Journal: BMC Gastroenterology

Article Title: Mitochondrial uncouplers inhibit hepatic stellate cell activation

doi: 10.1186/1471-230X-12-68

Figure Lengend Snippet: FCCP and Valinomycin reduce HSC ATP levels, ROS production and stimulate mitochondrial biogenesis related genes. (A) Cells were treated with 5 μM of FCCP or Valinomycin for 24 hours and ATP levels were measured using a luciferase based ATP determination kit. (B) ROS levels were analyzed in mHSCs treated for different time points with chemical uncouplers by DCF fluorescence. HSCs were treated with 5 μM of FCCP or Valinomycin for 7 days and the expression of genes involved in mitochondrial biogenesis PGC-1α (C) , Tfam, Ndufs7 and CoxIV (D) were analyzed by RT-qPCR. * indicates P < 0.05 compared to control groups. Data are expressed as means of 3 independent experiments ± SEM.

Article Snippet: 3,750 cells per cm 2 were plated in the presence or absence of FCCP or Valinomycin in combination with Tin protoporphyrin IX dichloride (SnPP) (Tocris Bioscience, USA) from the first day of culture.

Techniques: Luciferase, Fluorescence, Expressing, Quantitative RT-PCR, Control

Journal: BMC Gastroenterology

Article Title: Mitochondrial uncouplers inhibit hepatic stellate cell activation

doi: 10.1186/1471-230X-12-68

Figure Lengend Snippet: FCCP and Valinomycin inhibit the expression of HSC pro-fibrogenic genes. (A) Bright field images of mHSCs treated with FCCP (5 μM) or Valinomycin (5 μM) every two days for a period of seven days. qHSCs and aHSCs indicate quiescent and activated HSCs respectively. Dashed lines indicate the blow-up of area in inserts. (B) Expression of pro-fibrogenic genes during HSC activation in vitro and effect of uncoupler treatment, left and right graphics respectively. Mouse HSCs were treated for seven days with FCCP (5 μM) or Valinomycin (5 μM) and after this period mRNA levels of the indicated genes were analyzed by RT-qPCR. (C) α-SMA protein expression in cells treated with chemical uncouplers. Isolated mouse HSCs were cultivated on cover slips for 7 days and then fixed with 4% formaldehyde and stained with α-SMA antibodies. * indicates P < 0.05 compared to control groups. Data are expressed as means of 3 independent experiments ± SEM.

Article Snippet: 3,750 cells per cm 2 were plated in the presence or absence of FCCP or Valinomycin in combination with Tin protoporphyrin IX dichloride (SnPP) (Tocris Bioscience, USA) from the first day of culture.

Techniques: Expressing, Activation Assay, In Vitro, Quantitative RT-PCR, Isolation, Staining, Control

Journal: BMC Gastroenterology

Article Title: Mitochondrial uncouplers inhibit hepatic stellate cell activation

doi: 10.1186/1471-230X-12-68

Figure Lengend Snippet: Chemical uncoupling does not induce cytotoxicity on concentrations that inhibit HSC in vitro activation. (A) Cytotoxic effect of mitochondrial uncouplers, as measured by fluorescence of bis-alanylalanyl-phenylalanyl-rhodamine 110, a peptide substrate of cytoplasmic peptidases. Insert shows cells treated with cycloheximide as positive control (B) Caspase 3/7 activity after treatment of cells with different concentrations of Valinomycin and FCCP on different time points. Enzyme activity was measured as described in material and methods. * indicates P < 0.05 compared to control group.

Article Snippet: 3,750 cells per cm 2 were plated in the presence or absence of FCCP or Valinomycin in combination with Tin protoporphyrin IX dichloride (SnPP) (Tocris Bioscience, USA) from the first day of culture.

Techniques: In Vitro, Activation Assay, Fluorescence, Positive Control, Activity Assay, Control

Journal: BMC Gastroenterology

Article Title: Mitochondrial uncouplers inhibit hepatic stellate cell activation

doi: 10.1186/1471-230X-12-68

Figure Lengend Snippet: FCCP inhibits HSC proliferation through HO-1. (A) Representative images of cells stained with DAPI (blue fluorescence, left column) and EdU, as a measurement of DNA synthesis (green fluorescence, right column) at day 5. Cells were treated for 5 days with uncouplers (5 μM of each) alone or in combination with the heme-oxygenase inhibitor SnPP as indicated. (B) Quantification of proliferation (EdU positive) as percentage of DAPI positive cells visualized in (A). ( C ) HO-1 gene expression determined by RT-qPCR during in vitro activation of primary mouse HSCs and comparing 7 day old control cells versus cells treated for 7 days with FCCP or Valinomycin (D) . Data are expressed as means of 3 independent experiments ± SEM.

Article Snippet: 3,750 cells per cm 2 were plated in the presence or absence of FCCP or Valinomycin in combination with Tin protoporphyrin IX dichloride (SnPP) (Tocris Bioscience, USA) from the first day of culture.

