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: Nature Structural & Molecular Biology

Article Title: Structural basis of the specificity of USP18 toward ISG15

doi: 10.1038/nsmb.3371

Figure Lengend Snippet: ( a ) Immunoblot of lysates from HEK 293T cells transfected with S-tagged versions of mouse USP18 (mUSP18), mouse USP18 with the active site cysteine replaced by alanine (mUSP18 C61A), human USP21 (hUSP21), zebrafish USP18 ( dr USP18) and zebrafish USP18 with the active site cysteine replaced by an alanine ( dr USP18 C38A) or from untransfected cells (control). Protein expression was visualized with an antibody directed against the S tag. ( b ) Immunoblot of lysates from cells transfected with the indicated expression constructs, incubated with the active site–directed probe ISG15-PA. Complex formation was monitored on the basis of a size shift detected with an anti-S-tag antibody. ( c ) Immunoblot analyses of protein lysates from cells transfected with the indicated expression constructs, incubated with the active site–directed probe Ub-PA. Complex formation was monitored on the basis of a size shift detected with an anti-S-tag antibody. USP21 is cross-reactive for ISG15, and ubiquitin served as a positive control for Ub binding. Results shown in a – c are representative of three independent experiments.

Article Snippet: The vector pTriEx2-USP21cd encoding residues 197–565 of human USP21 was generated by amplification of USP21 cDNA from the vector Flag-HA-USP21 (Addgene) with the primers KpnI-USP21cd-for and XhoI-USP21cd-rev.

Techniques: Western Blot, Transfection, Expressing, Construct, Incubation, Positive Control, Binding Assay

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Neutrophils express membrane Orai1. Isolated mouse and human PMNs, or 2-day differentiated HL-60 were assessed for RNA, DNA, or protein expression. (A) Protein expression levels were measured from the average mean fluorescence intensity (MFI) of paired flow cytometry samples of HL-60 cells transfected with negative control or Orai1-specific siRNA (n = 10) and PMNs (n = 4). Background fluorescence resulting from nonspecific antibody binding was measured by analyzing cells treated with a blocking decoy peptide against which the Orai1 antibody was originally raised. (B) Relative expression levels of Orai1 mRNA compared with RPL mRNA were measured for siRNA-transfected, control siRNA-transfected, or mock-transfected HL-60 cells by quantitative PCR. Data expressed as Orail mRNA copy number relative to RPL after PCR amplification. (C) Relative genomic DNA expression of Orai1 in WT versus Orai1−/+ mice measured by electrophoresis of amplified DNA from mouse tissues. Bars represent average densitometry measurements from 18 Orai1−/+ and 6 littermate WT mice ± SE. (D) Orai1 protein expression measured from the average MFI of paired cytometric measurements in WT and Orai1+/− mouse PMNs (n = 3). *P < .05; ***P < .001.

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Membrane, Isolation, Expressing, Fluorescence, Flow Cytometry, Transfection, Negative Control, Binding Assay, Blocking Assay, Control, Real-time Polymerase Chain Reaction, Amplification, Electrophoresis, FACS, Knockdown

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Calcium dynamics via Orai1 supports GPCR-mediated PMN recruitment. PMNs isolated from murine Orai1+/+ (WT), or Orai1−/+ littermates, 2-day differentiated HL-60 cells, or freshly isolated human PMNs were loaded with fura-2 AM, perfused at a shear stress of 2 dyne/cm2 into a microfluidic flow chamber consisting of an L-cell monolayer substrate expressing E-selectin, and then 0.1μM fMLP was perfused just after time 0 and ratio-imaged by fluorescence microscopy. (A) Representative images of a human PMN decelerating, arresting, and undergoing shape polarization show fura-2 emission, with the intensity of red representing 340 nm excitation (calcium bound) and green representing 380nM excitation (calcium unbound). The average calcium concentration is shown in 1-second increments, and the percentage of cells arrested is shown in 10-second increments. (B) Percentage of arrested and polarized in WT and Orai1−/+ PMNs is based on analysis of more than 150 cells from 4 paired experiments. WT cells had significantly higher arrest and polarization than Orai1−/+ at time points beyond 30 seconds. **P < .01. (C) HL-60 were transfected with negative control, or Orai1-specific siRNA as indicated, were differentiated for 2 days with 1.3% DMSO and were imaged in the flow channels responding to 0.1μM fMLP as in panel A (n = 10 separate experiments). Negative control transfectants were treated with 2-APB or U73122 to completely block SOCE or store release. For Ca2+ quantified at 60 seconds. *P < .05. **P < .01. (D) Human PMNs were observed in absence of inhibition (n = 6), in the presence of 100μM 2-APB (n = 4), or in the presence of 1μM U73122 (n = 4) as indicated. Cells treated with 2-APB had significantly lower arrest fraction at 30 seconds (P = .003) and significantly lower polarization between 50 seconds and 100 seconds (P values range from .007 to .045 compared with the controls). *P < .05; #P < .05 (1-tailed t test, P = .059 by 2-tailed t test).

