Journal: The Journal of Biological Chemistry

Article Title: T Cell Receptor-induced Nuclear Factor κB (NF-κB) Signaling and Transcriptional Activation Are Regulated by STIM1- and Orai1-mediated Calcium Entry *

doi: 10.1074/jbc.M115.713008

Figure Lengend Snippet: Orai1-mediated Ca2+ entry is required for TCR- but not TNF-induced NF-κB activation. A, Jurkat T cells were incubated with (bottom panel) and without (top panel) Synta66 (50 μm) and then activated with thapsigargin (1 μm) under Ca2+-free (0 mm) and Ca2+-replete (2 mm) conditions. B, Jurkat T cells were either untreated (−) or incubated for 15 min (+) with Synta66 and then stimulated with anti-CD3/28 or TNF for the times indicated. Time-dependent changes in IκBα were determined by immunoblotting, and anti-α-tubulin was used as a loading control. C, Ca2+ traces in Jurkat T cells stably overexpressing the dominant negative E106A Orai1 that were stimulated with P/I, 3/28, or TNF as shown. D, Jurkat T cells stably overexpressing either wild-type Orai (Orai-cyan fluorescent protein, Orai-CFP) or the dominant negative Orai1-E106A were stimulated with 3/28, P/I, or TNF for the times indicated, and immunoblot analysis was performed to quantify IκBα and α-tubulin.

Article Snippet: In some experiments, cells were pretreated for 15 min with the Orai-1 inhibitor Synta66 (50 μ m , Aobious, Gloucester, MA) prior to stimulation.

Techniques: Activation Assay, Incubation, Western Blot, Stable Transfection, Dominant Negative Mutation

Journal: Cancers

Article Title: Blockage of Store-Operated Ca 2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca 2+ Selective Orai1 Pore

doi: 10.3390/cancers12102876

Figure Lengend Snippet: The inhibitory action of Synta66 is affected by Orai1 pore geometry: ( A ) Time course of normalized whole-cell inward rectifying currents at −86 mV, maximally activated upon passive store depletion of HEK293 cells co-expressing STIM1 and Orai1 (black) or Orai1 E106D (red), upon perfusion of 10 μM Synta66 and subsequent block by 10 μM La 3+ (n = 6 − 10 cells, from at least two individual transfections). ( B , C ) Corresponding I/V relationships of STIM1 and Orai1 (black, B ) or Orai1 E106D (red, C ) after maximal store-operated activation and upon addition of 10 μM Synta66 (blue). ( D , E ) Top view ( D ) and side-view ( E ) of the Orai channel showing docking configuration of Synta66 with the most negative glide score. In ( E ), Synta66 is shown as spheres, the selectivity filter is highlighted as red sticks. ( F ) Synta66 in docking position 1. Black lines resemble the protein backbone. Two H-bonds are formed between Synta66 and amino acids of Orai at Asp110 and His113.

Article Snippet: Synta66 (Merck Millipore, Burlington, MA, USA) was stored as a 50 mM stock solution in DMSO at -80°C until dilution to a maximum DMSO concentration of 0.1% in cell culture experiments.

Techniques: Expressing, Blocking Assay, Transfection, Activation Assay

Journal: Cancers

Article Title: Blockage of Store-Operated Ca 2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca 2+ Selective Orai1 Pore

doi: 10.3390/cancers12102876

Figure Lengend Snippet: Interactions between free ligands with human Orai1: ( A , B ) Starting configuration ( A , t = 0 ns) and ( B ) MD simulations after 300 ns with three molecules of Synta66 present at the entry of the pore. The Orai channel protein is represented as a grey glassy ribbon with the selectivity filter highlighted in red. Individual molecules of Synta66 are represented in orange, violet and green. ( C ) Projection of the distance along the z-axis of the distances between the centre-of-mass of each molecule of Synta66 (colour code as in A , B ) and the selectivity filter as a function of the simulation time. ( D–E ) Molecular Dynamic (MD) simulations of a single docked Synta66 compound with start position of pose 1 (stick model) and after 200 ns of simulations (sphere model). ( e ) Projection of distances of a single Synta66 to the selectivity filter as in ( C ). ( F ) Interactions (in %) for a single Synta66 with Orai1 residues.

Article Snippet: Synta66 (Merck Millipore, Burlington, MA, USA) was stored as a 50 mM stock solution in DMSO at -80°C until dilution to a maximum DMSO concentration of 0.1% in cell culture experiments.

Techniques:

Journal: Cancers

Article Title: Blockage of Store-Operated Ca 2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca 2+ Selective Orai1 Pore

doi: 10.3390/cancers12102876

Figure Lengend Snippet: Synta66 inhibition is impaired in Orai1 mutants that yield non-selective permeation: ( A ) Snapshot of Orai1 channel (top view) with a single Synta66 bound to loop1 and loop3 residues of Orai1. Residues that are mutated in ( B–E ) are highlighted as red sticks. ( B , D , H ) Time course of store-operated whole-cell currents at −86 mV for a co-expression of STIM1 and ( B ) Orai1 loop1 mutant (L109D, H113G, Y115G), ( D ) Orai1 loop3 mutant (F199G, P201G, L202G) or ( H ) Orai H134A mutant. Upon maximum Orai1 activation, 10 µM Synta66 was added to the extracellular solution followed by 10 µM La 3+ . ( C , E , I ) Current voltage relationships from representative experiments from ( B , D , H ) upon maximal store-operated Ca 2+ activation and of steady state currents upon perfusion with a 10 µM Synta66 containing solution. ( F ) Side view of the wild-type Orai1 channel (left) and Orai1-H134A mutant (right) with residues from ( A ) and H134/A134 highlighted as stick model. ( G ) Structure of the Orai1-H134A simulations (top view) with residues from ( A ) highlighted. ( J , L ) Time course of store-operated whole-cell currents at −86 mV by STIM1 and Orai1 in a standard Ca 2+ based extracellular solution. After reaching a maximum current plateau, extracellular solution was substituted to a Na + based divalent ion free solution (Na + DVF). Currents were subsequently monitored in a ( J ) Na + DVF with 10 µM Synta66 or ( L ) Ca 2+ based extracellular solution with 10 µM Synta66. ( K ) Current voltage relationships from representative experiments from (I) in a Na-DVF solution (black) or with 10 µM Synta66 (blue).

