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  • 77
    Promega atp celltitre glo
    Atp Celltitre Glo, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp glo assay
    In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by <t>ATP-Glo</t> assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b
    Atp Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega bactiter glo atp reagent
    In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by <t>ATP-Glo</t> assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b
    Bactiter Glo Atp Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp celltitre glo reagent
    In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by <t>ATP-Glo</t> assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b
    Atp Celltitre Glo Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo atp assay
    In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by <t>ATP-Glo</t> assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b
    Celltiter Glo Atp Assay, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp based celltiter glo kit
    Loss of SETD2 increases the metabolic rate in ccRCC cells. The cell metabolic rate was measured by (A) MTT (n=3) and (B) Alamar Blue assays (n=3) at 3 hours after cell plating. (C) Cellular <t>ATP</t> production was measured by <t>CellTiter-Glo</t> reagent (n=3). (D-E) Western blot and quantitative analysis for H3K36me3 histone protein (*p
    Atp Based Celltiter Glo Kit, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo atp measurement system
    Cellular antisense activity in HeLa pLuc705 cells of PNAs 4305, 4263, 4265, 4306, 2787 and 2534. After transfection of the cells with 3 µM PNA, irradiation of cells was carried out with the excitation wavelength specific for each fluorophore for 1, 5, 10, 20, 25 and 30 min. Cells were incubated further for 24 h after irradiation. All the samples were subjected to the luciferase analysis. Luciferase activities were analysed by <t>Bright-Glo</t> reagent (Promega) and are expressed as relative light units (RLU/well) normalized by the <t>ATP.</t> Each data set represents the mean ± SD of triplicate experiment. CQ: chloroquine. TM-Rhodamine: Tetramethylrhodamine.
    Celltiter Glo Atp Measurement System, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo luminescent atp assay
    Evaluation of BKM120-mediated cytotoxicity in twelve MB cell lines. (A) MB cells were treated with BKM120 ranging from 31.25 nM to 4 μM over a course of 48 hours. Cell viability was determined by <t>CellTiter-Glo</t> assay. Percent cell viability was plotted and IC 50 values were calculated by fitting the data to a four-parameter, variable slope dose-response model in GraphPad. Experiments were averages of three replicates. (B) PIK3CA gene expression MAS5 scores and BKM120 IC 50 values in all MB cell lines.
    Celltiter Glo Luminescent Atp Assay, supplied by Promega, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp assay cell titer glo
    Evaluation of BKM120-mediated cytotoxicity in twelve MB cell lines. (A) MB cells were treated with BKM120 ranging from 31.25 nM to 4 μM over a course of 48 hours. Cell viability was determined by <t>CellTiter-Glo</t> assay. Percent cell viability was plotted and IC 50 values were calculated by fitting the data to a four-parameter, variable slope dose-response model in GraphPad. Experiments were averages of three replicates. (B) PIK3CA gene expression MAS5 scores and BKM120 IC 50 values in all MB cell lines.
    Atp Assay Cell Titer Glo, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega celltiter glo luminescent atp assay kit
    Effects of APE1 overexpression on mitochondrial function in H/R-treated H9c2 cells. H9c2 cells were transfected with LV-APE1 or LV-Scr and subjected to H/R. (A) The mitochondrial membrane potential was detected using JC-1 staining. (B) Complex I, (C) complex II, (D) complex III and (E) complex IV activities were detected using microplate assay kits. (F) <t>ATP</t> production was assessed using the <t>CellTiter-Glo</t> luminescent ATP assay kit. (G) Bax/Bcl-2 ratio was measured using western blotting and (H) quantified. *P
    Celltiter Glo Luminescent Atp Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega cellular adp atp glo assay
    Effects of APE1 overexpression on mitochondrial function in H/R-treated H9c2 cells. H9c2 cells were transfected with LV-APE1 or LV-Scr and subjected to H/R. (A) The mitochondrial membrane potential was detected using JC-1 staining. (B) Complex I, (C) complex II, (D) complex III and (E) complex IV activities were detected using microplate assay kits. (F) <t>ATP</t> production was assessed using the <t>CellTiter-Glo</t> luminescent ATP assay kit. (G) Bax/Bcl-2 ratio was measured using western blotting and (H) quantified. *P
    Cellular Adp Atp Glo Assay, supplied by Promega, used in various techniques. Bioz Stars score: 84/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biotium atp glo bioluminometric cell viability assay
    Trehalose promotes autophagic flux and attenuates mitochondrial dysfunction in chondrocytes. ( a and b ) Mitochondrial membrane potential was detected using Mitotracker and the nuclei were stained with Hoechst. (bar: 10 μ m). ( c ) <t>ATP</t> content was assessed by <t>ATP-Glo</t> <t>Bioluminometric</t> Cell Viability Assay. ( d ) Immunofluorescence double-labeled staining for co-localization of Drp-1 with Tom20 (Green: drp-1, red: Tom 20, bar: 10 μ m). ( e ) TEM images of the mitochondria and autophaghic change in chondrocytes (× 15 000 or 50 000). Lightning mark: mitochondrial fission; Asterisk: swollen mitochondira with fractured cristae; Double arrow: autophagosome with double membrane structure; single arrow: autophagolysosome). ( f and g ) The protein levels of SOD2, Bax, Bcl-2 and Cyt C in chondrocytes treated as indicated. ( h and i ) Double-labeled immunofluorescence staining of p62 and Cyt C in chondrocytes. (Green: p62, red: Cyt C, bar: 10 μ m). All data represent mean±S.D ( n =5). ** P
    Atp Glo Bioluminometric Cell Viability Assay, supplied by Biotium, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biotium atp glo detection cocktail
    Trehalose promotes autophagic flux and attenuates mitochondrial dysfunction in chondrocytes. ( a and b ) Mitochondrial membrane potential was detected using Mitotracker and the nuclei were stained with Hoechst. (bar: 10 μ m). ( c ) <t>ATP</t> content was assessed by <t>ATP-Glo</t> <t>Bioluminometric</t> Cell Viability Assay. ( d ) Immunofluorescence double-labeled staining for co-localization of Drp-1 with Tom20 (Green: drp-1, red: Tom 20, bar: 10 μ m). ( e ) TEM images of the mitochondria and autophaghic change in chondrocytes (× 15 000 or 50 000). Lightning mark: mitochondrial fission; Asterisk: swollen mitochondira with fractured cristae; Double arrow: autophagosome with double membrane structure; single arrow: autophagolysosome). ( f and g ) The protein levels of SOD2, Bax, Bcl-2 and Cyt C in chondrocytes treated as indicated. ( h and i ) Double-labeled immunofluorescence staining of p62 and Cyt C in chondrocytes. (Green: p62, red: Cyt C, bar: 10 μ m). All data represent mean±S.D ( n =5). ** P
    Atp Glo Detection Cocktail, supplied by Biotium, used in various techniques. Bioz Stars score: 84/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp assay cell titer glo reagent
    Trehalose promotes autophagic flux and attenuates mitochondrial dysfunction in chondrocytes. ( a and b ) Mitochondrial membrane potential was detected using Mitotracker and the nuclei were stained with Hoechst. (bar: 10 μ m). ( c ) <t>ATP</t> content was assessed by <t>ATP-Glo</t> <t>Bioluminometric</t> Cell Viability Assay. ( d ) Immunofluorescence double-labeled staining for co-localization of Drp-1 with Tom20 (Green: drp-1, red: Tom 20, bar: 10 μ m). ( e ) TEM images of the mitochondria and autophaghic change in chondrocytes (× 15 000 or 50 000). Lightning mark: mitochondrial fission; Asterisk: swollen mitochondira with fractured cristae; Double arrow: autophagosome with double membrane structure; single arrow: autophagolysosome). ( f and g ) The protein levels of SOD2, Bax, Bcl-2 and Cyt C in chondrocytes treated as indicated. ( h and i ) Double-labeled immunofluorescence staining of p62 and Cyt C in chondrocytes. (Green: p62, red: Cyt C, bar: 10 μ m). All data represent mean±S.D ( n =5). ** P
    Atp Assay Cell Titer Glo Reagent, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp based cell viability assay celltiter glo