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Isolation, Shear, Expressing, Fluorescence, Microscopy, Concentration Assay, Analysis, Transfection, Negative Control, Blocking Assay, Inhibition, Knockdown

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Calcium influx through SOCE and Orai1 facilitates neutrophil arrest. (A) PMNs isolated from murine Orai1−/+, WT littermates (ie, Orai1+/+), 2-day differentiated HL-60 cells, or freshly isolated human PMNs were loaded with fura-2, incubated with 1 μM thapsigargin as indicated, and then perfused over a monolayer of L cells expressing E-selectin and ICAM-1 in the presence of 0.1mM ethyleneglycoltetraacetic acid. At time 0, 1.5mM CaCl2 was injected into the inlet reservoir triggering selectin-dependent rolling and calcium influx through store-operated calcium channels. (A) Panels indicate intracellular calcium concentration and arrested fraction from WT or Orai1−/+ PMNs as indicated. (B) HL-60 cells were transfected with scrambled control or specific Orai1 siRNA as indicated (both n = 10). Orai1 siRNA-transfected cells had significantly higher rolling fractions at 150 seconds, 160 seconds, and 170 seconds (P = .03, .01, and .03, respectively) than control transfected cells. (C) Human PMNs were analyzed in the presence of vehicle control (n = 6), 100μM 2-APB (n = 4), or 1μM U73122 (n = 4) as a function of time. Cells treated with 2-APB had significantly lower arrest fraction than control at 60 seconds (P = .01). All statistical comparisons in this figure are by 2-tailed t test: *P < .05; **P < .01.

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Isolation, Incubation, Expressing, Injection, Concentration Assay, Transfection, Control

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Intracellular calcium rises in synchrony with PMN arrest and polarization. (A) Data for human PMNs responding to fMLP stimulation are compiled from more than 200 individual observations onto 1 plot to compare the dynamics of calcium, arrest, and polarization. Arrest is represented here as the fraction of cells remaining rolling (ie, the inverse of arrest). (B) Data for human PMNs treated with thapsigargin and stimulated by the addition of 1.5mM CaCl2 are also compiled onto 1 plot to compare the dynamics of calcium arrest and polarization. Arrest is represented as in panel A. (C) Human and mouse PMNs were loaded with fluo-5f, perfused over a glass coverslip coated with recombinant E-selectin-Fc and ICAM-1-Fc, and imaged by TIRF microscopy. Representative images from 3 separate experiments are indicated. (D) Calcium (fluo-5F) intensity over 60 seconds, untreated PMNs had significantly higher relative fluo-5f signal than 2-APB treated cells at 15 seconds and higher time points (P < .01). (E) Calcium (fluo-5F) signal in mouse WT versus Orai1−/+ PMNs over 60 seconds. **P < .01. ***P < .001. Error bars represent the SE for each measured time point.

