Journal: Cancers

Article Title: Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

doi: 10.3390/cancers11030291

Figure Lengend Snippet: Effect of GA101 on intracellular Ca 2+ concentration in SU-DHL-4 ( A ) and BL2 ( B ) cell lines. Ca 2+ responses to GA101 (10 µg/mL) were measured using Fluo2-Leak Resistant-Acetoxy Methyl ester (Fluo2-LR-AM) Ca 2+ dye and recorded by videomicroscopy (Zeiss LSM 510) using a 25× objective. Black arrows indicate GA101 addition. Each trace represents the response of one cell and data are representative of at least three independent experiments. Data were processed using OriginPro 7.5 (Origin Lab) or GraphPad prism. Cells were recorded in extracellular Hank’s Balanced Salt Solution (HBSS) containing 2 mM Ca 2+ (2Ca) or in Ca 2+ -free HBSS (0Ca). Cells were preincubated with 100 nM thapsigargin (TG) for 45 min and recorded in Ca 2+ -free HBSS (0Ca + TG) or with 10 µM Ned-19 for 1 h and recorded in Ca 2+ -free HBSS (0Ca + Ned19). Calcium responses to GA101 in cells expressing Non Targeting shRNA (sh NT) or sh Orai1 were recorded in HBSS containing 2 mM Ca 2+ . Histograms represent areas under curves (AUC) calculated, under various recording conditions, between the application time of GA101 and t = 2000 s; * p < 0.05.

Article Snippet: Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Concentration Assay, Expressing, shRNA

Journal: Cancers

Article Title: Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

doi: 10.3390/cancers11030291

Figure Lengend Snippet: Involvement of store-operated Ca 2+ entry (SOCE) in GA101-induced cell death. ( A ) BL2 cells. ( B ) SU-DHL-4 cells. Left panels: Cells were incubated with GA101 in the presence or absence of BTP2 (10 µM) for 24 h. Right panels: Cells expressing sh NT or sh Orai1 were treated with GA101 for 24 h. Cell death was assessed by measuring the loss of mitochondrial membrane potential (Δψm), using tetramethylrhodamine methyl ester (TMRM) as a fluorescent dye, or by caspase 3 activation, measured by the FAM-FLICA in vitro caspase detection kit and both analyzed by flow cytometry; * p < 0.05.

Article Snippet: Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Incubation, Expressing, Activation Assay, In Vitro, Flow Cytometry

Journal: Cancers

Article Title: Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

doi: 10.3390/cancers11030291

Figure Lengend Snippet: Differential activation of endoplasmic reticulum (ER) stress in SU-DHL-4 and BL2 cell lines. ( A ) Cells expressing sh NT or sh Orai1 were treated with GA101 (1 µg/mL) for varying lengths of time. After lysis, phosphorylated eukaryotic translation initiation factor alpha (P-eIF2α), eIF2α, and BIM expression levels were assessed by immunoblot analysis. GAPDH was used as a loading control. ( B ) Quantification of Western blots is given as means ± SE of three to five independent experiments; * p < 0.05.

Article Snippet: Anti-human Orai1 rabbit polyclonal antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Activation Assay, Expressing, Lysis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: Cytosolic and mitochondrial calcium concentration of TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM cisplatin. ( A ) Immunostaining of transient receptor potential ankyrin 1 (TRPA1)- or transient receptor potential vanilloid 1 (TRPV1)-positive DRG neurons and live imaging of Fluo-4 (green) and Rhod-2 (red). ( B ) The cytosolic calcium of TRPV1-positive DRG neurons concentration instantly increased to 1.067 ± 0.018 (* p < 0.05) after 5 min and continuously increased to 1.394 ± 0.068 (** p < 0.01) after 70 min. The mitochondrial calcium concentration declined to 1.095 ± 0.009 (* p < 0.05) after 5 min for TRPV1-positive DRG neurons and steadily declined to 0.577 ± 0.044 after 70 min (** p < 0.01). ( C ) During the exposure of 10 µM cisplatin, the cytosolic calcium concentration of TRPA1-positive neurons increased after 5 min to 1.024 ± 0.011 (** p < 0.01) until 70 min at 1.240 ± 0.074 (*** p < 0.001). The mitochondrial calcium concentration was lower after 10 min at 0.928 ± 0.029 (*** p < 0.001) until it reached 0.790 ± 0.050 at 70 min (*** p < 0.001). ( D ) The cytosolic calcium concentration of TRPV1-positive DRG neurons was increased after 15 min to 65 min compared to TRPA1-positive DRG neurons (* p < 0.05, ** p < 0.01). ( E ) The mitochondrial calcium concentration of TRPA1- or TRPV1-positive DRG neurons was different after 5 min until the end of the experiment (* p < 0.05, ** p < 0.01). TRPV1-positive sensory neurons had a lower mitochondrial calcium concentration during exposure to 10 µM cisplatin after 15 min (* p < 0.05, ** p < 0.01). n = 6 cells per condition. * = significant effect between TRPA1+ and TRPV1+ DRG neurons. Scale = 50 µm.

