apoptotic cell death of pancreatic cancer cells. Our study shows that treatment with pan-caspase inhibitor Z-VAD.fmk prevented the cleavage of caspase 3 but had no effects on AMPK, autophagy, or ER stress. Zyflamend treatment activated AMPK; however, treatment with AMPK inhibitor CC did not result in changes to ER stress, apoptosis, autophagy, or JNK signaling pathways. Autophagy inhibition with 3-MA inhibited autophagy and apoptosis only. Zyflamend activated both the IRE-1 and PERK branches of the ER stress mediated UPR and resulted in the subsequent upregulation of the downstream JNK signaling pathway. ER stress inhibition with 4-PBA resulted in decreased JNK activation, autophagy, and apoptosis. While inhibition of JNK with SP600125 significantly reduced the the level of both, apoptosis and autophagy

Article Snippet: Finally, AMPK inhibitor (BML-275; a.k.a. compound C) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Protein-Protein interactions, Inhibition