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Glass, Recombinant, Microscopy

Journal: Blood

Article Title: Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

doi: 10.1182/blood-2009-05-224659

Figure Lengend Snippet: Conceptual model of calcium regulation and integrin engagement during PMN recruitment in shear flow. (A) During rolling, release of calcium stores initiated by GPCR and PLC-β signals IP3-sensitive calciosomes that cooperate with Orai1 SOCE eliciting an upshift in β2-integrin activation via Rap-1 that is sensitive to the rapid and local release of calcium stores resulting in PMN arrest. (B) As the PMN transitions to arrest, integrin engagement under stress stabilizes a high affinity conformation, which triggers activation of PLC-γ. The additional calcium release caused by integrin engagement sustains SOCE-mediated calcium influx and reinforces signaling for strengthening of arrest. (C) Calcium signaling causes rearrangement of the actin cytoskeleton and activation of secondary messengers (PKC) necessary for shape polarization. Electrical depolarization of the membrane and refilling of calcium stores through SERCA pumps slowly diminish intracellular calcium.

Article Snippet: Polyclonal rabbit anti–human Orai1 was purchased from Alomone Labs. Polyclonal anti–mouse Orai1 was purchased from Abcam.

Techniques: Shear, Activation Assay, Membrane

Journal: Journal of Immunology Research

Article Title: Genetic Manipulation of Calcium Release-Activated Calcium Channel 1 Modulates the Multipotency of Human Cartilage-Derived Mesenchymal Stem Cells

doi: 10.1155/2019/7510214

Figure Lengend Snippet: Modulation of Ca 2+ in CRAC-manipulated MSCs. The following experiments were conducted at 7 days after gene transfection of wild-type MSCs, pcDNA3.1-Orai1-transfected MSCs (M1-MSCs), and CRACM1-specific gRNA vector and linear EF1a-GFP-P2A-Puro donor-cotransfected MSCs (KOM1-MSCs). (a) PCR amplification of reverse transcription products produced the expected band following genetic modification. Molecular marker (lane 1); CARCM1 expression (523 bp) in MSCs, M1-MSCs, and KOM1-MSCs (lanes 3, 4, and 5, respectively); and GAPDH expression (214 bp) in MSCs, M1-MSCs, and KOM1-MSCs (lanes 7, 8, and 9, respectively) are shown. (b) CRACM1 mRNA expression in MSCs, M1-MSCs, and KOM1-MSCs (a.u. (arbitrary units); ∗ P < 0.05 and ∗∗∗ P < 0.001). Results are expressed as mean ± SEM ( n = 4). (c) The relative expression of CRACM1 to housekeeping GAPDH in MSCs, M1-MSCs, and KOM1-MSCs using quantitative real-time PCR. Relative fold of CRACM1 expression was achieved using the comparative Ct method (2 -ΔΔCt ) ( ∗∗ P < 0.01 and ∗∗∗ P < 0.001). (d) Time sequential patterns of Ca 2+ imaging in single MSCs, M1-MSCs, and KOM1-MSCs. The imaging period was 200 s without stimulation, followed by 500 s after stimulation. After a 200 s baseline measurement, cells were slowly perfused with TG (0.5 μ M) and then perfused with a CaCl 2 solution (2 mM) at the 400 s time point (scale bar: 10 μ m). (e) Typical Ca 2+ influx patterns of MSCs, M1-MSCs, and KOM1-MSCs shown in (c). (f) Maximum increases in fluorescent intensity values of MSCs, M1-MSCs, and KOM1-MSCs. Quantification was performed using images acquired from 100–120 cells of each group ( ∗ P < 0.05). Results are expressed as mean ± SEM. (g) Initial rate of Ca 2+ influx (in the first 15 s after Ca 2+ addition) into MSCs, M1-MSCs, and KOM1-MSCs. Quantification was performed using images acquired from 100–120 cells of each group ( ∗ P < 0.05 and ∗∗ P < 0.01). Results are expressed as mean ± SEM.

