Journal: PLoS ONE

Article Title: Effect of mitochondrially targeted carboxy proxyl nitroxide on Akt-mediated survival in Daudi cells: Significance of a dual mode of action

doi: 10.1371/journal.pone.0174546

Figure Lengend Snippet: Daudi cells and PBMC were treated with and without Mito-CP under hypoxia (5% O 2 ) and normoxia. ( A ) Shows percentage of cell viability after 6 h in Daudi and PBMC treated with and without Mito-CP and Dec-TPP + under hypoxia and normoxia. Bar graph plotted represents percentage of viable cells normalised value to percent control. Data were obtained from three separate experiments and were expressed as by mean ± SEM. * and ** denotes significantly different compared to control p<0.05 and p<0.01 respectively. ( B ) Shows percentage of cell proliferation after 24 h in Daudi and PBMC treated with and without Mito-CP under hypoxia and normoxia. Each bar graph represented was normalised to percent control. Data were obtained from three separate experiments and are expressed as mean ± SEM. * and **, significantly different compared to control p<0.05 and p<0.01 respectively.

Article Snippet: Daudi cells (human B lymphoblast cells) were purchased from the National Centre for Cell Science (Maharashtra, India) and maintained in Gibco RPMI-1640 (Life Technologies, Grand Island, New York, USA) medium with 100 U penicillin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India), 100 μg streptomycin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India) and 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, New York, USA), Dec-TPP + (Sigma Aldrich, St.louis, Missouri.)

Techniques: Control

Journal: PLoS ONE

Article Title: Effect of mitochondrially targeted carboxy proxyl nitroxide on Akt-mediated survival in Daudi cells: Significance of a dual mode of action

doi: 10.1371/journal.pone.0174546

Figure Lengend Snippet: Characteristic phenomenon of Phosphotidyl serine externalisation and disruption in cell membrane in apoptotic cells were observed by staining cells with Annexin V FITC and Propidium Iodide. Daudi cells and PBMCs were treated with and without Mito-CP (1μM) under hypoxia (5% O 2 ) and normoxia for a period of 6 h. Cells were visualised under confocal laser scanning microscope and photographed. Images obtained were analysed by Image J software and the data is expressed as bar graph. Data were obtained from five different experiments and are expressed as mean ±SEM. * and **, significantly different compared to control p<0.05 and p<0.01 respectively.

Article Snippet: Daudi cells (human B lymphoblast cells) were purchased from the National Centre for Cell Science (Maharashtra, India) and maintained in Gibco RPMI-1640 (Life Technologies, Grand Island, New York, USA) medium with 100 U penicillin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India), 100 μg streptomycin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India) and 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, New York, USA), Dec-TPP + (Sigma Aldrich, St.louis, Missouri.)

Techniques: Disruption, Membrane, Staining, Laser-Scanning Microscopy, Software, Control

Journal: PLoS ONE

Article Title: Effect of mitochondrially targeted carboxy proxyl nitroxide on Akt-mediated survival in Daudi cells: Significance of a dual mode of action