    Effect of HSA levels on the in vitro sensitivity of JAK2wt K562 and JAK2V617F mutated HEL and SET-2 cells to ruxolitinib. JAK2wt K562 (circles) and JAK2V617F + HEL (squares) and SET-2 (triangles) myeloid cell lines were cultured in 10% FBS medium or serum-free medium containing HSA (0–80 µM) and treated with the indicated doses of ruxolitinib for 72 hours. Cell viability was determined by the <t>ATP-based</t> cell-viability assay <t>CellTiter-Glo.</t> Data are the percent of untreated control cells. All data are presented as means of three independent experiments ± SD.
    Atp Based Cell Viability Assay Celltiter Glo, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biotium atp glo bioluminometric cell viability assay kit
    Therapeutic intervention of NTRK1 fusion-driven cell growth A. Therapeutic intervention of NTRK1 fusion-positive colon cancer cells. Therapeutic intervention of NTRK1 fusion-driven cell growth was evaluated in KM12 cells with Lestaurtinib, Crizotinib, and ARRY-470. KM12 was treated at 0.64 nM to 10 μM of each compounds for 4 days, and cell growth was evaluated by <t>ATP-Glo</t> <t>Bioluminometric</t> Cell Viability Assay kit (Biotium Inc.). KM12 was sensitive to Lestaurtinib and ARRY-470 with 10.7 nM and 3.2 nM of CC50, respectively. Crizotinib was less potent to inhibit KM12 proliferation with CC50 = 184.8 nM. B. Therapeutic intervention of NTRK1 fusion-negative colon cancer cells. HCT116 was resistant to ARRY-470. Crizotinib was more potent to inhibit HCT116 proliferation with CC50 = 568 nM.
    Atp Glo Bioluminometric Cell Viability Assay Kit, supplied by Biotium, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp glo kinase kit
    Therapeutic intervention of NTRK1 fusion-driven cell growth A. Therapeutic intervention of NTRK1 fusion-positive colon cancer cells. Therapeutic intervention of NTRK1 fusion-driven cell growth was evaluated in KM12 cells with Lestaurtinib, Crizotinib, and ARRY-470. KM12 was treated at 0.64 nM to 10 μM of each compounds for 4 days, and cell growth was evaluated by <t>ATP-Glo</t> <t>Bioluminometric</t> Cell Viability Assay kit (Biotium Inc.). KM12 was sensitive to Lestaurtinib and ARRY-470 with 10.7 nM and 3.2 nM of CC50, respectively. Crizotinib was less potent to inhibit KM12 proliferation with CC50 = 184.8 nM. B. Therapeutic intervention of NTRK1 fusion-negative colon cancer cells. HCT116 was resistant to ARRY-470. Crizotinib was more potent to inhibit HCT116 proliferation with CC50 = 568 nM.
    Atp Glo Kinase Kit, supplied by Promega, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp based celltiter glo luminescent cell viability assay
    miR-34c in human ovarian cancer. A ) Human normal fallopian tubes have a much higher miR-34c level than human serous ovarian cancer ( P = 0.0029). Taqman QPCR identified relative miR-34c levels in human normal fallopian tubes (n = 6), human serous ovarian cancer cell lines (n = 2), and human serous ovarian adenocarcinomas (n = 12). B and D ) miR-34c overexpression inhibited the proliferation of human ovarian cancer cell OVCAR8 and OVCAR5. Cell viability was measured by <t>Promega</t> <t>CellTiter-Glo</t> assay and expressed as mean values ± SEM for three determinations (* P
    Atp Based Celltiter Glo Luminescent Cell Viability Assay, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp based celltiter glo luminescent cell viability kit
    Cultured primary neurons derived from ALDH2*2/*2 mice are more sensitive to ethanol-induced toxicity relative to primary neurons of WT mice. a ) LDH release a measure of cell death, was determined using LDH assay kit in primary neurons in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM); Veh – Vehicle; A – Alda-1. b ) Cellular ROS production was determined using 2,7 dichloro- fluorescein diacetate (DCFDA) in primary neurons, treated as in a; Veh – Vehicle; A – Alda-1. c ) Measurement of cellular <t>ATP</t> levels using <t>CellTiter-Glo</t> Luminescent Cell Viability kit; Veh – Vehicle; A – Alda-1. d ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in primary neurons treated as in a; Veh – Vehicle; A – Alda-1. e ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux in primary neuron, treated as in a; Veh – Vehicle; A – Alda-1. f ) Levels of LC3B, TOM20, p53 and phosphorylation of JNK (Thr183/Tyr185) were determined in primary neurons, treated as in a, by immunoblotting. β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/ fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)
    Atp Based Celltiter Glo Luminescent Cell Viability Kit, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega kinase glo atp assay system
    Cultured primary neurons derived from ALDH2*2/*2 mice are more sensitive to ethanol-induced toxicity relative to primary neurons of WT mice. a ) LDH release a measure of cell death, was determined using LDH assay kit in primary neurons in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM); Veh – Vehicle; A – Alda-1. b ) Cellular ROS production was determined using 2,7 dichloro- fluorescein diacetate (DCFDA) in primary neurons, treated as in a; Veh – Vehicle; A – Alda-1. c ) Measurement of cellular <t>ATP</t> levels using <t>CellTiter-Glo</t> Luminescent Cell Viability kit; Veh – Vehicle; A – Alda-1. d ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in primary neurons treated as in a; Veh – Vehicle; A – Alda-1. e ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux in primary neuron, treated as in a; Veh – Vehicle; A – Alda-1. f ) Levels of LC3B, TOM20, p53 and phosphorylation of JNK (Thr183/Tyr185) were determined in primary neurons, treated as in a, by immunoblotting. β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/ fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)
    Kinase Glo Atp Assay System, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega bac titer glo atp cell viability solution
    Cultured primary neurons derived from ALDH2*2/*2 mice are more sensitive to ethanol-induced toxicity relative to primary neurons of WT mice. a ) LDH release a measure of cell death, was determined using LDH assay kit in primary neurons in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM); Veh – Vehicle; A – Alda-1. b ) Cellular ROS production was determined using 2,7 dichloro- fluorescein diacetate (DCFDA) in primary neurons, treated as in a; Veh – Vehicle; A – Alda-1. c ) Measurement of cellular <t>ATP</t> levels using <t>CellTiter-Glo</t> Luminescent Cell Viability kit; Veh – Vehicle; A – Alda-1. d ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in primary neurons treated as in a; Veh – Vehicle; A – Alda-1. e ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux in primary neuron, treated as in a; Veh – Vehicle; A – Alda-1. f ) Levels of LC3B, TOM20, p53 and phosphorylation of JNK (Thr183/Tyr185) were determined in primary neurons, treated as in a, by immunoblotting. β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/ fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)
    Bac Titer Glo Atp Cell Viability Solution, supplied by Promega, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega atp adp ratio glo assay kit
    Effects of Nrf1 silencing on glucose metabolism and <t>ATP</t> production in MIN6 β-cells and isolated mouse islets. (A, B, D) NADPH/NADP ratio (A) , <t>ATP/ADP</t> ratio (B), and calcium levels (D) in MIN6 cells. Cells were treated with 3 and 20 m M
    Atp Adp Ratio Glo Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega cell titer glo atp measurement kit
    Effects of Nrf1 silencing on glucose metabolism and <t>ATP</t> production in MIN6 β-cells and isolated mouse islets. (A, B, D) NADPH/NADP ratio (A) , <t>ATP/ADP</t> ratio (B), and calcium levels (D) in MIN6 cells. Cells were treated with 3 and 20 m M
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    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using <t>Caspase-Glo</t> 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic <t>ATP</t> levels were measured using ATP-based <t>CellTiter-Glo</t> Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P
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    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using <t>Caspase-Glo</t> 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic <t>ATP</t> levels were measured using ATP-based <t>CellTiter-Glo</t> Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P
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    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using <t>Caspase-Glo</t> 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic <t>ATP</t> levels were measured using ATP-based <t>CellTiter-Glo</t> Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P
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    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using <t>Caspase-Glo</t> 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic <t>ATP</t> levels were measured using ATP-based <t>CellTiter-Glo</t> Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P
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    Image Search Results