Article Snippet: PcDNA3.1-Orai1 carrying CRACM1 was a gift from Anjana Rao (Addgene plasmid # 21638) [ ], which was transfected into MSCs using Lipofectamine® 3000 Reagent (Thermo Fisher Scientific K.K., Tokyo, Japan), according to the manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation, Amplification, Reverse Transcription, Produced, Modification, Marker, Expressing, Real-time Polymerase Chain Reaction, Imaging

Journal: Journal of Immunology Research

Article Title: Genetic Manipulation of Calcium Release-Activated Calcium Channel 1 Modulates the Multipotency of Human Cartilage-Derived Mesenchymal Stem Cells

doi: 10.1155/2019/7510214

Figure Lengend Snippet: Overexpression of CRACM1 inhibits MSC differentiation to adipocytes. MSCs, M1-MSCs, and KOM1-MSCs were cultured in adipogenic differentiation medium for 7 days. Cells were then stained for mFABP (red) as a marker of adipocytes and counterstained with Hoechst® 33342 for nuclear staining (blue). (a) Typical images of MSCs, M1-MSCs, and KOM1-MSCs observed by fluorescence microscopy (×200; scale bar: 50 μ m). (b) Typical imaging screening panel for quantification of mFABP4 expression. MSCs, M1-MSCs, and KOM1-MSCs were seeded on 8-well plates, and 32 fields were captured in each well using a high-throughput image quantitation system. One of 32 fields is shown. Well number and average intensity are indicated on the image. (c) FABP expression in MSCs, M1-MSCs, and KOM1-MSCs. The average fluorescent intensity was obtained from 256 images for each group ( ∗ P < 0.05). Results are expressed as mean ± SEM. (d) Typical images of lipid droplets analysis. Lipid droplets, which were stained using Oil Red O, present as bright refractive round structures. (×400; scale bar: 25 μ m). (e) Relative positive area of lipid droplet analysis. The red-stained area was segmented from the background, and the relative positive area was quantified. More than four fields per section and an average of five sections from each sample were used for semiquantitative analysis ( ∗∗∗ P < 0.001). Results are expressed as mean ± SEM.

Article Snippet: PcDNA3.1-Orai1 carrying CRACM1 was a gift from Anjana Rao (Addgene plasmid # 21638) [ ], which was transfected into MSCs using Lipofectamine® 3000 Reagent (Thermo Fisher Scientific K.K., Tokyo, Japan), according to the manufacturer's instructions.

Techniques: Over Expression, Cell Culture, Staining, Marker, Fluorescence, Microscopy, Imaging, Expressing, High Throughput Screening Assay, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Mitofusin 2 Regulates STIM1 Migration from the Ca 2+ Store to the Plasma Membrane in Cells with Depolarized Mitochondria *

doi: 10.1074/jbc.M110.174029

Figure Lengend Snippet: Mitochondrial depolarization inhibits ICRAC following overexpression of STIM1 and Orai1. A, in fura 2-loaded RBL-1 cells co-expressing eYFP-STIM1 and Orai1, readmission of external Ca2+ to cells treated with thapsigargin (Thap) (2 μm) results in Ca2+ influx, and this is inhibited by depolarizing mitochondria with antimycin A (Anti. A) (5 μg/ml) and oligomycin (oligo) (0.5 μg/ml), applied to cells 10 min before thapsigargin. B, rate of Ca2+ entry (measured from experiments as in A) is compared (each bar represents >60 cells). C, large ICRAC in cells co-expressing eYFP-STIM1 and Orai1 is substantially reduced by mitochondrial depolarization, and aggregate data are plotted in D. Pipette solution contained InsP3 + 10 mm EGTA. Number of cells is 11 for STIM1 + Orai1 alone and 9 in the presence of antimycin A and oligomycin. E, images from confocal microscopy showing the distribution of eYFP-STIM1 in cells before (labeled Control) and then after exposure to 2 μm thapsigargin in Ca2+-free solution for the various conditions shown. F, distribution of eYFP-STIM1 and mitochondria (detected with MitoTracker red) are compared. Co-localization is indicated in yellow.

Article Snippet: HEK293 cells were co-transfected with cDNA encoding Orai1 (OriGene) and eYFP-STIM1 (gift from Dr. T. Meyer) using two independent methods, the Lipofectamine and Amaxa systems, as described previously ( 39 ). eYFP-mutant STIM1 was a gift from Dr. J. Putney.

