Journal: Blood Cancer Journal

Article Title: Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production

doi: 10.1038/bcj.2011.38

Figure Lengend Snippet: Niclosamide treatment results in loss of mitochondrial membrane potential (Δψ) and an increase in respiration rate. ( a ) Myeloma cells treated with niclosamide for 4 h were stained with JC-1 and analysed by flow cytometry. Red is indicative of cells with polarised mitochondrial membranes. Green staining indicates cells that have lost Δψ. Data shown is representative of n =3 experiments. ( b , c ) H929 and JJN3 cells were prestained with 10 n TMRE and baseline fluorescence intensity (geometric mean GM) determined by flow cytometry. Niclosamide (3.2 μ) was added and readings taken at 15, 30 s and every minute for 5 min. Representative histograms ( b ) are shown together with mean data ( c ) from n =4 experiments±s.e.m. ( d ) Respiration rate in 5 × 10 7 myeloma cells was measured using a Clark oxygen electrode. Baseline respiration rate was established as μmol O 2 /min/10 6 cells before the addition of either 3.2 μ niclosamide or 6.7 μ p -trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP). Representative plots are shown together with a histogram of mean data from a minimum of n =4 experiments±s.e.m.; * P

Article Snippet: Measurements of mitochondrial membrane potential (Δψ) 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma Aldrich) was dissolved at 1 mg/ml in dimethyl sulfoxide and stored at −20 °C.

Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Caveolin‐3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function) Caveolin‐3 deficiency associated with the dystrophy P104L mutation impairs skeletal muscle mitochondrial form and function

doi: 10.1002/jcsm.12541

Figure Lengend Snippet: Effects of Cav3 re‐expression on mitochondrial protein expression, membrane potential, superoxide and mitochondrial respiration. Myoblasts in which Cav3 had been deleted by CRISPR/Cas9 (Cav3KO) were infected with retroviral particles containing an empty vector (Cav3KO + EV) or a vector containing HA‐GFP tagged WT‐Cav3 (Cav3KO + Cav3) and stable transformants isolated by antibiotic selection prior to: (A) immunoblotting whole cell lysates (WCL) with antibodies against Cav3 or actin (used as a gel loading control) antibodies; (B) isolation of cytosolic (Cyto) and mitochondrial membranes and immunoblotting using antibodies to proteins indicated; (C) lysis and immunoblotting of WCL with antibodies to proteins shown and quantification of their abundance relative to GAPDH (used as a gel loading control) using Image J software; (D) determination of superoxide content using fluorescence intensity (FI) of MitoSOX and mitochondrial membrane potential monitored by spectral analysis of JC‐10 aggregate:monomer content from three separate experiments (for this experiment GFP would interfere with the JC‐10 probe and therefore wild type Cav3 was cloned into pcDNA3.1 using HindIII and XhoI Restriction sites and an empty vector or a vector containing Cav3 were transiently transfected in L6 muscle cells and selected for using G418 before JC‐10 probe incubation); and (E) analysis of mitochondrial respiration to assess basal oxygen consumption rate using Seahorse technology. Oligomycin (1 μM), FCCP (1 μM) and a mixture of Rotenone (1 μM)/Antimycin (2 μM) were added at the times indicated by dotted lines to determine basal, ATP‐linked and maximal respiration. The Seahorse trace shown in (E) is from a single experiment in which each point represents the mean ± SEM from triplicate analyses. All bar graph data in (E) represent the analysis of three individual experiments (values are mean ± SEM). Asterisks indicate significant change ( P

Article Snippet: Measurement of mitochondrial membrane potential, mitochondrial DNA, and citrate synthase activity For measurement of mitochondrial membrane potential, muscle cells were seeded and grown in dark‐walled 96‐well dishes until confluent for 4 days.