    In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by ATP-Glo assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b

    Journal: Nature

    Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

    doi: 10.1038/nature23291

    Figure Lengend Snippet: In hypoactive BRAF-mutant tumours with wild-type RAS/NF1 ERK signalling is sensitive to upstream inhibition of RAS a . b , RTK phosphorylation profile of the cells isolated from the ovarian metastatisis derived PDX (BRCC-OVA) was assessed with Human Phospho-RTK arrays. The elevated p-EGFR and p-MET bands are within the red and green rectangles, respectively. c . d , e . f , The growth inhibition of BRCC-OVA cells with indicated drugs at 0, 1, 3, 10, 30, 100, 300 and 1,000 nM of trametinib or 0, 10, 30, 100, 300, 1,000, 3,000 and 10,000 nM of INC280 or combination of increasing dose of trametinib with 100 nM INC280 was determined by ATP-Glo assay after 3 days of treatment. Graphs were generated using Prism 6 (mean ±s.d., n = 8). Fig. 4b

    Article Snippet: Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

    Techniques: Mutagenesis, Inhibition, Isolation, Derivative Assay, Glo Assay, Generated

    ERK signalling in hypoactive BRAF-mutant cells is sensitive to trametinib a . b , The cell lines as indicated in a were treated with different concentrations of trametinib for 3 days. Cell viability was determined by ATP-Glo assay. Dose-dependent inhibition curves were generated using Prism 6 (mean ± s.d., n =8).

    Journal: Nature

    Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

    doi: 10.1038/nature23291

    Figure Lengend Snippet: ERK signalling in hypoactive BRAF-mutant cells is sensitive to trametinib a . b , The cell lines as indicated in a were treated with different concentrations of trametinib for 3 days. Cell viability was determined by ATP-Glo assay. Dose-dependent inhibition curves were generated using Prism 6 (mean ± s.d., n =8).