Article Snippet: Anti-TRPV1 (extracellular) , 1:200, rabbit , Alomone Labs, Israel.

Techniques: Concentration Assay, Immunostaining, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: Cytosolic and mitochondrial calcium concentration of TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM oxaliplatin. ( A ) Immunostaining of TRPA1- or TRPV1-positive DRG neurons and live imaging of Fluo-4 (green) and Rhod-2 (red) before and after application of 10 µM cisplatin or oxaliplatin. ( B ) TRPA1-positive DRG neurons showed an immediate increase of the cytosolic calcium concentration to 1.037 ± 0.006 (** p < 0.01) after 5 min and continuously increased to 1.372 ± 0.062 after 70 min (*** p < 0.001). The mitochondrial calcium concentration decreased after 5 min to 0.967 ± 0.008 (*** p < 0.001) and declined to 0.712 ± 0.010 after 70 min (*** p < 0.001). ( C ) The TRPV1-positive DRG neurons showed an immediate increase of the cytosolic calcium concentration to 1.049 ± 0.005 (*** p < 0.001) after 5 min and increased to 1.480 ± 0.034 after 70 min (*** p < 0.001). The mitochondrial calcium concentration decreased to 0.959 ± 0.020 (* p < 0.05) after 5 min and to 0.725 ± 0.077 (** p < 0.01) after 70 min (* p < 0.05). ( D ) No difference could be determined in the cytosolic calcium concentration of TRPA1- or TRPV1-positive DRG neurons ( p > 0.05). ( E ) No difference could be determined in the mitochondrial calcium concentration of TRPA1- or TRPV1-positive DRG neurons ( p > 0.05). n = 6 cells per condition. * = significant effect between TRPA1+ and TRPV1+ DRG neurons. Scale = 50 µm.

Article Snippet: Anti-TRPV1 (extracellular) , 1:200, rabbit , Alomone Labs, Israel.

Techniques: Concentration Assay, Immunostaining, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: Relative cytosolic and mitochondrial calcium concentration during exposure to 10 µM cis- or oxaliplatin of TRPA1- or TRPV1-positive DRG neurons. ( A ) No differences in the cytosolic calcium concentration of TRPA1-positive DRG neurons after exposure to 10 µM cis- or oxaliplatin could be determined ( p > 0.05). ( B ) The cytosolic calcium of TRPV1-positive DRG neurons concentration during exposure to cis- or oxaliplatin showed no difference ( p > 0.05). ( C ) During exposure to 10 µM cis- or oxaliplatin, no difference in the mitochondrial calcium concentration of TRPA1-positive DRG neurons could be determined ( p > 0.05). ( D ) While exposing TRPV1-positive DRG neurons, differences between the two chemotherapeutics on the mitochondrial calcium concentration could be determined after 20 min. The mitochondrial calcium concentration of TRPV1-positive DRG neurons exposed to oxaliplatin was higher at 0.856 ± 0.033 compared to 0.727 ± 0.040 (* p < 0.05). The mitochondrial calcium concentration was higher for oxaliplatin-exposed TRPV1-positive DRG neurons until 0.786 ± 0.058 after 40 min (* p < 0.05). n = 6 cells per condition. * = significant effect between cis- and oxaliplatin, ** p < 0.01