Article Snippet: Synta66 (Merck Millipore, Burlington, MA, USA) was stored as a 50 mM stock solution in DMSO at -80°C until dilution to a maximum DMSO concentration of 0.1% in cell culture experiments.

Techniques: Inhibition, Expressing, Mutagenesis, Activation Assay

Journal: Cancers

Article Title: Blockage of Store-Operated Ca 2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca 2+ Selective Orai1 Pore

doi: 10.3390/cancers12102876

Figure Lengend Snippet: STIM1 interaction is not affected by Synta66 : ( A ) Localization, overlay and calculated fluorescence resonance energy transfer (FRET) life cell image series of cyan fluorescent protein (CFP)-STIM1 and yellow fluorescent protein (YFP)-STIM1 in the absence (upper panel) and presence of Thapsigargin (TG, 1 µM) mediated endoplasmic reticulum (ER) Ca 2+ store-depletion (lower panel). ( B ) Time-course of calculated FRET between CFP- and YFP-STIM1 with and without pre-treatment with 10 µM Sytna66. ER Ca 2+ store-depletion was induced by 1 µM TG. ( C ) Analogous experiments as in ( A ) upon incubation with 10 µM Synta66 for 20 min.

Article Snippet: Synta66 (Merck Millipore, Burlington, MA, USA) was stored as a 50 mM stock solution in DMSO at -80°C until dilution to a maximum DMSO concentration of 0.1% in cell culture experiments.

Techniques: Fluorescence, Förster Resonance Energy Transfer, Incubation

Journal: Cancers

Article Title: Blockage of Store-Operated Ca 2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca 2+ Selective Orai1 Pore

doi: 10.3390/cancers12102876

Figure Lengend Snippet: STIM1-Orai1 interaction is not affected by Synta66: ( A ) Localization, overlay and calculated FRET life cell image series of STIM1-CFP and Orai1-YFP in the absence (upper panel) and presence of 1 µM TG mediated ER Ca 2+ store-depletion (lower panel). ( B ) Time-course of calculated FRET between STIM1-CFP and Orai1-YFP with and without treatment with 10 µM Sytna66. ER Ca 2+ store-depletion was induced by 1 µM TG. ( C ) Analogous experiments as in ( A ) upon pre-treatment with 10 µM Synta66 for 20 min.

Article Snippet: Synta66 (Merck Millipore, Burlington, MA, USA) was stored as a 50 mM stock solution in DMSO at -80°C until dilution to a maximum DMSO concentration of 0.1% in cell culture experiments.

Techniques:

Journal: Cancers

Article Title: Blockage of Store-Operated Ca 2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca 2+ Selective Orai1 Pore

doi: 10.3390/cancers12102876

Figure Lengend Snippet: Inhibition of SOCE in GBM cell lines by Synta66: ( A ) Western blot for detection of endogenously expressed Orai1 in lysates of A172, LN-18 and U-87 MG cells. Lysates were deglycosylated in order to achieve a single Orai1 band as in . ( B ) Western Blot for detection of endogenously expressed STIM1 in lysates of A172, LN-18 and U-87 MG cells. ( C ) Normalized maximal values of SOC activation from Fura-2 AM loaded GBM cells (A172, LN-18 and U-87) and comparison to SOC treated with 1 and 10 µM Synta66 (light and dark blue, respectively). Results shown as mean ± SEM from two independent experiments (n = 109–192 cells). A172 and LN-18 had no detectable SOCE peak, U-87 MG had 7.0± 0.6 % SOCE peak. ( D–F ) Time course experiments show mean values of cytosolic Ca 2+ measurements in Fura-2 AM loaded GBM cell lines. Cells are monitored initially in a Ca 2+ free extracellular solution followed by application of 30 µM BHQ and addition of 2 mM Ca 2+ to monitor SOCE (black). Analogous experiments with pre-treatment of 10 µM Synta66 immediately before the start of the experiment were used (blue).

Article Snippet: Synta66 (Merck Millipore, Burlington, MA, USA) was stored as a 50 mM stock solution in DMSO at -80°C until dilution to a maximum DMSO concentration of 0.1% in cell culture experiments.

Techniques: Inhibition, Western Blot, Activation Assay

Journal: Cancers

Article Title: Blockage of Store-Operated Ca 2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca 2+ Selective Orai1 Pore

doi: 10.3390/cancers12102876

Figure Lengend Snippet: Effect of Synta66 on cell growth: ( A – C ) Effect of Synta66 (1 and 10 µM, light and dark blue respectively) on proliferation of GBM cell lines ( A ) LN-18, ( B ) A172 and ( C ) U-87 MG was examined via Hoechst staining of nuclei. Signal was normalized to control signal at t = 0 h, results shown as mean ± SEM, n = 6 from two independent experiments; *: p -value < 0.05 (two-sided t -test).

Article Snippet: Synta66 (Merck Millipore, Burlington, MA, USA) was stored as a 50 mM stock solution in DMSO at -80°C until dilution to a maximum DMSO concentration of 0.1% in cell culture experiments.

Techniques: Staining