    Article Snippet: Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

    Techniques: Mutagenesis, Glo Assay, Inhibition, Generated

    Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the ATP-Glo assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d

    Journal: Nature

    Article Title: Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS

    doi: 10.1038/nature23291

    Figure Lengend Snippet: Activation of MEK/ERK signalling by hypoactive BRAF mutants is RAS-dependent a , V5-tagged wild-type (WT) or mutant BRAF kinases were expressed in 293H cells that stably express NRAS(Q61K). These BRAF protein kinases were isolated with anti-V5 agarose. The in vitro . b . c , In vitro kinase activity of the indicated BRAF proteins which were isolated from 293H cells that stably express NRAS(Q61K) was assessed as described in a . d . e , NIH3T3 cells expressing the indicated BRAF proteins were stimulated with 100 ng ml −1 . f were cultured in doxycycline (30 ng ml −1 . g . h . The P values were calculated by using a paired t -test. N.S., not significant. i , The phosphorylation of multiple RTKs in the indicated cell lines was assayed using the Human Phospho-RTK Array Kit. Phosphorylated RTKs are highlighted with boxes in different colours. j . k , Cells were cultured and exposed to cetuximab at concentrations of 0, 0.3, 1, 3, 10, 30, 100 and 300 nM for 3 days. The effects of drug on cell growth were quantified using the ATP-Glo assay. Graphs were generated using Prism 6 (mean ±s.d. are represented by the dots and error bars, n =8). l . m , The effects of cetuximab or trametinib on the growth of the cells described in l was determined by ATP-Glo assay after 3 days treatment. Graphs were generated using Prism 6 (mean ± s.d., n = 8). n . o , Growth inhibition of the cells described in n after three days exposure to varying doses of cetuximab on day 3 was determined by ATP-Glo assay. Graphs were generated using Prism 6 (mean ± s.d., n =8) Fig. 1d

    Article Snippet: Cell growth was quantified using the ATP-Glo assay (Promega, G7572) every 24 h. For each condition, 8 replicates at each concentration were measured.

    Techniques: Activation Assay, Mutagenesis, Stable Transfection, Isolation, In Vitro, Activity Assay, Expressing, Cell Culture, Glo Assay, Generated, Inhibition

    Loss of SETD2 increases the metabolic rate in ccRCC cells. The cell metabolic rate was measured by (A) MTT (n=3) and (B) Alamar Blue assays (n=3) at 3 hours after cell plating. (C) Cellular ATP production was measured by CellTiter-Glo reagent (n=3). (D-E) Western blot and quantitative analysis for H3K36me3 histone protein (*p

    Journal: Journal of proteome research

    Article Title: Loss of SETD2 induces a metabolic switch in renal cell carcinoma cell lines toward enhanced oxidative phosphorylation

    doi: 10.1021/acs.jproteome.8b00628

    Figure Lengend Snippet: Loss of SETD2 increases the metabolic rate in ccRCC cells. The cell metabolic rate was measured by (A) MTT (n=3) and (B) Alamar Blue assays (n=3) at 3 hours after cell plating. (C) Cellular ATP production was measured by CellTiter-Glo reagent (n=3). (D-E) Western blot and quantitative analysis for H3K36me3 histone protein (*p

    Article Snippet: Cellular ATP levels were measured using the CellTiter-Glo ATP assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

    Techniques: MTT Assay, Western Blot

    Viability of cells infected with ZP6248 and its mutants. The GHOST GPR15 cells were infected with ZP6248 and its mutants and cultured for 10(day 10) was determined by CellTiter-Glo Luminescent Cell Viability Assay. Each virus was assayed in triplicate and mean ± standard deviation is shown. Similar results were obtained in two independent experiments and the results from one experiment are shown.

    Journal: PLoS ONE

    Article Title: The Variable Loop 3 in the Envelope Glycoprotein Is Critical for the Atypical Coreceptor Usage of an HIV-1 Strain

    doi: 10.1371/journal.pone.0098058

    Figure Lengend Snippet: Viability of cells infected with ZP6248 and its mutants. The GHOST GPR15 cells were infected with ZP6248 and its mutants and cultured for 10(day 10) was determined by CellTiter-Glo Luminescent Cell Viability Assay. Each virus was assayed in triplicate and mean ± standard deviation is shown. Similar results were obtained in two independent experiments and the results from one experiment are shown.

    Article Snippet: Cell viability assay Cellular ATP was measured in white 96-well plates using the CellTiter Glo luminescence ATP assay kit (Promega, Madison, WI, USA).

    Techniques: Infection, Cell Culture, Cell Viability Assay, Standard Deviation

    Cellular antisense activity in HeLa pLuc705 cells of PNAs 4305, 4263, 4265, 4306, 2787 and 2534. After transfection of the cells with 3 µM PNA, irradiation of cells was carried out with the excitation wavelength specific for each fluorophore for 1, 5, 10, 20, 25 and 30 min. Cells were incubated further for 24 h after irradiation. All the samples were subjected to the luciferase analysis. Luciferase activities were analysed by Bright-Glo reagent (Promega) and are expressed as relative light units (RLU/well) normalized by the ATP. Each data set represents the mean ± SD of triplicate experiment. CQ: chloroquine. TM-Rhodamine: Tetramethylrhodamine.

    Journal: Scientific Reports

    Article Title: Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity

    doi: 10.1038/s41598-017-18947-x

    Figure Lengend Snippet: Cellular antisense activity in HeLa pLuc705 cells of PNAs 4305, 4263, 4265, 4306, 2787 and 2534. After transfection of the cells with 3 µM PNA, irradiation of cells was carried out with the excitation wavelength specific for each fluorophore for 1, 5, 10, 20, 25 and 30 min. Cells were incubated further for 24 h after irradiation. All the samples were subjected to the luciferase analysis. Luciferase activities were analysed by Bright-Glo reagent (Promega) and are expressed as relative light units (RLU/well) normalized by the ATP. Each data set represents the mean ± SD of triplicate experiment. CQ: chloroquine. TM-Rhodamine: Tetramethylrhodamine.

