Journal: Cancers

Article Title: Calcium Independent Effect of Orai1 and STIM1 in Non-Hodgkin B Cell Lymphoma Dissemination

doi: 10.3390/cancers10110402

Figure Lengend Snippet: Stromal Derived Factor 1 (SDF-1) provokes an intracellular Ca 2+ response in the HLY-1 diffuse large B cell lymphoma (DLBCL) cell line involving intracellular Ca 2+ pool mobilization and Orai1/STIM1 extracellular Ca 2+ influx. Ca 2+ responses to SDF-1 (100 ng/mL) were measured using Fluo2-LR-AM Ca 2+ dye and recorded by videomicroscopy (Zeiss LSM 510) using ×25 objective. Black arrows indicate SDF-1 addition. Each trace represents the response of one cell and data are representative of at least three independent experiments. Typical response of unique cell (peak or peak follow by sustained plateau phase) are present as example ( Ab ). Data were processed using GraphPad prism. ( A ) Pharmacological characterization of SDF-1-induced Ca 2+ increase. Cells were recorded in extracellular saline solution (HBSS) containing 2 mM Ca 2+ (2 Ca, ( Aa )) or in Ca 2+ -free HBSS (0 Ca, ( Ac )). Cells were pre-incubated with BTP2 ( Ae ) or GSK7975A ( Af ) at 10 µM for 30 min and recorded in 2 mM Ca 2+ HBSS containing inhibitors. ( Ad , Ag ) Histograms represent areas under curves (AUC) calculated, under various recording conditions, between the application time of SDF-1 and t = 2050 s, and normalized compared to control (2 Ca or shNT). Data are expressed as mean ± SEM, * p < 0.05. ( B ) Effect of Orai1 or STIM1 expression knock-down on SDF-1-induced Ca 2+ response. The stable modified HLY-1 cell line established after lentiviral transduction with plasmid containing non targeting shRNA (shNT), shRNA against Orai1 or STIM1 were recorded in extracellular saline solution (HBSS) containing 2 mM Ca 2+ .

Article Snippet: BTP2 was from Interchim (Montluçon, France).

Techniques: Derivative Assay, Incubation, Expressing, Modification, Transduction, Plasmid Preparation, shRNA

Journal: Cancers

Article Title: Calcium Independent Effect of Orai1 and STIM1 in Non-Hodgkin B Cell Lymphoma Dissemination

doi: 10.3390/cancers10110402

Figure Lengend Snippet: Orai1 and STIM1 regulate basal and SDF-1-induced DLBCL cell migration in a Ca 2+ independent manner in vitro. Cell migration was assessed in 96-transwell chemotaxis chambers assay. Histograms represent mean ± SEM from at least 3 independent experiments, * p < 0.05. ( A ) Ca 2+ is not necessary for DLBCL cell migration. To test the effect of the pharmacological agents on chemotaxis induced by SDF-1 (100 ng/mL), cells were pre-treated during 20 min in the presence or not of the agents before to be loaded to upper transwell chambers and pharmacological agents were maintained in medium during the experiment. BAPTA-AM, intracellular Ca 2+ chelator, 5 µM; EGTA, extracellular Ca 2+ chelator, 1 mM; BTP2 and GSK7975A, CRAC inhibitors, 10 µM. ( B ) Orai1 and STIM1 are required for DLBCL migration. Basal and SDF-1 (100 ng/mL)-induced migration were measured (as described above) in stable modified HLY-1 and SU-DHL-4 cells established after lentiviral transduction with plasmid containing non targeting shRNA (shNT), shRNA against Orai1 or STIM1.

Article Snippet: BTP2 was from Interchim (Montluçon, France).

Techniques: Migration, In Vitro, Chemotaxis Assay, Modification, Transduction, Plasmid Preparation, shRNA

Journal: Cancers

Article Title: Calcium Independent Effect of Orai1 and STIM1 in Non-Hodgkin B Cell Lymphoma Dissemination

doi: 10.3390/cancers10110402

Figure Lengend Snippet: STIM1, but not Ca 2+ , regulate DLBCL dissemination in vivo. ( A ) Effect of STIM1 under-expression on HLY-1 cell dissemination. ( B ) Effect of intraperitoneal injection of BTP2 (12 µg/kg) or vehicle, three times per week on HLY-1 cell dissemination. Images were captured with a Nikon Eclipse Ci microscope equipped with a Plan Fluor 10× 0.3 NA objective. Scale bars = 150 μm. Histograms represent the quantification of positive surface for HLA-ABC staining. All tissues were delimited and to evaluate the percentage of positive surface for HLA-ABC staining on tissue, thresholding on positive and negative staining was done using Mercator software. Data are represented as mean ± SEM (n = 10), * p < 0.05.