Article Snippet: Anti-TRPV1 (extracellular) , 1:200, rabbit , Alomone Labs, Israel.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: ROS production of TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM cisplatin. ( A ) Fluorescence of CellRox (green) in TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM cisplatin at different time points. ( B ) TRPA1-positive sensory neurons showed an instant increase of the ROS production to 1.431 ± 0.042 (*** p < 0.001) after 5 min and reached its peak at 2.100 ± 0.197 after 30 min (*** p < 0.001). After 50 min, the ROS production dropped below the control level at 50 min to 0.607 ± 0.036 (*** p < 0.001) and further declined to 0.370 ± 0.035 (*** p < 0.001) after 70 min. ( C ) After exposure to 10 µM cisplatin of the TRPV1-positive DRG neurons, the ROS production increased to 1.238 ± 0.042 (*** p < 0.001) after 5 min and dropped below control level at 50 min at 0.873 ± 0.068 (* p < 0.05) and steadily declined to 0.756 ± 0.072 (** p < 0.01) after 70 min. n = 6 cells per condition. * = significant effect compared to control level. Scale = 50 µm.

Article Snippet: Anti-TRPV1 (extracellular) , 1:200, rabbit , Alomone Labs, Israel.

Techniques: Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: ROS production of TRPA1- or TRPV1-positive DRG neurons during exposure to 10 µM oxaliplatin. ( A ) Fluorescence of CellRox (green) of TRPA1- or TRPV1-positive DRG neurons at different time points. ( B ) The ROS production of TRPA1-positive DRG neurons increased after 5 min to 1.421 ± 0.103 (** p < 0.01) during exposure to 10 µM oxaliplatin and dropped to control level after 40 min at 1.31 ± 0.194 ( p > 0.05). ( C ) The TRPV1-positive sensory neurons had an increased ROS production after 5 min at 1.080 ± 0.020 (** p < 0.01) and dropped to the control level after 10 min at 1.033 ± 0.036 ( p > 0.05). After 30 min, the ROS production declined below the control level at 0.810 ± 0.079 (* p < 0.05) and steadily declined to 0.429 ± 0.050 after 70 min (*** p < 0.001). n = 6 cells per condition. * = significant effect compared to control level. Scale = 20 µm.

Article Snippet: Anti-TRPV1 (extracellular) , 1:200, rabbit , Alomone Labs, Israel.

Techniques: Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: The ROS production of TRPA1- or TRPV1-positive DRG neurons during exposure to cis- or oxaliplatin. ( A ) A difference could be determined for TRPA1-positive DRG neurons at 30 min with a higher ROS production for cisplatin-exposed DRG neurons at 2.100 ± 0.197 (* p < 0.05). After 45 min (1.194 ± 0.223; * p < 0.05) until 70 min (1.022 ± 0.233; * p < 0.05), the ROS production was higher for oxaliplatin-exposed neurons. ( B ) The ROS production of TRPV1-positive DRG neurons was higher for cisplatin-exposed neurons over the entire experimental period (* p < 0.05, ** p < 0.01). n = 6 cells per condition. * = significant effect compared to control level.

Article Snippet: Anti-TRPV1 (extracellular) , 1:200, rabbit , Alomone Labs, Israel.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Platinum-Based Drugs Cause Mitochondrial Dysfunction in Cultured Dorsal Root Ganglion Neurons

doi: 10.3390/ijms21228636

Figure Lengend Snippet: Antibodies and dilution used for the TRPA1- and TRPV1-positive staining.

Article Snippet: Anti-TRPV1 (extracellular) , 1:200, rabbit , Alomone Labs, Israel.

Techniques: Staining

Journal: Cell reports

Article Title: NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca 2+ entry in T cells

doi: 10.1016/j.celrep.2021.109603

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human ORAI1 (flow cytometry) , Alomone Labs , Cat# ACC-060; ACC-060-F.

Techniques: FLAG-tag, Western Blot, Flow Cytometry, Recombinant, Staining, Plasmid Preparation, Software, Imaging, Microscopy