Journal: Nature Communications

Article Title: UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

doi: 10.1038/s41467-017-01601-5

Figure Lengend Snippet: Reduced Unc93b1 3 d -STIM1 association compromises STIM1 function. a Fibroblasts expressing STIM1-WT-GFP-tagged and UNC93B1 WT or UNC93B1 3d -FLAG-tagged, plasmids were lysed and STIM1 was immunoprecipitated with GFP beads and immunoblotted with anti-GFP and anti-FLAG antibodies. b In WT and 3d/3d DCs transfected with STIM1-WT-GFP-plasmid, STIM1–UNC93B1 interaction was detected using Duolink proximity ligation assay with anti-GFP and anti-UNC93B1-specific antibodies. Cells are seen in brightfield (top panel). PLA signals are shown in red and nuclei in blue (bottom panel) and quantified with Image J ( n = 11 cells; * P < 0.05). Bars = 10 μm. c Representative figures and quantification of PLA signals (dots/μm 2 ) after 60 min of phagocytosis of 3 μm beads by WT and 3d/3d DCs transfected with STIM1-WT-GFP cDNA ( n = 12 cells; *** P < 0.001). Bars = 10 μm. d Averaged Ca 2+ signals in WT and 3d/3d DCs loaded with fluo4-AM as described in Methods section. Cells were stimulated with 1 μM TG (thapsigargin) in Ca 2+ -free medium before addition of 2 mM Ca 2+ for the time periods indicated by the horizontal bars. One representative experiment is presented. Histograms summarize five separate experiments (triplicates) showing the area of the fluorescence signal under TG-induced response in the absence of external Ca 2+ and percentage of influx following addition of 2 mM Ca 2+ (mean ± S.E.M.; ** P < 0.01). e Fibroblasts were co-transduced with STIM1-WT-GFP- and UNC93B1 WT or UNC93B1 3d -Cherry-tagged plasmids followed by treatment with TG in Ca 2+ -free Ringer’s solution for the time points indicated or left untreated. Representative TIRF images of STIM1-WT-GFP (left panel) and quantification of percentage of fluorescence intensity per cell area using Image J ( n = 15 cells; * P < 0.05; ** P < 0.01). Bars = 10 μm. f Localized Ca 2+ traces near phagosomes (green dots, white arrow) were measured in WT and 3d/3d DC loaded with 4 μM Fluo-8 beads for 30 min uptake and 2.5 μM BAPTA. The color-coded ratio images represent Fluo-8 fluorescence divided by the average cytosolic Fluo-8 fluorescence (left panels) and quantification shows percentage of periphagosomal Ca 2+ hotspots ( n = 4 experiments/462–660 phagosomes for WT and 136–200 phagosomes for 3d/3d DCs) (right panel; mean ± S.E.M.; *** P < 0.001). Bars = 10 μm. For a , b , c , e one experiment out of three is shown. Statistics are performed via unpaired t -test

Article Snippet: STIM1 plasmids (pEX-SP-YFP-STIM1(23-685), pEX-SP-YFP-STIM1(D76A), pEX-SP-YFP-STIM1(1-237), pEX-SP-YFP-STIM1(342-448)) were purchased from Addgene or were a kind gift from Dr. R. Lewis (Stanford University, USA).

Techniques: Expressing, Immunoprecipitation, Transfection, Plasmid Preparation, Proximity Ligation Assay, Fluorescence, Transduction

Journal: Nature Communications

Article Title: UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

doi: 10.1038/s41467-017-01601-5

Figure Lengend Snippet: UNC93B1 association with STIM1 ER-luminal is required for STIM1 oligomerization. a Schematic representation of STIM1 full-length protein (WT) and STIM1 mutants (STIM1-ΔCt and STIM1-CAD) used in this study. b Fibroblasts expressing GFP-tagged STIM1- WT, −ΔCT and −CAD and UNC93B1 WT -FLAG-tagged plasmids were immunoprecipitated with GFP beads and immunoblotted with anti-GFP and anti-FLAG antibodies. One experiment representative out of two is shown. c STIM1 oligomerization was followed in HeLa cells in the presence of either Cherry-tagged WT or 3d-mutated UNC93B1 by measuring the increase in the FRET efficiency of YFP-tagged and CFP-tagged STIM1 upon stimulation with 1 μM TG over time. The V50 parameter is reported as the time-to-half–maximum FRET efficiency. Graph shows mean ± S.E.M. ( n = 3 experiments); * P < 0.05 by paired t -test

Article Snippet: STIM1 plasmids (pEX-SP-YFP-STIM1(23-685), pEX-SP-YFP-STIM1(D76A), pEX-SP-YFP-STIM1(1-237), pEX-SP-YFP-STIM1(342-448)) were purchased from Addgene or were a kind gift from Dr. R. Lewis (Stanford University, USA).

