Journal: Haematologica

Article Title: ASLAN003, a potent dihydroorotate dehydrogenase inhibitor for differentiation of acute myeloid leukemia

doi: 10.3324/haematol.2019.230482

Figure Lengend Snippet: ASLAN003-induced apoptosis involves both mitochondrial and death receptor pathways. (A) Whole-cell lysates or cytosolic fractions from KG- 1 and MOLM-14 cells treated with dimethylsulfoxide (DMSO), 0.5 μM or 1 μM ASLAN003 for 48 h were used to measure apoptosis-related proteins by western blot analyses. (B) THP-1, MOLM-14 and KG- 1 cells were exposed to 1 μM or 2 μM ASLAN003 or DMSO as the control for 48 h and then stained with JC-10 and analyzed by flow cytometry for quantification of intrinsic mitochondrial membrane potential (MMP). Representative FACS plots are shown. The bars in the bar chart represent the means of the increased depolarization of MMP of the three cell lines after exposure to ASLAN003 in two independent experiments, the error bars denote the standard deviation.

Article Snippet: Assessment of mitochondrial membrane potential The mitochondrial membrane potential was determined using a JC-10 Assay Kit (Sigma, MAK160).

Techniques: Western Blot, Staining, Flow Cytometry, FACS, Standard Deviation

Journal: Aging (Albany NY)

Article Title: SIN-3 as a key determinant of lifespan and its sex dependent differential role on healthspan in Caenorhabditis elegans

doi: 10.18632/aging.101682

Figure Lengend Snippet: Loss of sin-3 causes reproductive defects in C. elegans. Graphical representation of ( a ) total number of eggs laid in the entire reproductive span of worms, ( b ) total number of eggs that were viable and ( c ) percentage of males in the viable progeny after self-fertilization and mating. ( d ) Loss of sin-3 causes AVID phenotype defect in C. elegans measured as percentage of worms showing ruptured vulva and protrusion of intestine and uterus. ( e-g ) Graphical representation for the quantification of red, green and the ratio of red/green for JC-1 staining respectively depicting the hypopolarisation of mitochondrial membrane potential in case in sin-3 deletion in C. elegans (For figure a, b and e-f: ns, non-significant; * p

Article Snippet: Briefly, assay of ΔΨm was performed using JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetra-ethyl benzimid-azolylcarbocyanine iodide, catalog no. T-3168, Molecular Probes) dissolved in DMSO at a concentration of one mg/ml.

Techniques: Staining

Journal: Antioxidants

Article Title: Mitochondrial Activation and Reactive Oxygen-Species Overproduction during Sperm Capacitation are Independent of Glucose Stimuli

doi: 10.3390/antiox9080750

Figure Lengend Snippet: Evaluation of human spermatozoa mitochondrial membrane polarization at baseline (T = 0 h) and without or with glucose (0, 5.5, 11 and 22 mM) stimuli following 6 h in capacitation conditions, using the dye JC-1. Results are expressed as mean ± SEM ( n = 15 for each condition). Significantly different results are expressed as: ( a ) Relative to T = 0 h group.

Article Snippet: JC-1 Assay for Mitochondrial Membrane Potential The mitochondrial membrane potential of spermatozoa was evaluated using the fluorogenic dye JC-1 (T3168, Invitrogen™, Carlsbad, CA, USA).

Techniques:

Journal: Antioxidants

Article Title: Mitochondrial Activation and Reactive Oxygen-Species Overproduction during Sperm Capacitation are Independent of Glucose Stimuli

doi: 10.3390/antiox9080750

Figure Lengend Snippet: Analysis of JC-1 J-aggregates (red) or monomers (green) in human spermatozoa at baseline (T = 0 h) and without or with glucose (0, 5.5, 11, and 22 mM) stimuli following 6 h in capacitation conditions, by fluorescence microscopy. In ( A , D , G , J , M ) are represented JC-1 monomers (green, FITC filter) and nuclei (DAPI filter) for T = 0 h group and glucose concentrations (0, 5.5, 11 and 22 mM), respectively. In ( B , E , H , K , N ) are represented JC-1 J-aggregates (red, G-2A filter) and nuclei (DAPI filter) for T = 0 h group and glucose concentrations (0, 5.5, 11 and 22 mM), respectively. In ( C , F , I , L , O ) are represented the overlap between JC-1 J-aggregates and monomers for T = 0 h group and glucose concentrations (0, 5.5, 11 and 22 mM), respectively.

