Journal: Blood

Article Title: Tcl1 interacts with Atm and enhances NF-κB activation in hematologic malignancies

doi: 10.1182/blood-2011-08-374561

Figure Lengend Snippet: Tcl1 and Atm are crucial for growth and proliferation of lymphoma cells in vitro and in vivo. (A) Daudi cells were mock-transfected or transfected with si-TCL1 or si-Scr and then untreated or treated with Kudos 55933 or DMSO. Cells were counted at 24-hour intervals. Results represent the average of 3 independent experiments. (B) Total lysates collected were analyzed by Western blot and tested with indicated antibodies. (C) Graph representing tumor volumes at indicated days during the experiment for the 5 groups indicated (4 mice/group). Tumor size was measured daily until the tumor reached 50 mm3. Then, 5 μg of synthetic si-TCL1or si-Scr diluted in Lipofectamine and with or without Kudos (50 μL total volume) were injected directly into the tumors and at 3, 7, and 10 days. Tumors were measured on the day of the injections and 4 days after the last injection. (D) Total lysates from tumors were analyzed by Western blot and tested with indicated antibodies. (E) Xenograft tumors were weighed. Data are mean ± SEM of 3 independent experiments, and each is measured in triplicate. *P < .05.

Article Snippet: Tumors were established by injecting Daudi lymphoma cells (200 μL PBS, 7.5 × 10 7 cells/mouse) into the right flanks of female nude mice at 6 weeks of age (Charles River Breeding Laboratories).

Techniques: In Vitro, In Vivo, Transfection, Western Blot, Injection

Journal: Yonsei Medical Journal

Article Title: Methionyl-tRNA Synthetase is a Useful Diagnostic Marker for Lymph Node Metastasis in Non-Small Cell Lung Cancer

doi: 10.3349/ymj.2019.60.11.1005

Figure Lengend Snippet: Immunoblotting of methionyl-tRNA synthetase (MRS)-positive H460 cells, CD45/CD3-positive Molt-4 cells, and CD45/CD20-positive Daudi cells (A). Fluorescent immunochemical staining for MRS and CD45 in the reference samples containing a fixed ratio of H460, Daudi, and Molt-4 cells (B and C) and endobronchial ultrasound-guided transbronchial needle aspirate-derived samples (D and E). Images were taken using a fluorescent microscope (B and D) and confocal microscope (C and E). Blue represents nucleus (4′, 6-Diamidine-2′-phenylindole dihydrochloride: DAPI); green, MRS; and red, CD45. B and D confocal images show merged images only. TTF-1, thyroid transcription factor 1.

Article Snippet: Molt-4, Daudi, and H460 cells were obtained from the Korean Cell Line Bank (Seoul, Korea).

Techniques: Western Blot, Staining, Derivative Assay, Microscopy

Journal: Scientific Reports

Article Title: Antibody-targeted paclitaxel loaded nanoparticles for the treatment of CD20 + B-cell lymphoma

doi: 10.1038/srep45682

Figure Lengend Snippet: ( a ) A schematic representation of AR160. ( b ) Rituximab was tagged with AlexaFluor 488 and co-incubated with ABX for visualization. ImageStream reveals fluorescently labeled nanoparticles of approximately 0.1 uM. ( c ) ABX was tagged with AlexaFluor 488 and co-incubated with rituximab and Daudi cells were stained with either PE anti-human CD19, fluorescent AR160, or both. Cells were analyzed by Guava flow cytometery. Daudi cells were about 75% positive for CD19, AR160 or both. ( d ) The labeled Daudi cells were also run by ImageStream and an image of a doubly stained Daudi cell is shown. ( e ) AR160 was separated into 3 fractions: the particulate, and proteins greater than and less than 100 kD. Paclitaxel concentration in each fraction was determined by HPLC and showed about 69.2% of paclitaxel is in the particulate and the remaining paclitaxel is among proteins greater the 100 kD. Paclitaxel was measured in AR160 fractions after 24 ( f ), 48 hours ( g ), and 60 minutes in AB serum ( h ). Data shows a shift of the majority of paclitaxel from the particulate to the proteins >100 kD. ( i ) Western blot was performed on the greater than 100 kD fraction and rituximab, paclitaxel and albumin co-localized in a band of approximately 200 kD. Biacore screening of an albumin peptide library for binding to riruximab reveals 3 binding peptides ( j ) HSA peptide 4 binds to rituximab with a Kd of 5.7 × 10 −8 . ( k ) HSA peptide 13 binds to rituximab with a Kd of 4.0 × 10 −7 . ( l ) HSA peptide 40 binds to rituximab with a Kd of 5.2 × 10 −10 . ( m ) ABX was incubated for 30 minutes with 4 mg/ml rituximab and either no peptide (AR160), 10x molar excess, relative to antibody, of control peptide (ABX+Rit+control) or HSA peptide 40 (ABX+Rit+HSA peptide 40). Particle sizes were determined by nanoparticle tracking analysis utilizing the NS300. HSA peptide 40 was shown to block the formation of AR160 suggesting the peptide blocks rituximab from binding ABX. Results are representative of 3 experiments.

Article Snippet: Five million Daudi cells were implanted into the right flank of athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN).

Techniques: Incubation, Labeling, Staining, Concentration Assay, Western Blot, Binding Assay, Blocking Assay

Journal: Scientific Reports

Article Title: Antibody-targeted paclitaxel loaded nanoparticles for the treatment of CD20 + B-cell lymphoma

doi: 10.1038/srep45682

Figure Lengend Snippet: ( a ) ABX and AR160 was prepared and incubated for 24 hours in saline at room temperature and size and particle number was measured at 0, 1, 2, 4, 6, and 24 hours by NS300 nanoparticle tracking system. AR160 remained stable over 24 hours as particle size and number remained unchanged during that time. The particle size distributions and particle size and numbers are shown. ( b , c ) ABX and AR160 were prepared, and 30 × 108 particles were added to human AB serum and incubated for 60 minutes in human AB serum. Particle size and numbers were determined at 5, 15, 30, and 60 minutes after being added to the AB serum. Particle size distributions for ABX ( b ) and AR160 ( c ) are shown. ( d ) Graphical representation of the particle number of ABX relative to AR160 after incubation in AB serum. ( e ) To verify toxicity of AR160, we treated CD20+ Daudi cells with AR160, ABX and rituximab. Cells were treated overnight with the drugs at concentrations from 0–200 ug/ml with the addition of EdU, a thymidine analog. The level of proliferation was determined by staining cells with FITC labeled anti-EdU. The proliferation index was calculated by normalization to the untreated positive control. ( f ) Rituximab ligand binding was confirmed by flow cytometry in which the Daudi cells were pretreated with rituximab, ABX, and AR160 and then stained with PE anti-human CD20. The drug-pretreated samples were compared to isotype control and PE anti-human CD20 alone serving as negative and positive controls, respectively. Rituximab and AR160, but not ABX alone blocked subsequent PE anti-human CD20 binding. Data is representative of 3 experiments.

Article Snippet: Five million Daudi cells were implanted into the right flank of athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN).

Techniques: Incubation, Staining, Labeling, Positive Control, Ligand Binding Assay, Flow Cytometry, Binding 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: 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