Techniques: Expressing, Immunoprecipitation

Journal: Nature Communications

Article Title: UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

doi: 10.1038/s41467-017-01601-5

Figure Lengend Snippet: Impaired antigen degradation and cross-presentation in cells with STIM1 ablation. a Quantitative PCR analysis of Stim1 and Stim2 gene expression in DCs nucleofected with Stim1-specific siRNA (mean ± S.E.M.; n = 3 experiments). b Immunoblot of STIM1 and actin expression in WT (control siRNA) and Stim1-silenced (siRNA #5) DCs and quantification of actin/STIM1 ratio of three independent experiments (** P < 0.01 via unpaired t -test). c Averaged Ca 2+ signals in control and STIM1-silenced DCs loaded with fluo-4AM and stimulated with TG in Ca 2+ -free solution before adding 2 mM Ca 2+ . One representative experiment is shown (left panel) ( n = 3 × 10 5 cells) and graph shows Ca 2+ influx (right) from three independent experiments normalized to siRNA control cells (**** P < 0.0001 via paired t -test). d Proteolysis was measured by quantifying the degradation of OVA in control and STIM1-silenced phagosomes for the indicated time points ( n = 3 experiments; mean ± S.E.M.; * P < 0.05, *** P < 0.001 via paired t -test). e Endosomal pH in control and STIM1-silenced DCs pulsed for 10 min with FITC-coupled and Alexa-647-coupled dextrans and chased for 50 min ( n = 3; mean ± S.E.M.). f DCs knocked down for STIM1 or not from WT and 3d/3d mice were challenged with BSAb, OVAb, or OVA-transfected splenocytes as sources of exogenous antigen before co-culture with CFSE-labeled OT-I T cells. T cell proliferation was monitored by flow cytometry 3 days later. Representative histogram plots are shown (left). Quantification (right) of OT-I cell division is shown as mean percentage of proliferating cells ( n = 3; mean ± S.E.M.; * P < 0.05; ** P < 0.01; **** P < 0.0001 using unpaired t -test). g Immunoblot analysis of STIM1 and actin protein expression in control human fibroblasts and fibroblasts from a patient with STIM1 deficiency and quantification of actin/STIM1 ratio. **** P < 0.01; n = 3 (unpaired t -test). h Control and STIM1-deficient FcγRIIA-EGFP-K b -transduced human fibroblasts were stimulated with precipitated OVA immunocomplexes (OVA pICs) or BSA pICs (none) as control (upper panel) or SIINFEKL peptide (lower panel) before co-culture with B3Z hybridoma. IL-2 secretion was measured by ELISA ( n = 3; mean ± S.E.M.; *** P < 0.001 via unpaired t -test)

Article Snippet: STIM1 plasmids (pEX-SP-YFP-STIM1(23-685), pEX-SP-YFP-STIM1(D76A), pEX-SP-YFP-STIM1(1-237), pEX-SP-YFP-STIM1(342-448)) were purchased from Addgene or were a kind gift from Dr. R. Lewis (Stanford University, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Co-Culture Assay, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

doi: 10.1038/s41467-017-01601-5

Figure Lengend Snippet: Active STIM1 restores antigen proteolysis and cross-presentation in 3d/3d DCs. a Detection of STIM1 active (STIM1-D76A) and UNC93B1 interaction using Duolink proximity ligation assay (PLA) with anti-GFP (STIM1-D76A) and anti-UNC93B1-specific antibodies in WT or 3d/3d DCs. Quantification of mean fluorescence using ImageJ software ( n = 12 cells; mean ± S.E.M.; ns for non-significant). One experiment out of three is shown. Bars = 10 μm. b Fibroblasts expressing GFP-tagged STIM1-D76A and WT or 3d-mutated UNC93B1-FLAG-tagged plasmids were immunoprecipitated with GFP beads (STIM1-D76A) and immunoblotted with anti-GFP and anti-FLAG antibodies. One experiment representative out of two is shown. c Fibroblasts were transiently co-transduced with STIM1-D76A-GFP and WT or 3d-mutated UNC93B1-Cherry-tagged plasmids. Representative TIRF and EPI images of STIM1 active and UNC93B1 (left panel) and quantification of percentage of GFP fluorescence intensity per cell area using ImageJ software ( n = 15 cells). One experiment out of two is shown. Bars = 10 μm. d DCs from WT and 3d/3d mice, transfected with STIM1-D76A-GFP or control GFP plasmids, were challenged with OVAb for 6 h before co-culture with CFSE-labeled OT-I T cells. T cell proliferation was monitored by flow cytometry 3 days later ( n = 3; mean ± S.E.M.; * P < 0.05, *** P < 0.001 via unpaired t -test). e Phagosomal OVA degradation was measured at different time points in WT and 3d/3d DCs transfected with control GFP or with STIM1-D76A-GFP plasmids ( n = 3 experiments; mean ± S.E.M.; * P < 0.05, ** P < 0.01 via unpaired t -test). f WT and 3d/3d DCs were treated with ConB (20 nM) 10 min prior addition of OVAb (left panel) or SIINFEKL peptide (right panel) for 6 h. Cells were then extensively washed and co-cultured with B3Z CD8 + T cell hybridoma. T cell activation was monitored by measuring β-galactosidase activity ( n = 3 experiments; mean ± S.E.M.; *** P < 0.001, **** P < 0.0001 via unpaired t -test)