    Article Snippet: Luminescent readings obtained by the Bright-Glo luciferase assay system were background-subtracted and normalized for the cell viability by the CellTiter-Glo ATP measurement system (Promega) based on the manufacturer protocol.

    Techniques: Activity Assay, Transfection, Irradiation, Incubation, Luciferase

    Evaluation of BKM120-mediated cytotoxicity in twelve MB cell lines. (A) MB cells were treated with BKM120 ranging from 31.25 nM to 4 μM over a course of 48 hours. Cell viability was determined by CellTiter-Glo assay. Percent cell viability was plotted and IC 50 values were calculated by fitting the data to a four-parameter, variable slope dose-response model in GraphPad. Experiments were averages of three replicates. (B) PIK3CA gene expression MAS5 scores and BKM120 IC 50 values in all MB cell lines.

    Journal: PLoS ONE

    Article Title: BKM120 induces apoptosis and inhibits tumor growth in medulloblastoma

    doi: 10.1371/journal.pone.0179948

    Figure Lengend Snippet: Evaluation of BKM120-mediated cytotoxicity in twelve MB cell lines. (A) MB cells were treated with BKM120 ranging from 31.25 nM to 4 μM over a course of 48 hours. Cell viability was determined by CellTiter-Glo assay. Percent cell viability was plotted and IC 50 values were calculated by fitting the data to a four-parameter, variable slope dose-response model in GraphPad. Experiments were averages of three replicates. (B) PIK3CA gene expression MAS5 scores and BKM120 IC 50 values in all MB cell lines.

    Article Snippet: Measurement of cellular ATP level CellTiter GLO luminescent assay (Promega), which measures total ATP levels, was combined with the CyQuant fluorescent DNA assay (Life Technologies, Grand Island, NY) to measure ATP level per cell.

    Techniques: Glo Assay, Expressing

    Effects of APE1 overexpression on mitochondrial function in H/R-treated H9c2 cells. H9c2 cells were transfected with LV-APE1 or LV-Scr and subjected to H/R. (A) The mitochondrial membrane potential was detected using JC-1 staining. (B) Complex I, (C) complex II, (D) complex III and (E) complex IV activities were detected using microplate assay kits. (F) ATP production was assessed using the CellTiter-Glo luminescent ATP assay kit. (G) Bax/Bcl-2 ratio was measured using western blotting and (H) quantified. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) alleviates myocardial hypoxia-reoxygenation injury by inhibiting oxidative stress and ameliorating mitochondrial dysfunction

    doi: 10.3892/etm.2019.7212

    Figure Lengend Snippet: Effects of APE1 overexpression on mitochondrial function in H/R-treated H9c2 cells. H9c2 cells were transfected with LV-APE1 or LV-Scr and subjected to H/R. (A) The mitochondrial membrane potential was detected using JC-1 staining. (B) Complex I, (C) complex II, (D) complex III and (E) complex IV activities were detected using microplate assay kits. (F) ATP production was assessed using the CellTiter-Glo luminescent ATP assay kit. (G) Bax/Bcl-2 ratio was measured using western blotting and (H) quantified. *P

    Article Snippet: ATP generation was detected using a CellTiter-Glo luminescent ATP assay kit (Promega Corp., Madison, WI, USA) according to the manufacturer's protocol.

    Techniques: Over Expression, Transfection, Staining, ATP Assay, Western Blot

    Trehalose promotes autophagic flux and attenuates mitochondrial dysfunction in chondrocytes. ( a and b ) Mitochondrial membrane potential was detected using Mitotracker and the nuclei were stained with Hoechst. (bar: 10 μ m). ( c ) ATP content was assessed by ATP-Glo Bioluminometric Cell Viability Assay. ( d ) Immunofluorescence double-labeled staining for co-localization of Drp-1 with Tom20 (Green: drp-1, red: Tom 20, bar: 10 μ m). ( e ) TEM images of the mitochondria and autophaghic change in chondrocytes (× 15 000 or 50 000). Lightning mark: mitochondrial fission; Asterisk: swollen mitochondira with fractured cristae; Double arrow: autophagosome with double membrane structure; single arrow: autophagolysosome). ( f and g ) The protein levels of SOD2, Bax, Bcl-2 and Cyt C in chondrocytes treated as indicated. ( h and i ) Double-labeled immunofluorescence staining of p62 and Cyt C in chondrocytes. (Green: p62, red: Cyt C, bar: 10 μ m). All data represent mean±S.D ( n =5). ** P

    Journal: Cell Death & Disease

    Article Title: Trehalose ameliorates oxidative stress-mediated mitochondrial dysfunction and ER stress via selective autophagy stimulation and autophagic flux restoration in osteoarthritis development

    doi: 10.1038/cddis.2017.453

    Figure Lengend Snippet: Trehalose promotes autophagic flux and attenuates mitochondrial dysfunction in chondrocytes. ( a and b ) Mitochondrial membrane potential was detected using Mitotracker and the nuclei were stained with Hoechst. (bar: 10 μ m). ( c ) ATP content was assessed by ATP-Glo Bioluminometric Cell Viability Assay. ( d ) Immunofluorescence double-labeled staining for co-localization of Drp-1 with Tom20 (Green: drp-1, red: Tom 20, bar: 10 μ m). ( e ) TEM images of the mitochondria and autophaghic change in chondrocytes (× 15 000 or 50 000). Lightning mark: mitochondrial fission; Asterisk: swollen mitochondira with fractured cristae; Double arrow: autophagosome with double membrane structure; single arrow: autophagolysosome). ( f and g ) The protein levels of SOD2, Bax, Bcl-2 and Cyt C in chondrocytes treated as indicated. ( h and i ) Double-labeled immunofluorescence staining of p62 and Cyt C in chondrocytes. (Green: p62, red: Cyt C, bar: 10 μ m). All data represent mean±S.D ( n =5). ** P

    Article Snippet: ATP Assay The ATP-Glo Bioluminometric Cell Viability Assay (Biotium, Hayward, CA, USA) was used to assess cellular ATP levels according to the manufacturer’s protocol.