Techniques: Over Expression, Expressing, Transferring, Confocal Microscopy, Labeling, Control

Journal: The Journal of Biological Chemistry

Article Title: Mitofusin 2 Regulates STIM1 Migration from the Ca 2+ Store to the Plasma Membrane in Cells with Depolarized Mitochondria *

doi: 10.1074/jbc.M110.174029

Figure Lengend Snippet: STIM1 migration does not depend on mitochondrial Ca2+ buffering or ATP production. A, loading cells with BAPTA impaired the cytoplasmic Ca2+ rise evoked by thapsigargin. Cells were loaded with either fura 2-AM and 0.1% DMSO (control) or fura 2-AM and BAPTA-AM (10 μm) prior to stimulation with thapsigargin. B, confocal images from a nontreated cell showing eYFP-STIM1 distribution at rest and then following stimulation with thapsigargin in the absence and then presence of antimycin A (Anti) and oligomycin (oligo). Images represent different cells. C, loading cells with BAPTA does not impair migration of eYFP-STIM1 to the cell periphery, but mitochondrial depolarization still reduces translocation in BAPTA-loaded cells. D, time course of Na+ current through CRAC channels in whole-cell patch clamp recording from cells overexpressing Orai1 and eYFP-STIM1. Filled circles denote a control cell and open circles denote a cell after exposure to antimycin A and oligomycin. E, corresponding current-voltage relationships from D are shown, taken when the currents had peaked. F, aggregate data from six control cells and five antimycin A/oligomycin-treated cells are compared. Amplitude was measured at −80 mV. G, mitochondrial depolarization (labeled +Anti./oligo) does not affect intracellular Mg-ATP levels (measured through Mg2+ concentration) provided glycolysis is intact. 2-DOX denotes 10 mm 2-deoxyglucose. H, time course of decay of the Ca2+ signal to thapsigargin is unaffected by mitochondrial depolarization. This time course represents Ca2+ removal by the ATP-dependent plasma membrane Ca2+ pump. I, aggregate data from several experiments as in H are summarized (control denotes 76 cells and Anti./oligo 85 cells).

Article Snippet: HEK293 cells were co-transfected with cDNA encoding Orai1 (OriGene) and eYFP-STIM1 (gift from Dr. T. Meyer) using two independent methods, the Lipofectamine and Amaxa systems, as described previously ( 39 ). eYFP-mutant STIM1 was a gift from Dr. J. Putney.

Techniques: Migration, Control, Translocation Assay, Patch Clamp, Labeling, Concentration Assay, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Mitofusin 2 Regulates STIM1 Migration from the Ca 2+ Store to the Plasma Membrane in Cells with Depolarized Mitochondria *

doi: 10.1074/jbc.M110.174029

Figure Lengend Snippet: Store-operated Ca2+ entry can be partially rescued by overexpressing STIM1 in HEK293 cells with depolarized mitochondria. A–C, HEK293 cells were stimulated with thapsigargin in Ca2+-free solution (at t = 0) and then 2 mm external Ca2+ was readmitted as shown. Only the Ca2+ entry component is shown for simplicity. The extent of Ca2+ release was similar for the different conditions. A, store-operated entry in wild type cells is inhibited by antimycin A and oligomycin. B, overexpression of eYFP-STIM1 results in modest recovery of store-operated Ca2+ entry in cells with depolarized mitochondria. C, overexpression of eYFP-STIM1 and Orai1 results in substantial rescue of store-operated Ca2+ entry in the presence of antimycin A and oligomycin. For each graph, the response in antimycin A and oligomycin has been normalized to the corresponding control response. Control response has been re-calculated as ((R − R0)/Rpeak)·100%, where R is the measured ratio (356/380) at any time point following Ca2+ readmission; Rpeak is the peak ratio measured in the cell (maximal response); and R0 is the basal ratio (resting Ca2+). D, Ca2+ influx following overexpression of Orai1 and STIM1 is compared before and after mitochondrial depolarization in cells loaded with fura 5F. E–H, TIRF images comparing puncta formation in a resting HEK cell shown in (E), and then after store depletion with thapsigargin (Thap) for 4 min (F) and after store depletion following exposure to antimycin A (Anti) and oligomycin (oligo) for 15 min (G). H, histograms plot the absolute fluorescence intensity measured in 1-μm2 segments against the frequency of occurrence. Upper panel represents cells exposed to thapsigargin, and the lower panel analyses cells pre-exposed to antimycin A and oligomycin prior to thapsigargin challenge.