Article Snippet: BTP2 was from Interchim (Montluçon, France).

Techniques: In Vivo, Expressing, Injection, Microscopy, Staining, Negative Staining, Software

Journal: International Journal of Molecular Sciences

Article Title: Internalized Amyloid-β (1-42) Peptide Inhibits the Store-Operated Calcium Entry in HT-22 Cells

doi: 10.3390/ijms232012678

Figure Lengend Snippet: Effect of the SOCE inhibitor BTP2 in untreated (control) and treated HT-22 cells with Aβ(1-42). Untreated HT-22 cells (control) and treated cells with 2 μM Aβ(1-42) for 2 h were incubated with 3 μM of BTP2 for 15 min at 37 °C. Panel ( A ): representative fluorescence microscopy images of untreated HT-22 (control), cells with 3 μM BTP2, and cells treated with Aβ(1-42) plus 3 μM BTP2. (a,f,k) Bright field images (BF) of the fields selected for fluorescence images of Fluo3-loaded cells obtained before addition of 2 μM Tg (b,g,l), at the peak fluorescence after the addition of Tg (c,h,m), after completion of the Ca 2+ release from the ER (d,i,n), and at the peak fluorescence after the addition of 3 mM Ca 2+ (e,j,o). Scale bar = 20 μm. Panel ( B ): representative kinetic traces of HT-22 control with (blue trace line) or without BTP2 (black trace line) and HT-22 cells incubated with 2 μM Aβ(1-42) for 2 h plus BTP2 (red trace line) after addition of 2 μM Tg (indicated by the blue arrow) for Ca 2+ release from stores, and addition of 3 mM Ca 2+ to evaluate the extension of Ca 2+ entry in HT-22 cells (indicated by the green arrow). Panel ( C ): means of the average fluorescent intensity (ΔF/F 0 ) represented by percentage (%), relative to control cells. BTP2 does not elicit ER Ca 2+ depletion but inhibits the Ca 2+ influx in 71 ± 8% in control cells and HT-22 cells incubated with 2 μM Aβ(1-42) for 2 h, compared with the respective controls. Data are presented as the mean ± SEM of experiments done at least in 10 Petri plates in 5 independent assays (n > 80 cells for each condition) (* p < 0.05, relatively to each control). n.s.—non-significant.

Article Snippet: Thapsigargin (Tg) was obtained from Sigma-Aldrich (Madrid, Spain) and BTP2 was from Merck Roche–Merck (Darmstadt, Germany).

Techniques: Incubation, Fluorescence, Microscopy

Journal: Molecular Medicine

Article Title: Lansoprazole-induced osteoporosis via the IP3R- and SOCE-mediated calcium signaling pathways

doi: 10.1186/s10020-022-00448-x

Figure Lengend Snippet: Relative calcium fluorescence change curves. A Relative [Ca 2+ ] i response in MC3T3-E1 cells that were preincubated with 2-APB in Ca 2+ -free medium. B Relative [Ca 2+ ] i response in MC3T3-E1 cells that were preincubated with ryanodine in Ca 2+ -free medium. C Relative [Ca 2+ ] i response in MC3T3-E1 cells that were preincubated with verapamil in Ca 2+ -containing medium. D Relative [Ca 2+ ] i response in MC3T3-E1 cells that were preincubated with BTP-2 in Ca 2+ -containing medium (n = 3)

Article Snippet: Cells were then promptly treated with LPZ, 2-APB (ApexBio, B6643), ryanodine (ApexBio, B5092), thapsigargin (TG) (Sigma, T9033), verapamil (ApexBio, B1687), and BTP-2 (ApexBio, B7542).

Techniques: Fluorescence

Journal: Molecular Medicine

Article Title: Lansoprazole-induced osteoporosis via the IP3R- and SOCE-mediated calcium signaling pathways

doi: 10.1186/s10020-022-00448-x

Figure Lengend Snippet: [Ca 2+ ] i after longterm treatment with LPZ. A [Ca 2+ ] i levels in MC3T3-E1 cells that were preincubated with BTP-2 in calcium-containing medium, as detected by flow cytometry. B [Ca 2+ ] i levels in MC3T3-E1 cells that were preincubated with TG in calcium-free medium, detected by flow cytometry. C Quantitative analysis of [Ca 2+ ] i levels. (n = 3)

Article Snippet: Cells were then promptly treated with LPZ, 2-APB (ApexBio, B6643), ryanodine (ApexBio, B5092), thapsigargin (TG) (Sigma, T9033), verapamil (ApexBio, B1687), and BTP-2 (ApexBio, B7542).

Techniques: Flow Cytometry