    Techniques: Staining, Viability Assay, Immunofluorescence, Labeling, Transmission Electron Microscopy

    Effect of HSA levels on the in vitro sensitivity of JAK2wt K562 and JAK2V617F mutated HEL and SET-2 cells to ruxolitinib. JAK2wt K562 (circles) and JAK2V617F + HEL (squares) and SET-2 (triangles) myeloid cell lines were cultured in 10% FBS medium or serum-free medium containing HSA (0–80 µM) and treated with the indicated doses of ruxolitinib for 72 hours. Cell viability was determined by the ATP-based cell-viability assay CellTiter-Glo. Data are the percent of untreated control cells. All data are presented as means of three independent experiments ± SD.

    Journal: Scientific Reports

    Article Title: Ruxolitinib binding to human serum albumin: bioinformatics, biochemical and functional characterization in JAK2V617F+ cell models

    doi: 10.1038/s41598-019-52852-9

    Figure Lengend Snippet: Effect of HSA levels on the in vitro sensitivity of JAK2wt K562 and JAK2V617F mutated HEL and SET-2 cells to ruxolitinib. JAK2wt K562 (circles) and JAK2V617F + HEL (squares) and SET-2 (triangles) myeloid cell lines were cultured in 10% FBS medium or serum-free medium containing HSA (0–80 µM) and treated with the indicated doses of ruxolitinib for 72 hours. Cell viability was determined by the ATP-based cell-viability assay CellTiter-Glo. Data are the percent of untreated control cells. All data are presented as means of three independent experiments ± SD.

    Article Snippet: Cell viability The viability of cells was determined using the ATP-based cell-viability assay CellTiter-Glo (Promega, Madison, WI, USA) according to the manufacturer’s instruction.

    Techniques: In Vitro, Cell Culture, Viability Assay

    Therapeutic intervention of NTRK1 fusion-driven cell growth A. Therapeutic intervention of NTRK1 fusion-positive colon cancer cells. Therapeutic intervention of NTRK1 fusion-driven cell growth was evaluated in KM12 cells with Lestaurtinib, Crizotinib, and ARRY-470. KM12 was treated at 0.64 nM to 10 μM of each compounds for 4 days, and cell growth was evaluated by ATP-Glo Bioluminometric Cell Viability Assay kit (Biotium Inc.). KM12 was sensitive to Lestaurtinib and ARRY-470 with 10.7 nM and 3.2 nM of CC50, respectively. Crizotinib was less potent to inhibit KM12 proliferation with CC50 = 184.8 nM. B. Therapeutic intervention of NTRK1 fusion-negative colon cancer cells. HCT116 was resistant to ARRY-470. Crizotinib was more potent to inhibit HCT116 proliferation with CC50 = 568 nM.

    Journal: Oncotarget

    Article Title: NTRK1 fusions for the therapeutic intervention of Korean patients with colon cancer

    doi: 10.18632/oncotarget.6724

    Figure Lengend Snippet: Therapeutic intervention of NTRK1 fusion-driven cell growth A. Therapeutic intervention of NTRK1 fusion-positive colon cancer cells. Therapeutic intervention of NTRK1 fusion-driven cell growth was evaluated in KM12 cells with Lestaurtinib, Crizotinib, and ARRY-470. KM12 was treated at 0.64 nM to 10 μM of each compounds for 4 days, and cell growth was evaluated by ATP-Glo Bioluminometric Cell Viability Assay kit (Biotium Inc.). KM12 was sensitive to Lestaurtinib and ARRY-470 with 10.7 nM and 3.2 nM of CC50, respectively. Crizotinib was less potent to inhibit KM12 proliferation with CC50 = 184.8 nM. B. Therapeutic intervention of NTRK1 fusion-negative colon cancer cells. HCT116 was resistant to ARRY-470. Crizotinib was more potent to inhibit HCT116 proliferation with CC50 = 568 nM.

    Article Snippet: KM12 and HCT116 (KCLB) were treated at 0.64 nM to 10 μM of each compounds for 4 days, and cell growth was evaluated by the ATP-Glo Bioluminometric Cell Viability Assay Kit (Biotium Inc., USA).

    Techniques: Viability Assay

    miR-34c in human ovarian cancer. A ) Human normal fallopian tubes have a much higher miR-34c level than human serous ovarian cancer ( P = 0.0029). Taqman QPCR identified relative miR-34c levels in human normal fallopian tubes (n = 6), human serous ovarian cancer cell lines (n = 2), and human serous ovarian adenocarcinomas (n = 12). B and D ) miR-34c overexpression inhibited the proliferation of human ovarian cancer cell OVCAR8 and OVCAR5. Cell viability was measured by Promega CellTiter-Glo assay and expressed as mean values ± SEM for three determinations (* P

    Journal: Biology of Reproduction

    Article Title: Functional Analysis of miR-34c as a Putative Tumor Suppressor in High-Grade Serous Ovarian Cancer 1

    doi: 10.1095/biolreprod.114.121988

    Figure Lengend Snippet: miR-34c in human ovarian cancer. A ) Human normal fallopian tubes have a much higher miR-34c level than human serous ovarian cancer ( P = 0.0029). Taqman QPCR identified relative miR-34c levels in human normal fallopian tubes (n = 6), human serous ovarian cancer cell lines (n = 2), and human serous ovarian adenocarcinomas (n = 12). B and D ) miR-34c overexpression inhibited the proliferation of human ovarian cancer cell OVCAR8 and OVCAR5. Cell viability was measured by Promega CellTiter-Glo assay and expressed as mean values ± SEM for three determinations (* P

    Article Snippet: The viability of DKO tumor cells transfected with miR-34c or let-7b mimics was determined by ATP level quantitation-based Promega CellTiter-Glo Luminescent Cell Viability Assay.