Article Snippet: HEK293 cells were co-transfected with cDNA encoding Orai1 (OriGene) and eYFP-STIM1 (gift from Dr. T. Meyer) using two independent methods, the Lipofectamine and Amaxa systems, as described previously ( 39 ). eYFP-mutant STIM1 was a gift from Dr. J. Putney.

Techniques: Over Expression, Control, Fluorescence

Journal: eLife

Article Title: S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse

doi: 10.7554/elife.72051

Figure Lengend Snippet: Figure 1. ORAI1 is S-acylated at cysteine C143. (A) ORAI1 immunoblot of HeLa cells treated with PEG-5k to label S-acylation sites after exposure to NEM to block free thiols and then to hydroxylamine (HA) to break acyl-thioester bonds. (B) Western blot and corresponding autoradiogram of HeLa cells labelled for 2 hr with 3H-palmitic acid with or without HA and immunoprecipitated with anti-ORAI1. (C, D) Western blots and corresponding autoradiograms of HeLa (C) and RPE-1 (D) cells expressing the indicated YFP-tagged ORAI1 mutants labelled with 3H-palmitic acid and

Article Snippet: For protein detection either on western blot or immunofluorescence, we used; NFATc1 (clone 7A6, MABS409, Sigma), TCR alpha/beta- PE (12- 9986- 42, eBioscience), AntiCholera Toxin, B- Subunit (227040, Sigma), Myc- Tag (9B11) (2276, Cell- Signalling), gamma Tubulin (4D11) (MA1- 850, Thermofisher), ANTI- FLAG M2 (F1804, Sigma), anti- ORAI1 (600–401- DG9, rockland immunochemicals Inc), anti- GFP (SAB4301138, Sigma), anti- mouse- HRP, and rabbit- HRP (1706516 and 172101, Bio- Rad (USA).

Techniques: Western Blot, Blocking Assay, Immunoprecipitation, Expressing

Journal: eLife

Article Title: S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse

doi: 10.7554/elife.72051

Figure Lengend Snippet: Figure 3. Preventing ORAI1 S-acylation reduces channel clustering and affinity for lipid rafts. (A) Cartoon of the YFP-HA-ORAI WT or C143A constructs with HA exposed extracellularly between TM3 and TM4. HEK-293 cells transiently expressing the indicated constructs were stained for HA in non- permeabilising conditions to decorate the ORAI1 PM fraction. Graph bars represent the mean ± SEM of 15 (WT), 17 (C143A) and 10 (WT+ NP-40) cells. Right, Representative confocal images of YFP (green) and HA (red) staining in the indicated conditions. Scale bar = 25 µm. (B) Representative

Article Snippet: For protein detection either on western blot or immunofluorescence, we used; NFATc1 (clone 7A6, MABS409, Sigma), TCR alpha/beta- PE (12- 9986- 42, eBioscience), AntiCholera Toxin, B- Subunit (227040, Sigma), Myc- Tag (9B11) (2276, Cell- Signalling), gamma Tubulin (4D11) (MA1- 850, Thermofisher), ANTI- FLAG M2 (F1804, Sigma), anti- ORAI1 (600–401- DG9, rockland immunochemicals Inc), anti- GFP (SAB4301138, Sigma), anti- mouse- HRP, and rabbit- HRP (1706516 and 172101, Bio- Rad (USA).

Techniques: Construct, Expressing, Staining

Journal: eLife

Article Title: S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse

doi: 10.7554/elife.72051

Figure Lengend Snippet: Figure 4. PAT20 S-acylates ORAI1 and modulates its activity. (A) Western blot and matching autoradiogram of RPE-1 cells expressing ORAI1-YFP plus the indicated PAT isoform, labelled with 3H-palmitic acid and immunoprecipitated with anti-GFP. Graph bar (right) shows densitometry analysis of the tritiated bands relative to GFP in RPE1 (Blue, N = 2) and HeLa cells (yellow, N = 1). (B) Functional effect of PAT3, 7, and 20 expression. Representative western blot of HeLa cells expressing Myc-tagged PAT isoforms (left), averaged SOCE responses (middle), and peak amplitude (right). Data are mean