Article Snippet: JC-1 Assay for Mitochondrial Membrane Potential The mitochondrial membrane potential of spermatozoa was evaluated using the fluorogenic dye JC-1 (T3168, Invitrogen™, Carlsbad, CA, USA).

Techniques: Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Co-administration of iRGD with peptide HPRP-A1 to improve anticancer activity and membrane penetrability

doi: 10.1038/s41598-018-20715-4

Figure Lengend Snippet: Mitochondrial depolarization, reactive oxygen species (ROS) generation and co-localization of HPRP-A1 with mitochondrial membrane. ( A ) Mitochondrial depolarization of cells. A549 cells were cultured with 4 μM or 8 μM HPRP-A1 with or without 64 μM iRGD for 5 min, 15 min, 30 min or 60 min. JC-1 probe was added according to the manufacturer’s instruction and the fluorescence was detected by flow cytometry. ( B ) ROS generation in cells. A549 cells were treated with 4 μM HPRP-A1 with or without 64 μM iRGD for 5 and 15 min, and ROS probe was added according to the manufacturer’s instructions. The fluorescence was detected using flow cytometry. ( C ) Co-localization of 8 μM FITC-labeled HPRP-A1 with or without 64 μM iRGD incubated with A549 cells for 30 min. Nuclei (blue color) and mitochondria (red color) were stained with Hoechst 33258 and Mito-Tracker ® Red, respectively, and cells were examined by laser scanning confocal microscopy. * P

Article Snippet: The mitochondrial membrane potential was measured using the fluorescent dye JC-1 (Molecular Probes, Beijing, China) .

Techniques: Cell Culture, Fluorescence, Flow Cytometry, Cytometry, Labeling, Incubation, Staining, Confocal Microscopy

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Colchicine Alleviates Cholesterol Crystal-Induced Endothelial Cell Pyroptosis through Activating AMPK/SIRT1 Pathway

doi: 10.1155/2020/9173530

Figure Lengend Snippet: Colchicine plays an antipyroptosis role by inhibiting intracellular oxidative stress. HUVECs were subjected to H 2 O 2 (300 μ M), cholesterol crystal (0.5 mg/ml) and colchicine (10 nM), or N-acetyl-cysteine (NAC, 5 mM) for 10 h or 24 h. (a) ROS level was detected using a DCFH-DA probe, and (b) mitochondrial membrane potential was tested by JC-1 (scale bars = 100 μ m). (c, d) The indicators of oxidative stress GSSG and MDA were tested by assay kits. (e) Western blotting was dedicated to examine the protein expression levels of NLRP3, ASC-1, procaspase-1, and caspase-1. (f–h) Quantitative analysis of pyroptosis-associated protein expression. (i, j) Real-time PCR analysis of the mRNA levels of GSDMD and proinflammatory cytokines (IL-18 and IL-1 β ). Data was expressed as the mean ± SD of three separate experiments. ∗∗ P

Article Snippet: Detection of Mitochondrial Membrane Potential The mitochondrial membrane potential was measured by the fluorescence probe JC-1 using the mitochondrial membrane potential assay kit (C2006, Beyotime Institute of Biotechnology).

Techniques: Multiple Displacement Amplification, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Colchicine Alleviates Cholesterol Crystal-Induced Endothelial Cell Pyroptosis through Activating AMPK/SIRT1 Pathway

doi: 10.1155/2020/9173530

Figure Lengend Snippet: Colchicine plays an antipyroptosis role by inhibiting intracellular oxidative stress. HUVECs were treated for 10 or 24 h with different doses of colchicine (0–10 nM) in the presence of cholesterol crystal (0.5 mg/ml) or were left untreated (control). (a) Intracellular ROS level was detected using a DCFH-DA probe. (b) JC-1 Staining Kit was used for the detection of mitochondrial membrane potential. (c, d) The indicators of oxidative stress GSSG and MDA were tested by assay kits in endothelial cells. Endothelial cells treated with increasing doses of H 2 O 2 (0-400 μ M); (e) the cell viability was detected using a CCK8 assay. HUVECs were subjected to H 2 O 2 (300 μ M), cholesterol crystal (0.5 mg/ml) and colchicine (10 nM), or N-acetyl-cysteine (NAC, 5 mM) for 24 hrs. (f) The cell viability was detected using a CCK8 assay. Pyroptotic cell death was evaluated with (g) LDH release and (h) Hoechst 33342/PI staining. Scale bars = 100 μ m. Data was expressed as the mean ± SD of three separate experiments. ∗∗ P

Article Snippet: Detection of Mitochondrial Membrane Potential The mitochondrial membrane potential was measured by the fluorescence probe JC-1 using the mitochondrial membrane potential assay kit (C2006, Beyotime Institute of Biotechnology).