Techniques: Expressing, CRISPR, Infection, Plasmid Preparation, Isolation, Selection, Lysis, Software, Fluorescence, Clone Assay, Transfection, Incubation

Journal: International Journal of Nanomedicine

Article Title: Competitive inhibition of survivin using a cell-permeable recombinant protein induces cancer-specific apoptosis in colon cancer model

doi: 10.2147/IJN.S73916

Figure Lengend Snippet: SR9-mediated cytotoxicity is due to mitochondrial depolarization and not autophagy. Notes: ( A ) The clonogenic potential of both Caco-2 and SW480 cells was significantly lowered post-treatment with SR9 and CHNP–SR9 at 24 hours. ( B ) SR9 induced mitochondrial depolarization, which is an early sign of apoptosis and which further led to release of cytochrome-C from the mitochondria into the cytoplasm, and then went on to activate caspase-3, which is the final caspase in inducing apoptosis. ( C ) The 3D tumor spheroid assay was performed to mimic the tumor polyp, and it was observed that both SR9 and CHNP–SR9 were highly effective in significantly reducing the tumor spheroid size within 24 hours. ( D ) Thirty-five key molecules involved in the apoptosis pathway were evaluated using the protein apoptotic array kit, and 17 of those key molecules are shown in the figure. * P

Article Snippet: Apoptosis studies The mitochondrial membrane potential assay kit (Sigma-Aldrich Co., St Louis, MO, USA) was used to calculate the mitochondrial potential of SR9 and CHNP–SR9-treated Caco-2 and SW480 cells.

Techniques:

Journal: Scientific Reports

Article Title: Adiponectin modulates oxidative stress-induced mitophagy and protects C2C12 myoblasts against apoptosis

doi: 10.1038/s41598-017-03319-2

Figure Lengend Snippet: Proposed model of action of APN on oxidative stress. H 2 O 2 administration to C2C12 cells causes reductions in mitochondrial membrane potential, ROS production and apoptosis. High levels of ROS and depolarized mitochondria induced mitophagy. APN pretreatment suppressed the over-production of ROS by activating the Nrf2/HO-1 pathway, protecting mitochondria, inhibiting Parkin E3 ubiquitin ligase and modulating mitophagy by partially blocking the colocalization of mitochondria with autophagosomes/lysosomes. In addition, APN downregulates the transcript levels of both Bax and Bax/Bcl-2 protein induced by oxidative stress.

Article Snippet: Measurement of mitochondrial membrane potential Mitochondrial membrane potential was measured using the fluorescent strain TMRE (Sigma, 87917).

Techniques: Blocking Assay

Journal: Scientific Reports

Article Title: Adiponectin modulates oxidative stress-induced mitophagy and protects C2C12 myoblasts against apoptosis

doi: 10.1038/s41598-017-03319-2

Figure Lengend Snippet: Effect of APN on H 2 O 2 -induced mitochondrial membrane potential. ( A ) C2C12 cells were transfected with 30 μg/mL APN, and control cells were treated with 5 mM H 2 O 2 for 1 h. Representative fluorescence images of TMRE and MitoTracker Green FM (MTR) are shown. Scale bar, 5 μm. ( B ) Quantification of TMRE signals in cells before and after H 2 O 2 treatment (mean ± SEM; n = 100 cells from 3 independent experiments, ** p

Article Snippet: Measurement of mitochondrial membrane potential Mitochondrial membrane potential was measured using the fluorescent strain TMRE (Sigma, 87917).

Techniques: Transfection, Fluorescence

Journal: Oncotarget

Article Title: Reactive oxygen/nitrogen species contribute substantially to the antileukemia effect of APO866, a NAD lowering agent

doi: 10.18632/oncotarget.27336

Figure Lengend Snippet: Deletion of PARP1 fully inhibits H 2 O 2 production, mitochondrial depolarization, caspase activation, and loss of ATP cell content in APO866-treated Jurkat cells. Detection of cytosolic ( A ), mitochondrial ( B ) superoxide anions, NO ( C ), H 2 O 2 generation ( D ) and MMP ( E ), and ATP cell content ( G ) in APO866-treated WT or KO Jurkat cells were monitored as described in Figure 1. ( F ) Caspase 3 activation was assessed in WT (or PARP1KO) Jurkat cells treated with 10 nM APO866 for 96 h using a fluorescent specific probe for activated forms of CAS P3 and flow cytometry.

Article Snippet: Assessment of mitochondrial membrane potential MMP was determined using flow cytometry after cell staining with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimididazolylcarbocyanine iodide (JC-1, Calbiochem, 420200-5).

Techniques: Activation Assay, Flow Cytometry, Cytometry