Article Snippet: For protein detection either on western blot or immunofluorescence, we used; NFATc1 (clone 7A6, MABS409, Sigma), TCR alpha/beta- PE (12- 9986- 42, eBioscience), AntiCholera Toxin, B- Subunit (227040, Sigma), Myc- Tag (9B11) (2276, Cell- Signalling), gamma Tubulin (4D11) (MA1- 850, Thermofisher), ANTI- FLAG M2 (F1804, Sigma), anti- ORAI1 (600–401- DG9, rockland immunochemicals Inc), anti- GFP (SAB4301138, Sigma), anti- mouse- HRP, and rabbit- HRP (1706516 and 172101, Bio- Rad (USA).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Functional Assay

Journal: eLife

Article Title: S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse

doi: 10.7554/elife.72051

Figure Lengend Snippet: Figure 5. ORAI1 S-acylation promotes Jurkat T cell activation. (A) Averaged fura-2 responses, their peak amplitude (middle graph bar) and slope (right graph bar) evoked by Ca2+ re-addition in Tg treated (1 µM, 8 min) Jurkat cells lines generated by CRISPR with control or ORAI1-targeted guiding sequences and stably re-expressing either WT or C143A ORAI1-YFP. Data are mean ± SEM of 210 (Control), 242 (KO), 189 (WT), and 203 (C143A) cells from three independent experiments (B) Western blot showing the amount of biotinylated GFP immunoreactivity in the PM in Jurkat CRISPR ORAI1

Article Snippet: For protein detection either on western blot or immunofluorescence, we used; NFATc1 (clone 7A6, MABS409, Sigma), TCR alpha/beta- PE (12- 9986- 42, eBioscience), AntiCholera Toxin, B- Subunit (227040, Sigma), Myc- Tag (9B11) (2276, Cell- Signalling), gamma Tubulin (4D11) (MA1- 850, Thermofisher), ANTI- FLAG M2 (F1804, Sigma), anti- ORAI1 (600–401- DG9, rockland immunochemicals Inc), anti- GFP (SAB4301138, Sigma), anti- mouse- HRP, and rabbit- HRP (1706516 and 172101, Bio- Rad (USA).

Techniques: Activation Assay, Generated, CRISPR, Control, Stable Transfection, Expressing, Western Blot

Journal: eLife

Article Title: S-acylation by ZDHHC20 targets ORAI1 channels to lipid rafts for efficient Ca2+ signaling by Jurkat T cell receptors at the immune synapse

doi: 10.7554/elife.72051

Figure Lengend Snippet: Figure 6. ORAI1 S-acylation regulates TCR enrichment and signaling at the immune synapse. (A) Conditions used to study synapse formation between Raji and Jurkat T cells. Raji are pulsed with SEE prior to co-culture with Jurkat cells, IS formation evaluated in living (25 min) and fixed cells (1 H) and Jurkat activation at 24 hr. (B) Relative changes in NFAT-Luciferase vs. housekeeping- Renilla luminescence evoked by coculture of the indicated cell lines for 24 hr with naïve or SEE (1 µg/ml) pulsed Raji. Data are mean ± SEM of 15–20 biological replicates in cells from four independent experiments.

Article Snippet: For protein detection either on western blot or immunofluorescence, we used; NFATc1 (clone 7A6, MABS409, Sigma), TCR alpha/beta- PE (12- 9986- 42, eBioscience), AntiCholera Toxin, B- Subunit (227040, Sigma), Myc- Tag (9B11) (2276, Cell- Signalling), gamma Tubulin (4D11) (MA1- 850, Thermofisher), ANTI- FLAG M2 (F1804, Sigma), anti- ORAI1 (600–401- DG9, rockland immunochemicals Inc), anti- GFP (SAB4301138, Sigma), anti- mouse- HRP, and rabbit- HRP (1706516 and 172101, Bio- Rad (USA).

Techniques: Co-Culture Assay, Activation Assay, Luciferase