Techniques: Staining, Multiple Displacement Amplification, CCK-8 Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Colchicine Alleviates Cholesterol Crystal-Induced Endothelial Cell Pyroptosis through Activating AMPK/SIRT1 Pathway

doi: 10.1155/2020/9173530

Figure Lengend Snippet: Silencing of AMPK-SIRT1 signaling eliminates the protective effect of colchicine on endothelial cells. HUVECs were transfected with siRNA targeting AMP α 1 (si-AMPK α 1) and SIRT1 (si-SIRT1) or a control siRNA (Si NC) or were not transfected (-). Cells were then added to cholesterol crystal (0.5 mg/ml) in the presence of colchicine (10 nM) for 10 or 24 hrs. Real-time PCR analysis of the mRNA expression of AMPK α and SIRT1 after transfection with si-AMPK α 1, si-SIRT1, and si-NC for 24 hrs without (a) or with (b) the presence of cholesterol crystals and colchicine. (c) Intracellular ROS level was detected using a DCFH-DA probe, and (d) mitochondrial membrane potential was tested by JC-1. (e) Pyroptotic cell death was measured with Hoechst 33342 (blue)/PI (red) double-fluorescent staining. (f, g) The indicators of oxidative stress GSSG and MDA were tested by assay kits. (h) Real-time PCR analysis of the mRNA levels of GSDMD and proinflammatory cytokines (IL-18, IL-1 β , IL-6, IL-8, and MCP-1). Scale bars = 100 μ m. Data was expressed as the mean ± SD of three independent experiments. ∗ P

Article Snippet: Detection of Mitochondrial Membrane Potential The mitochondrial membrane potential was measured by the fluorescence probe JC-1 using the mitochondrial membrane potential assay kit (C2006, Beyotime Institute of Biotechnology).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Expressing, Staining, Multiple Displacement Amplification

Journal: Cell Death & Disease

Article Title: TMEM59 interacts with TREM2 and modulates TREM2-dependent microglial activities

doi: 10.1038/s41419-020-02874-3

Figure Lengend Snippet: TMEM59 downregulation attenuates elevated autophagy and impaired mitochondrial functions in Trem2 KO microglia. Primary microglia derived from wild type (WT) and Trem2 KO mice were transfected with a scramble control (SCR) or Tmem59 siRNA (SI59) for 48–72 h. a Cells were lysed, and equal protein amounts of cell lysates were immunoblotted for proteins indicated. Protein levels were quantified by densitometry and compared to controls (WT microglia transfected with SCR, set to one arbitrary units, AU). Data represent mean ± SEM ( n = 3). b Cells were measured for ATP levels for comparison. Data represent mean ± SEM ( n = 3). c Cells were assayed for baseline oxygen consumption rate (OCR) using a Seahorse analyzer. Data represent mean ± SEM ( n = 5). d Cells were subjected to JC-1 staining to measure mitochondrial membrane potential. The fluorescence intensities of JC‑1 monomers that dominate in depolarized mitochondria (in green) and JC-1 aggregates that dominate in polarized mitochondria (in red) were visualized under a confocal microscope. Red and green color intensities were quantified by ImageJ, and the ratio of red/green signal was calculated and compared. Data represent mean ± SEM ( n = 3). Scale bars: 45 μm. * p

Article Snippet: Mitochondrial membrane potential measurementMitochondrial membrane potential was assayed using the JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime, Shanghai, China), following the manufacturer’s protocols.