doi: 10.1371/journal.pone.0174546

Figure Lengend Snippet: (A) Mitochondrial membrane potential in Daudi cells and PBMCs with and without Mito-CP (1μM) and Dec-TPP + (1μM) under hypoxia (5%O 2 ) and normoxia were measured using JC-1 dye. Data were obtained from three separate experiments and are expressed as mean ± SEM. * and **, significantly different when compared to control p<0.05 and p<0.01 respectively. ( B ) Cellular ATP levels in Daudi cells and PBMC were determined using a bioluminescence based assay kit with or without Mito-CP (1μM) and Dec-TPP + (1μM) treatment under hypoxia (5%O 2 ) and normoxia. ATP concentration were determined using the standard ATP provided with the manufacturer’s kit (Molecular Probes, Eugene, OR, USA). Bar graph represented was normalised to protein concentration. Data were obtained from three separate experiments and are expressed as mean ± SEM. * and **, significantly different when compared to control p<0.05 and p<0.01 respectively. ( C ) Hydrogen peroxide levels in Daudi cells and PBMCs with and without Mito-CP (1μM) and Dec-TPP + (1μM) under hypoxia (5%O 2 ) and normoxia were measured using Amplex red assay. Concentration of hydrogen peroxide were obtained using the standard hydrogen peroxide provided with the amplex red reagent manufacturer’s kit (Molecular Probes, Eugene, OR, USA). Bar graph represented was normalised to protein concentration. Data were obtained from three separate experiments and are expressed as mean ± SEM. *, significantly different when compared to control p<0.05. ( D ) Shows EPR monitoring of mitochondrial localization of Mito-CP in Daudi cells and PBMC under hypoxia (5%O 2 ) and normoxia. (i) EPR spectra were obtained from mitochondrial fraction of Daudi cells and PBMCs treated with and without Mito-CP. (ii) As was done for (i), Daudi cells and PBMCs were treated with Mito-CP (1μm). (iii) As was done for (i), Daudi cells and PBMCs were treated with Mito-CP under hypoxia. The parameters used in EPR spectra follow: G xx = 2.0089, G yy = 2.0058, G zz = 2.0021, A xx = 5.6, A yy = 5.3, A zz = 34 G, β = 60°, R xx = 8.9×10 7 , r yy = 8.9x10 7 , r zz = 1.0x10 7 s -i , Ψ = 60°, C 20 = 2.00. ( E ) Shows quantified data of total EPR signal intensity of Mito-CP in Daudi cells and PBMC under normoxia and hypoxia. Concentration of cell lysate used for analysis was determined by Bradford method and equal amount of concentration in each sample was analysed in EPR spectrometer. EPR signal intensity was normalized to the peak intensity obtained by Mito-CP alone before treatment. Data were obtained from three separate experiments and are expressed as mean ± SEM. * and ** denotes significantly different when compared to control p<0.05 and p<0.01 respectively.

Article Snippet: Daudi cells (human B lymphoblast cells) were purchased from the National Centre for Cell Science (Maharashtra, India) and maintained in Gibco RPMI-1640 (Life Technologies, Grand Island, New York, USA) medium with 100 U penicillin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India), 100 μg streptomycin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India) and 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, New York, USA), Dec-TPP + (Sigma Aldrich, St.louis, Missouri.)

Techniques: Membrane, Control, ATP Bioluminescent Assay, Concentration Assay, Protein Concentration, Amplex Red Assay

Journal: PLoS ONE

Article Title: Effect of mitochondrially targeted carboxy proxyl nitroxide on Akt-mediated survival in Daudi cells: Significance of a dual mode of action

doi: 10.1371/journal.pone.0174546

Figure Lengend Snippet: (A) Real time polymerase chain reaction were performed to quantify BAX mRNA levels in Daudi and PBMC with and without Mito-CP (1μM) treatment under hypoxia (5%O 2 ) and normoxia. Amplified BAX mRNA was analysed by melting curve analysis and fold change in expression in each experimental group were calculated by 2 -ΔΔCT . Data were obtained from three separate experiments and are expressed as mean ± SEM. * and ** denotes significantly different when compared to control p<0.05 and p<0.01 respectively. ( B ) Daudi cells and PBMCs were treated with and without Mito-CP (1μM) under hypoxia (5%O 2 ) and normoxia. HIF-1α levels were measured by western blot and densitometric analysis are shown as bar graphs. Data were obtained from three separate experiments and were expressed as by mean ± SEM. * denotes significantly different when compared to control p<0.05.

Article Snippet: Daudi cells (human B lymphoblast cells) were purchased from the National Centre for Cell Science (Maharashtra, India) and maintained in Gibco RPMI-1640 (Life Technologies, Grand Island, New York, USA) medium with 100 U penicillin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India), 100 μg streptomycin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India) and 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, New York, USA), Dec-TPP + (Sigma Aldrich, St.louis, Missouri.)