Techniques: Staining, Fluorescence, DNA Synthesis, Gene Expression, Quantitative RT-PCR, In Vitro, Activation Assay, Control

Journal: BMC Gastroenterology

Article Title: Mitochondrial uncouplers inhibit hepatic stellate cell activation

doi: 10.1186/1471-230X-12-68

Figure Lengend Snippet: FCCP and Valinomycin inhibit TGF-β signaling in 2 day old mHSCs. (A) Expression of pro-fibrogenic genes in cells treated for 24 hours with 5 μM of FCCP and Valinomycin or together with TGF-β (10 ng/mL) as indicated. (B) Expression of TGF-β early immediate genes Smad6 and Smad7 on HSCs treated for 2 hours with TGF-β. * indicates P < 0.05 compared to control groups and ** indicates P < 0.05 compared to TGF-β group. Data are expressed as means of 3 independent experiments ± SEM.

Article Snippet: 3,750 cells per cm 2 were plated in the presence or absence of FCCP or Valinomycin in combination with Tin protoporphyrin IX dichloride (SnPP) (Tocris Bioscience, USA) from the first day of culture.

Techniques: Expressing, Control

Journal: BMC Gastroenterology

Article Title: Mitochondrial uncouplers inhibit hepatic stellate cell activation

doi: 10.1186/1471-230X-12-68

Figure Lengend Snippet: Mitochondrial uncouplers reduce activation markers and proliferation of human derived HSCs. (A) Human activated HSCs (passage 7) were treated for 5 days with FCCP or Valinomycin (10 μM, both compounds) followed by analysis of mRNA levels of pro-fibrogenic genes by RT-qPCR. Activated human HSCs treated for 5 days with mitochondrial uncouplers present reduced α-SMA protein expression as observed by western blot (B) and immunocytochemistry (C) . Numbers below Figure B represent densitometry values of α-SMA compared to control and normalized by β-Actin levels. (D) Representative images of cells stained with DAPI (blue fluorescence, left column) and EdU, as a measurement of DNA synthesis (green fluorescence, right column) at day 5. (B) Quantification of proliferation (EdU positive) as percentage of DAPI positive cells visualized in (D) .

Article Snippet: 3,750 cells per cm 2 were plated in the presence or absence of FCCP or Valinomycin in combination with Tin protoporphyrin IX dichloride (SnPP) (Tocris Bioscience, USA) from the first day of culture.

Techniques: Activation Assay, Derivative Assay, Quantitative RT-PCR, Expressing, Western Blot, Immunocytochemistry, Control, Staining, Fluorescence, DNA Synthesis

Journal: eLife

Article Title: PEBP1 amplifies mitochondrial dysfunction-induced integrated stress response

doi: 10.7554/eLife.102852

Figure Lengend Snippet:

Article Snippet: FCCP , TargetMol , T6834 , 370-86-5 , ETC uncoupler.

Techniques: Inhibition, Positive Control, Selection, Stable Transfection, Membrane, Infection

Journal: Life

Article Title: Utilization of Human Samples for Assessment of Mitochondrial Bioenergetics: Gold Standards, Limitations, and Future Perspectives

doi: 10.3390/life11090949

Figure Lengend Snippet: Vendor and catalog number information for reagents used in the proof of concept assays completed for this review.

Article Snippet: Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) , Enzo , BML-CM120.

Techniques: Modification, Saline, Clinical Proteomics, Membrane

Journal: Life

Article Title: Utilization of Human Samples for Assessment of Mitochondrial Bioenergetics: Gold Standards, Limitations, and Future Perspectives

doi: 10.3390/life11090949

Figure Lengend Snippet: The mitochondrial compounds/substrates used to investigate mitochondrial function in fresh and frozen samples. The compounds used to measure mitochondrial respiratory function and their corresponding functions are listed. Abbreviations: ADP: Adenosine diphosphate; ATP: Adenosine triphosphate; CI: Complex I; CII: Complex II; CIII: Complex III; CIV: Complex IV; CV: Complex V; ETC: Electron transport chain; NADH: Reduced nicotinamide adenine dinucleotide; OXPHOS: Oxidative phosphorylation; TMPD: N, N, N’, N’-tetramethyl-p-phenylenediamine.

Article Snippet: Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) , Enzo , BML-CM120.

Techniques: Phospho-proteomics, Membrane, Recombinant, Clinical Proteomics

Journal: Life

Article Title: Utilization of Human Samples for Assessment of Mitochondrial Bioenergetics: Gold Standards, Limitations, and Future Perspectives

doi: 10.3390/life11090949

Figure Lengend Snippet: Extracellular flux analysis in fresh and frozen lymphocytes from two donors. Respirometry was measured in fresh ( A , B ) and frozen lymphocytes ( C – E ). ( A ) Oxygen consumption was measured in intact cells before and after injection of oligomycin (CV inhibitor), FCCP (chemical uncoupler), and antimycin A (CIII inhibitor) and rotenone (CI inhibitor). ( B ) Respiration in permeabilized cells with CII substrates. A final injection of TMPD was included to measure CIV. ( C ) RIFS protocol with succinate and rotenone to drive respiration through CII. ( D ) CV activity measured by media acidification as a result of ATP hydrolysis. ( E ) Oligomycin-sensitivity in the CV measurement. Abbreviations: AA: Antimycin A; ATP: Adenosine triphosphate; FCCP: Carbonyl cyanide p-trifluoromethoxyphenylhydrazone; oligo: Oligomycin; Rot: Rotenone; Succ: Succinate; TMPD: N, N, N’, N’-tetramethyl-p-phenylenediamine.

Article Snippet: Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) , Enzo , BML-CM120.

Techniques: Injection, Activity Assay