Techniques: Derivative Assay, Mouse Assay, Transfection, Staining, Fluorescence, Microscopy

Journal: Biomolecules

Article Title: Chemopreventive Property of Sencha Tea Extracts towards Sensitive and Multidrug-Resistant Leukemia and Multiple Myeloma Cells

doi: 10.3390/biom10071000

Figure Lengend Snippet: Representative images of JC-1 fluorescence with flow cytometry and statistical analysis of mitochondrial membrane potential on KMS-12-BM cells. ( A ) Cells were treated with DMSO as control and 0.5-, 1-, 2-, and 4-fold IC 50 of sencha-MeOH/H 2 O extract and bortezomib respectively for 24 h. ( B ) Statistical results of the apoptotic cell were defined as MMP collapse after 24 h treatment. IC 50 values were 11.37 μg/mL for sencha-MeOH extract, 14.85 μg/mL for sencha-H 2 O extract, and 0.0019 μM for bortezomib towards KMS-12-BM cells. Asterisks above bars denote p -values for one-way ANOVA analysis: * p

Article Snippet: Assessment of MMP Aliquots of 105 cells (CCRF/CEM or KMS-12-BM)/well were seeded in 96-well flat-bottom plate in a volume of 200 µL and treated with DMSO as a negative control, doxorubicin/ bortezomib or 0.5-, 1-, or 2-, 4-fold IC50 of sencha-MeOH and -H2 O extracts for 24 h. JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical, Ann Arbor, Michigan, USA) was used to detect MMP by flow cytometry according to the manufacturer’s instructions.

Techniques: Fluorescence, Flow Cytometry

Journal: Biomolecules

Article Title: Chemopreventive Property of Sencha Tea Extracts towards Sensitive and Multidrug-Resistant Leukemia and Multiple Myeloma Cells

doi: 10.3390/biom10071000

Figure Lengend Snippet: Representative images of JC-1 fluorescence with flow cytometry and statistical analysis of mitochondrial membrane potential on CCRF/CEM cells. ( A ) Cells were treated with DMSO as control and 0.5-, 1-, 2-, and 4-fold IC 50 of sencha-MeOH/H 2 O extract and doxorubicin respectively for 24 h. ( B ) Statistical results of the apoptotic cell were defined as MMP collapse after 24 h treatment. IC 50 values were 8.38 μg/mL for sencha-MeOH extract, 11.50 μg/mL for sencha-H 2 O extract, and 0.0093 μM for doxorubicin towards CCRF/CEM cells. Asterisks above bars denote p -values for one-way ANOVA analysis: ** p

Article Snippet: Assessment of MMP Aliquots of 105 cells (CCRF/CEM or KMS-12-BM)/well were seeded in 96-well flat-bottom plate in a volume of 200 µL and treated with DMSO as a negative control, doxorubicin/ bortezomib or 0.5-, 1-, or 2-, 4-fold IC50 of sencha-MeOH and -H2 O extracts for 24 h. JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical, Ann Arbor, Michigan, USA) was used to detect MMP by flow cytometry according to the manufacturer’s instructions.

Techniques: Fluorescence, Flow Cytometry

Journal: Cancers

Article Title: Paroxetine Induces Apoptosis of Human Breast Cancer MCF-7 Cells through Ca2+-and p38 MAP Kinase-Dependent ROS Generation

