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

Journal: Molecular cell

Article Title: The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca 2+ Channels and Mast Cell Migration through PAR4 Receptors.

doi: 10.1016/j.molcel.2018.03.025

Figure Lengend Snippet: Figure 4. Ca2+ Influx to Mite Extract Requires STIM1 Gating of Orai1 (A) Western blots compare STIM1 and Orai1 protein expression before and after knockdown (KD). (B) Aggregate data from two independent experiments are shown. (C) Mite-induced Ca2+ influx is reduced following knockdown of either STIM1 or Orai1. (D) Aggregate data from several experiments as in (C) are compared. HDM (400 mg/mL) is the mean of 159 cells, KD STIM1 126 cells, and KD Orai1 81 cells. (E) TIRF images of STIM1-YFP puncta are compared between resting HEK293 cells and cells stimulated with mite extract. Images for the stimulated condition were taken 9 min after mite exposure in Ca2+-free solution. (F) Confocal images compare STIM1-YFP distribution in HEK293 cells at rest, after stimulation with thapsigargin (8 min), and after exposure to mite extract (15 min). (G) The rate of Ca2+ entry, normalized to control (mock-transfected cells), is compared for the conditions shown. HDM bar denotes mean of 44 cells, STIM1 38 cells, and STIM1+Orai1 30 cells. (H) Aggregate data are compared. Orai1 was knocked down and then the various constructs indicated were expressed (together with STIM1) 24 hr later. GFP bar denotes mean of 66 cells (which were transfected only with GFP plasmid and served as control), - 52 cells, Orai1 33 cells, L273S-Orai1 37 cells, and 81AARAE85- Orai1 15 cells. For the graphs, all data are presented as mean ± SEM.

Article Snippet: 16HBE Dr. Ling-Pei Ho Oxford University Jurkat T cells Dr. Daniel Bakowski Calcico Therapeutics BMMC isolated from C57BL/6 mice Department of Biomedical Services Oxford University Oligonucleotides siRNA for STIM1 Life Technologies 4390815 siRNA for Orai1 OriGene SR508429 (Continued on next page) e1 Molecular Cell 70, 228–241.e1–e5, April 19, 2018

Techniques: Western Blot, Expressing, Knockdown, Control, Transfection, Construct, Plasmid Preparation

Journal: Molecular cell

Article Title: The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca 2+ Channels and Mast Cell Migration through PAR4 Receptors.

doi: 10.1016/j.molcel.2018.03.025

Figure Lengend Snippet: Figure 5. Der p3 Regulates CRAC Channels (A) Boiling mite extract suppresses the second slow phase of Ca2+ release and store-operated Ca2+ influx. (B) Aggregate data are compared. HDM (400 mg/mL) trace is mean of 21 cells and boiled HDM 41 cells. (C) Serine protease inhibitors reduce mite-extract-evoked Ca2+ entry. Mite extract was pre-mixed with each inhibitor for 20 min before application to cells. HDM group denotes mean of 43 cells, nafamostat (10 mM) 57 cells, camostat (500 mM) 24 cells, aprotinin (10 mM) 95 cells, and E-64 (10 mM) 69 cells. (D) The effect of mite extract fractions on Ca2+ entry is compared. The background trace denotes Ca2+ influx in cells exposed to Ca2+-free solution for the same time. The background is the same in all three traces because each fractionation experiment was run on the same day. (E) Coomassie blue gel of the fractions shown in (D). (F) Heatmap summarizes which Der p proteins were present in each fraction, detected by mass spectrometry analysis. (G) Western blot shows cleavage of pro-Der p3 by a low dose of mite extract and that this is prevented by pre-treatment with E-64. (H) Bacteria-purified pro-Der p3 (6 mg) and a low dose of house dust mite extract (30–60 mg/mL) each fail to elicit Ca2+ influx, but their combination (pre-mixed at 37C for 10–20 min) evokes prominent Ca2+ entry. Pro-Der p3 trace is mean of 12 cells, low HDM 20 cells, and pro-Der p3 + low HDM 11 cells. (I) Ca2+ entry is inhibited when pro-Der p3 is mutated at the pro-cleavage site such that cleavage of the pro-form is impaired. Each bar is the mean of >15 cells. The cleavage sequences of original Der p3 and uncut Der p3 are shown at the top of the panel. Mutated amino acids are shown in red. The signal sequence has been removed after methionine for simplicity. (J) Ca2+ influx to the combination of pro-Der p3 and a low dose of mite extract is suppressed by either pre-treatment of mite extract with E-64 (labeled E-64+ Low HDM; pro-Der p3) or knockdown of Orai1. Addition of E-64 after pro-der p3 had been incubated with mite extract for 10 min had no inhibitory effect (labeled Low HDM + pro-Der p3; E-64). Each trace is the mean of between 8 and 23 cells. All data are from RBL-2H3 cells except (H), which was from HEK293 cells. For the graphs, all data are presented as mean ± SEM.

Article Snippet: 16HBE Dr. Ling-Pei Ho Oxford University Jurkat T cells Dr. Daniel Bakowski Calcico Therapeutics BMMC isolated from C57BL/6 mice Department of Biomedical Services Oxford University Oligonucleotides siRNA for STIM1 Life Technologies 4390815 siRNA for Orai1 OriGene SR508429 (Continued on next page) e1 Molecular Cell 70, 228–241.e1–e5, April 19, 2018

Techniques: Fractionation, Mass Spectrometry, Western Blot, Bacteria, Sequencing, Labeling, Knockdown, Incubation

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: Matrix biology : journal of the International Society for Matrix Biology

Article Title: A CNA-35-based high-throughput fibrosis assay reveals ORAI1 as a regulator of collagen release from pancreatic stellate cells.

doi: 10.1016/j.matbio.2024.12.004

Figure Lengend Snippet: Fig. 6. Orai1 is expressed in the fibrotic PDAC tissue. (A) Representative immunohistochemistry images of PDAC tissue and neighboring non-tumorous tissue (N = 4) stained for the CAF marker αSMA (cyan), the Ca2+

Article Snippet: Antigen retrieval was performed using 10 mM sodium citrate buffer (pH 6.0), followed by blocking with 1 % BSA-containing PBS (Sigma-Aldrich) for 1 h Slides were stained with primary anti-ORAI1 antibody (75,522, RRID: AB_11,007,920, 1:100, Novus Biologicals) in a humidified chamber at 4 ◦C overnight.

Techniques: Immunohistochemistry, Staining, Marker