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

Journal: PLoS ONE

Article Title: Effect of mitochondrially targeted carboxy proxyl nitroxide on Akt-mediated survival in Daudi cells: Significance of a dual mode of action

doi: 10.1371/journal.pone.0174546

Figure Lengend Snippet: (A) Daudi cells and PBMC were treated with and without Mito-CP (1μM) under hypoxia (5%O 2 ) and normoxia. AKT inhibitor wortmanin (1μM) was also used to show inhibition of p-AKT. Protein lysate concentration was determined by Bradford method. P-AKT, XIAP, cytochrome c, cleaved PARP were measured by western blot. β-actin was used to normalise of protein expression. ( B ) Shows densitometry analysis of p-AKT, XIAP, cytochrome c, cleaved PARP. Data were obtained from three separate experiments and were expressed as by mean ± SEM.

Article Snippet: Daudi cells (human B lymphoblast cells) were purchased from the National Centre for Cell Science (Maharashtra, India) and maintained in Gibco RPMI-1640 (Life Technologies, Grand Island, New York, USA) medium with 100 U penicillin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India), 100 μg streptomycin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India) and 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, New York, USA), Dec-TPP + (Sigma Aldrich, St.louis, Missouri.)

Techniques: Inhibition, Concentration Assay, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Effect of mitochondrially targeted carboxy proxyl nitroxide on Akt-mediated survival in Daudi cells: Significance of a dual mode of action

doi: 10.1371/journal.pone.0174546

Figure Lengend Snippet: ( A ) Daudi cells and PBMC were treated with and without Mito-CP (1μM) and Dec-TPP + (1μM) under hypoxia (5%O 2 ) and normoxia. P-AKT, AKT, P-AMPK, AMPK were measured by western blot. Β-actin was used to normalise of protein expression. ( B ) Densitometry analysis of P-AKT, AKT, P-AMPK, and AMPK were performed and the beta actin normalized P-AKT/Total-AKT and P-AMPK/Total-AMPK values were represented as bar graph. Data were obtained from three separate experiments and were expressed as by mean ± SEM. * and **, significantly different when compared to control p<0.05 and p<0.01 respectively.

Article Snippet: Daudi cells (human B lymphoblast cells) were purchased from the National Centre for Cell Science (Maharashtra, India) and maintained in Gibco RPMI-1640 (Life Technologies, Grand Island, New York, USA) medium with 100 U penicillin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India), 100 μg streptomycin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India) and 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, New York, USA), Dec-TPP + (Sigma Aldrich, St.louis, Missouri.)

Techniques: Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Effect of mitochondrially targeted carboxy proxyl nitroxide on Akt-mediated survival in Daudi cells: Significance of a dual mode of action

doi: 10.1371/journal.pone.0174546

Figure Lengend Snippet: Schematic representation of mechanism of anticancer activity of Mito-CP in Daudi cells.

Article Snippet: Daudi cells (human B lymphoblast cells) were purchased from the National Centre for Cell Science (Maharashtra, India) and maintained in Gibco RPMI-1640 (Life Technologies, Grand Island, New York, USA) medium with 100 U penicillin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India), 100 μg streptomycin/ml (HiMedia Laboratories, LBS Marg, Mumbai, India) and 10% fetal bovine serum (FBS) (Life Technologies, Grand Island, New York, USA), Dec-TPP + (Sigma Aldrich, St.louis, Missouri.)

Techniques: Activity Assay

Journal: eLife

Article Title: Caspase-mediated cleavage of IRE1 controls apoptotic cell commitment during endoplasmic reticulum stress

doi: 10.7554/eLife.47084

Figure Lengend Snippet:

Article Snippet: Cell line (human) , Daudi , Genentech , RRID: CVCL_0008 , Burkitt’s lymphoma.

Techniques: Knock-Out, Isolation, Clone Assay, CRISPR, Transfection, Construct, FLAG-tag, shRNA, Recombinant, Caspase-Glo Assay, Cell Viability Assay, Flow Cytometry, Fractionation, Cell Culture, Purification, Software