    Techniques: Real-time Polymerase Chain Reaction, Over Expression, Glo Assay

    Cultured primary neurons derived from ALDH2*2/*2 mice are more sensitive to ethanol-induced toxicity relative to primary neurons of WT mice. a ) LDH release a measure of cell death, was determined using LDH assay kit in primary neurons in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM); Veh – Vehicle; A – Alda-1. b ) Cellular ROS production was determined using 2,7 dichloro- fluorescein diacetate (DCFDA) in primary neurons, treated as in a; Veh – Vehicle; A – Alda-1. c ) Measurement of cellular ATP levels using CellTiter-Glo Luminescent Cell Viability kit; Veh – Vehicle; A – Alda-1. d ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in primary neurons treated as in a; Veh – Vehicle; A – Alda-1. e ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux in primary neuron, treated as in a; Veh – Vehicle; A – Alda-1. f ) Levels of LC3B, TOM20, p53 and phosphorylation of JNK (Thr183/Tyr185) were determined in primary neurons, treated as in a, by immunoblotting. β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/ fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Journal: Acta Neuropathologica Communications

    Article Title: Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

    doi: 10.1186/s40478-019-0839-7

    Figure Lengend Snippet: Cultured primary neurons derived from ALDH2*2/*2 mice are more sensitive to ethanol-induced toxicity relative to primary neurons of WT mice. a ) LDH release a measure of cell death, was determined using LDH assay kit in primary neurons in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM); Veh – Vehicle; A – Alda-1. b ) Cellular ROS production was determined using 2,7 dichloro- fluorescein diacetate (DCFDA) in primary neurons, treated as in a; Veh – Vehicle; A – Alda-1. c ) Measurement of cellular ATP levels using CellTiter-Glo Luminescent Cell Viability kit; Veh – Vehicle; A – Alda-1. d ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in primary neurons treated as in a; Veh – Vehicle; A – Alda-1. e ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux in primary neuron, treated as in a; Veh – Vehicle; A – Alda-1. f ) Levels of LC3B, TOM20, p53 and phosphorylation of JNK (Thr183/Tyr185) were determined in primary neurons, treated as in a, by immunoblotting. β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/ fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Article Snippet: ATP measurements Relative intracellular ATP levels were determined using ATP-based CellTiter-Glo Luminescent Cell Viability kit (Promega), which causes cell lysis and generates a luminescent signal proportional to the amount of ATP present.

    Techniques: Cell Culture, Derivative Assay, Mouse Assay, Lactate Dehydrogenase Assay, Quantitation Assay, Standard Deviation

    Cultured primary astrocyte derived from ALDH2*2/*2 mice are more activated in response to ethanol relative to primary astrocytes of WT mice. a ) Measurement of mitochondrial ROS using MitoSOX™ in primary astrocytes in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. b ) Heat map representation of qPCR analysis of genes associated with inflammation, apoptosis, autophagy and mitochondrial health; Veh – Vehicle; A – Alda-1. c ) Nitrite levels were determined in primary astrocytes using Griess reagent kit in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. d ) Measurement of cellular ATP levels using CellTiter-Glo Luminescent Cell Viability kit in primary astrocytes in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. e ) Levels of cellular COX-2 and interleukin-1β release at 6 h were determined by immunoblotting in primary astrocytes in the presence or absence of Alda-1 (20 μM; 50 mM Ethanol). β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Journal: Acta Neuropathologica Communications

    Article Title: Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

    doi: 10.1186/s40478-019-0839-7

    Figure Lengend Snippet: Cultured primary astrocyte derived from ALDH2*2/*2 mice are more activated in response to ethanol relative to primary astrocytes of WT mice. a ) Measurement of mitochondrial ROS using MitoSOX™ in primary astrocytes in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. b ) Heat map representation of qPCR analysis of genes associated with inflammation, apoptosis, autophagy and mitochondrial health; Veh – Vehicle; A – Alda-1. c ) Nitrite levels were determined in primary astrocytes using Griess reagent kit in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. d ) Measurement of cellular ATP levels using CellTiter-Glo Luminescent Cell Viability kit in primary astrocytes in the presence or absence of Alda-1 (20 μM/24 h; 50 mM Ethanol); Veh – Vehicle; A – Alda-1. e ) Levels of cellular COX-2 and interleukin-1β release at 6 h were determined by immunoblotting in primary astrocytes in the presence or absence of Alda-1 (20 μM; 50 mM Ethanol). β-actin was used as loading control; Veh – Vehicle; A – Alda-1. Data information: Mean, standard deviation, and p-values are shown. Results are presented as fold of control. n = 3–4 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Article Snippet: ATP measurements Relative intracellular ATP levels were determined using ATP-based CellTiter-Glo Luminescent Cell Viability kit (Promega), which causes cell lysis and generates a luminescent signal proportional to the amount of ATP present.

    Techniques: Cell Culture, Derivative Assay, Mouse Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    ALDH2*2 mutation is associated with increased oxidative stress in patient-derived fibroblasts with familial Alzheimer’s Disease (AD). a ) Genotyping of a Japanese AD patient-derived fibroblasts identifies heterozygous ALDH2*2/*1 mutation. ALDH2 protein expression in these cells was measured by immunoblotting three different culture passage numbers. b ) The levels of ALDH2 protein were determined in total lysates by immunoblotting of control (healthy subject; H)- and 2*2/*1 AD patient-derived fibroblasts. β-actin was used as loading control. c ) ALDH2 protein levels were quantified and represented as fold change of control. d ) Enzymatic activity of ALDH2 in lysates from ALDH2*2/*1 AD patient-derived fibroblasts relative to fibroblasts from control was measured over 5 min. e ) 4-HNE levels in control and AD patient-derived fibroblasts were measured using 4-HNE assay kit in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. f ) Mitochondrial ROS measured by MitoSOX™ in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/36 h) cultured in glucose free and galactose supplemented media. g ) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. h ) Mitochondrial ROS measured using MitoSOX™ in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. i ) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. j ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in one control- and one AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. k ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux as in panel j. Data information: Mean, standard deviation, and p -values are shown. Results are presented as fold of control. n = 3 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Journal: Acta Neuropathologica Communications