Article Snippet: STIM1 plasmids (pEX-SP-YFP-STIM1(23-685), pEX-SP-YFP-STIM1(D76A), pEX-SP-YFP-STIM1(1-237), pEX-SP-YFP-STIM1(342-448)) were purchased from Addgene or were a kind gift from Dr. R. Lewis (Stanford University, USA).

Techniques: Proximity Ligation Assay, Fluorescence, Software, Expressing, Immunoprecipitation, Transduction, Transfection, Co-Culture Assay, Labeling, Flow Cytometry, Cell Culture, Activation Assay, Activity Assay

Journal: Nature Communications

Article Title: UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

doi: 10.1038/s41467-017-01601-5

Figure Lengend Snippet: Function of UNC93B1 and STIM1 in Ca 2+ influx and antigen cross-presentation in DCs. In WT DCs, UNC93B1 associates with STIM1 through their ER-facing domains resulting in STIM1 oligomerization and activation. In DCs carrying the 3d mutation, UNC93B1-STIM1 association is compromised leading to decreased STIM1 oligomerization and activation, and to reduced SOCE and periphagosomal Ca 2+ signaling. UNC93B1 participates to events critical for efficient cross-presentation either STIM1 dependent (antigen degradation, phago-lysosomal fusion, phagosomal Ca 2+ hotspots, in red in the scheme) or STIM1 independent (endosomal/phagosomal pH, NOX2 recruitment and ROS production, cathepsins activity and antigen export to the cytosol, TLR folding and transport, highlighted in black). In 3d/3d cells, all these parameters are severely impaired resulting in inefficient antigen cross-presentation. In summary, UNC93B1, together with STIM1, are important regulators of antigen cross-presentation by modulating phagosomal homeostasis and calcium physiology in DCs. Ag: antigen, CysC: cystatin C, LRO: lysosome-related organelle

Article Snippet: STIM1 plasmids (pEX-SP-YFP-STIM1(23-685), pEX-SP-YFP-STIM1(D76A), pEX-SP-YFP-STIM1(1-237), pEX-SP-YFP-STIM1(342-448)) were purchased from Addgene or were a kind gift from Dr. R. Lewis (Stanford University, USA).

Techniques: Activation Assay, Mutagenesis, Activity Assay

Journal: Journal of Ginseng Research

Article Title: The purified extract of steamed Panax ginseng protects cardiomyocyte from ischemic injury via caveolin-1 phosphorylation-mediating calcium influx

doi: 10.1016/j.jgr.2023.07.003

Figure Lengend Snippet: Primer Sequence of qRT-PCR

Article Snippet: Antibodies of GAPDH, and ORAI1, TRPC1 and STIM1 were purchased from Servicebio, and Boster Biological Technology, Wuhan, China, respectively.

Techniques: Sequencing

Journal: Journal of Ginseng Research

Article Title: The purified extract of steamed Panax ginseng protects cardiomyocyte from ischemic injury via caveolin-1 phosphorylation-mediating calcium influx

doi: 10.1016/j.jgr.2023.07.003

Figure Lengend Snippet: EPG inhibited apoptosis and decreased STIM via p-caveolin-1 against MI injury in myocardium of rats (n = 4-6/group). (A) TUNEL staining, apoptotic nuclei (green) and DAPI-positive normal nuclei (blue). (B) Immuno-histochemical staining of p-caveolin-1 and STIM1. (C) Apoptotic rate (%). (D, E) Quantitative mean fluorescence intensity (MFI). ###P < 0.001 vs. sham, ∗∗∗P < 0.001 vs. model, & P < 0.05, &&&P < 0.001 vs. EPG.

Article Snippet: Antibodies of GAPDH, and ORAI1, TRPC1 and STIM1 were purchased from Servicebio, and Boster Biological Technology, Wuhan, China, respectively.

Techniques: TUNEL Assay, Staining, Fluorescence