doi: 10.3390/cancers11010064

Figure Lengend Snippet: Paroxetine-induced apoptosis and ROS generation are mediated by p38 activation. ( A ) Effect of paroxetine on the phosphorylation of MAPK. MCF-7 was treated with 30 μM paroxetine for the indicated time periods. Activated p38 MAPK (p-p38) and ERK (p-ERK) were detected in immunoblots using antibodies specific for the phosphorylated form of each kinase. The same blot was stripped and used to determine the amount of each kinase; ( B ) Effect of MAPK inhibitors on paroxetine-induced cell death. Cells were pretreated with 10 μM SB203580, 10 μM PD98059, or 10 μM SP600125 for 30 min before 12 h treatment with 30 μM paroxetine. Cell viability was determined by an MTT assay. ( Ca , Cb ) Effect of NAC and SB203580 on PARP cleavage ( Ca ) and p38 activation ( Cb ). Protein extracts were prepared after treatment with or without 10 μM SB203580 and 3 mM NAC for 30 min, followed by treatment with or without 10 μM or 30 μM paroxetine for 12 h. Immunoblotting assays were performed with antibodies against PARP, cleaved PARP, Bax, p-p38, p38, and α-tubulin; ( D ) Effect of SB203580 and NAC on paroxetine-induced cell death. Cells were pretreated with 10 μM SB203580 and 3 mM NAC for 30 min before 12 h treatment with 30 μM paroxetine. Cell viability was determined by an MTT assay; ( E , F ) Reduction of ROS in MCF-7 cells by pretreatment with NAC, SB203580, or NAC and SB203580. MCF-7 cells were incubated with the ROS indicator H 2 DCFDA and then pretreated with 10 μM SB203580 and/or NAC for 30 min before 3 h treatment with 30 μM paroxetine. The changes in ROS levels in the cells were analyzed using fluorescence microscopy ( E ) and quantified ( F ); ( G ) Reduction in MMP depolarization in MCF-7 cells by SB203580. Cells were incubated with a specific MMP dye, JC-1, in the presence or absence of paroxetine and/or SB203580. The cells were exposed to paroxetine and/or SB203580 for 3 h. SB203580 pretreatment was performed before 30 min of paroxetine treatment. Green and red indicate JC-1 monomers and JC-1 aggregates, respectively. Changes in the MMP of the cells by paroxetine were analyzed using fluorescence microscopy. The scale bar represents 50 μm. ‘Paro’ represents paroxetine. * p

Article Snippet: Measurement of Mitochondrial Membrane Potential (MMP) and Plasma Membrane Potential MMP changes were determined by JC-1 mitochondrial membrane potential detection kit (Biotium Inc.) according to the manufacturer’s protocol.

Techniques: Activation Assay, Western Blot, MTT Assay, Incubation, Fluorescence, Microscopy

Journal: Cancers

Article Title: Paroxetine Induces Apoptosis of Human Breast Cancer MCF-7 Cells through Ca2+-and p38 MAP Kinase-Dependent ROS Generation

doi: 10.3390/cancers11010064

Figure Lengend Snippet: Paroxetine-induced mitochondrial ROS generation and MMP depolarization. ( A ) Mitochondria were a source of ROS generation by paroxetine treatment. The cells were exposed to paroxetine (30 μM) for 12 h. The cells were incubated with MitoTracker Red dye and H 2 DCFDA for 30 min in the presence or absence of paroxetine (30 μM). Red, green, and yellow indicate mitochondrial regions, ROS generation, and mitochondrial regions showing ROS generation (merged color), respectively. The scale bar indicates 20 μm; ( B ) The mitochondrial ROS generation was confirmed using specific mitochondrial ROS dye, MitoSOX. Cells were incubated with MitoSOX after paroxetine (30 μM) treatment for indicated time periods. The scale bar represents 50 μm. ( C , D ) MCF-7 cells were incubated with a specific MMP dye, JC-1, in the presence or absence of paroxetine and/or NAC. The cells were exposed to paroxetine and/or NAC for 12 h. NAC pretreatment was performed before 2 h of paroxetine treatment. Green and red indicate JC-1 monomers and JC-1 aggregates, respectively. Changes in the MMP of the cells by paroxetine were analyzed using fluorescence microscopy ( C ) and quantified ( D ). The scale bar represents 100 μm; ( E ) Changes in the MMP of the cells by paroxetine were also analyzed using a FACSCalibur flow cytometer. The dot plots showed red fluorescence (JC-1 red aggregates) versus green fluorescence (JC-1 green monomer); ( F ) Changes in the plasma membrane potential (PMP) of cells by paroxetine treatment were measured with DiO membrane labeling dye. To induce depolarization of the membrane potential, KCl (25 mM) was used and compared to paroxetine’s effect (30 μM). The PMP of the cells was quantified by the Fluoview software program; ( G ) Effect of MitoTEMPO on paroxetine-induced cell death, ROS generation, and decrease in MMP. The scale bar represents 50 μm. ‘Paro’ and ‘Mito’ represent paroxetine and MitoTEMPO, respectively. Each bar is the mean ± SD obtained from three independent experiments. * p

Article Snippet: Measurement of Mitochondrial Membrane Potential (MMP) and Plasma Membrane Potential MMP changes were determined by JC-1 mitochondrial membrane potential detection kit (Biotium Inc.) according to the manufacturer’s protocol.

Techniques: Incubation, Fluorescence, Microscopy, Flow Cytometry, Cytometry, Labeling, Software