    Article Title: Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

    doi: 10.1186/s40478-019-0839-7

    Figure Lengend Snippet: ALDH2*2 mutation is associated with increased oxidative stress in patient-derived fibroblasts with familial Alzheimer’s Disease (AD). a ) Genotyping of a Japanese AD patient-derived fibroblasts identifies heterozygous ALDH2*2/*1 mutation. ALDH2 protein expression in these cells was measured by immunoblotting three different culture passage numbers. b ) The levels of ALDH2 protein were determined in total lysates by immunoblotting of control (healthy subject; H)- and 2*2/*1 AD patient-derived fibroblasts. β-actin was used as loading control. c ) ALDH2 protein levels were quantified and represented as fold change of control. d ) Enzymatic activity of ALDH2 in lysates from ALDH2*2/*1 AD patient-derived fibroblasts relative to fibroblasts from control was measured over 5 min. e ) 4-HNE levels in control and AD patient-derived fibroblasts were measured using 4-HNE assay kit in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. f ) Mitochondrial ROS measured by MitoSOX™ in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/36 h) cultured in glucose free and galactose supplemented media. g ) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in control- and patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) cultured in glucose free and galactose supplemented media. h ) Mitochondrial ROS measured using MitoSOX™ in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. i ) Cellular ATP levels measured using CellTiter-Glo Luminescent Cell Viability kit in 2 control- and 2 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. Each data point represents an average of 3 independent biological replicates from each subject. j ) Quantitation of basal respiration (OCR) as a measure of oxidative phosphorylation (OXPHOS) using Seahorse Extracellular Flux in one control- and one AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM) 48 h after transfection with ALDH2*2. k ) Quantitation of extracellular acidification rate (ECAR) as a measure of glycolytic dependence using Seahorse Extracellular Flux as in panel j. Data information: Mean, standard deviation, and p -values are shown. Results are presented as fold of control. n = 3 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Article Snippet: ATP measurements Relative intracellular ATP levels were determined using ATP-based CellTiter-Glo Luminescent Cell Viability kit (Promega), which causes cell lysis and generates a luminescent signal proportional to the amount of ATP present.

    Techniques: Mutagenesis, Derivative Assay, Expressing, Activity Assay, Cell Culture, Transfection, Quantitation Assay, Standard Deviation

    Ethanol increases metabolic dysfunction of Alzheimer’s disease (AD) patient-derived fibroblasts that is rescued by ALDH2 activation. a ) Measurement of mitochondrial ROS using MitoSOX™ in 4 control (healthy subject; H)- and 4 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. b ) 4-HNE levels were measured using 4-HNE Assay Kit in control (healthy subject; H)- and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. c ) Cellular ATP levels were analyzed using CellTiter-Glo Luminescent Cell Viability kit in control (healthy subject; H)- and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/36 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. d ) Cellular ROS production was measured using 2,7 dichloro-fluorescein diacetate (DCFDA) in control (healthy subject; H) and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/fold of control. n = 3 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Journal: Acta Neuropathologica Communications

    Article Title: Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

    doi: 10.1186/s40478-019-0839-7

    Figure Lengend Snippet: Ethanol increases metabolic dysfunction of Alzheimer’s disease (AD) patient-derived fibroblasts that is rescued by ALDH2 activation. a ) Measurement of mitochondrial ROS using MitoSOX™ in 4 control (healthy subject; H)- and 4 AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. b ) 4-HNE levels were measured using 4-HNE Assay Kit in control (healthy subject; H)- and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. c ) Cellular ATP levels were analyzed using CellTiter-Glo Luminescent Cell Viability kit in control (healthy subject; H)- and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/36 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. d ) Cellular ROS production was measured using 2,7 dichloro-fluorescein diacetate (DCFDA) in control (healthy subject; H) and AD patient-derived fibroblasts in the presence or absence of Alda-1 (20 μM/48 h) and ethanol (50 mM). Each data point represents an average of 3 independent biological replicates from individual lines. Data information: Mean, standard deviation, and p-values are shown. Results are presented as percent/fold of control. n = 3 independent biological replicates; probability by one-way ANOVA (with Holm-Sidak post hoc test)

    Article Snippet: ATP measurements Relative intracellular ATP levels were determined using ATP-based CellTiter-Glo Luminescent Cell Viability kit (Promega), which causes cell lysis and generates a luminescent signal proportional to the amount of ATP present.

    Techniques: Derivative Assay, Activation Assay, Standard Deviation

    Effects of Nrf1 silencing on glucose metabolism and ATP production in MIN6 β-cells and isolated mouse islets. (A, B, D) NADPH/NADP ratio (A) , ATP/ADP ratio (B), and calcium levels (D) in MIN6 cells. Cells were treated with 3 and 20 m M

    Journal: Antioxidants & Redox Signaling

    Article Title: CNC-bZIP Protein Nrf1-Dependent Regulation of Glucose-Stimulated Insulin Secretion

    doi: 10.1089/ars.2014.6017

    Figure Lengend Snippet: Effects of Nrf1 silencing on glucose metabolism and ATP production in MIN6 β-cells and isolated mouse islets. (A, B, D) NADPH/NADP ratio (A) , ATP/ADP ratio (B), and calcium levels (D) in MIN6 cells. Cells were treated with 3 and 20 m M

    Article Snippet: Resulting supernatants were used immediately for measurement of ATP and ATP/ADP ratio by using the ATP Bioluminescent Assay Kit (Sigma) and ATP/ADP Ratio-Glo™ Assay kit (Promega, Madison, WI), respectively, as per the manufacturer's protocols.

    Techniques: Isolation

    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: MTT Assay, Expressing, Western Blot, Activity Assay, Double Staining, FACS, Fluorescence, Staining

    Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: Activation Assay, Staining, FACS, Expressing, Western Blot, Microscopy, Double Staining

    Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: Inhibition, Activation Assay, Western Blot, Immunofluorescence, Staining, Double Staining, FACS, Real-time Polymerase Chain Reaction, Transfection, Cell Viability Assay, MTT Assay

    Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: MTT Assay, Expressing, Western Blot, Activity Assay, Staining, FACS