Fig 1: Transcriptional control of ORAI3.a Chromatin accessibility maps of naive CD4+ T cells from RA and PsA patients and from healthy individuals were generated by ATAC-seq. Accessible regions at ORAI3 TSS region (chr16:30,960,352–1215) and downstream of TSS (chr16:30,964,994–5367). b Accessible regions were analyzed for transcription factor (TF) motifs. Indicated TFs were silenced using siRNA transfected into HEK293T cells for 48 h, non-targeting siRNA was used as a negative control. ORAI3 transcripts were quantified by qPCR and normalized to β-actin. Data are presented as mean ± SEM from three independent experiments (n = 3). Knockdown efficiency is shown in Supplementary Fig. 6. c Purified CD4+ T cells were transfected with increasing concentrations of IKAROS siRNA smart pool for 48 h. ORAI3 transcription was analyzed by qPCR and normalized to β-actin. Results are triplicates from one of two independent experiments. Data are presented as mean ± SEM. d The sequence (860 bp) upstream of ORAI3 TSS was cloned into the PGL3-basic plasmid and transfected into HEK293T cells alone or with an IKAROS-expressing plasmid. Firefly luciferase activity relative to Renilla luciferase is shown. Data are presented as mean ± SEM from three independent experiments (n = 3). e ChIP assays of CD4+ T cells using anti-IKAROS antibodies and a primer set amplifying the indicated potential binding site. Data are presented as mean ± SEM from three independent experiments (n = 3). f Flow cytometry analysis of AA-induced Ca2+ influx in IKAROS siRNA smart pool- and control siRNA-transfected CD4+ naive T cells. Histograms are representative of two experiments. g Immunoblot analysis of AA-induced ERK phosphorylation in CD4+ naive T cells transfected with IKAROS siRNA smart pool; one of two experiments. Uncropped Western blots in Supplementary Fig. 10. h Flow cytometry analysis of AA-induced CD69 expression in purified naive CD4+ T cells transfected with IKAROS siRNA smart pool. Representative histograms and data from four experiments. Data in b, c were analyzed with one-way ANOVA followed by Tukey’s multiple comparison test with adjustment. Data in d, e, and h were analyzed with unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.
Fig 2: Silencing of ORAI3 in RA T cells reduces synovial inflammation.a, b NSG mice engrafted with human synovial tissue were reconstituted with CD45RO– PBMC from RA patients transfected with control or ORAI3 siRNA. Tissue was collected on day 7 after reconstitution and analyzed. a Three-color immunofluorescence staining of tissue sections for IFN-γ and CD3 (left). Scale bars, 20 µm. Frequencies of CD3+ and IFN-γ+CD3+ T cells in synovial tissues are shown as mean ± SEM of ten random fields of six synovial grafts for each treatment arm (right). Data were analyzed with unpaired two-tailed Student’s t test. b Expression of genes encoding lineage-determining transcription factors and key inflammatory markers as determined by RT-PCR. Results from tissues of mice (n = 6) adoptively transferred with control cells are shown as white bars and circles, and those from mice with ORAI3-silenced cells as gray bars and squares. Data are presented as mean ± SEM and analyzed with unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.
Fig 3: Arachidonic acid acts through ORAI3 to activate T cells.a Naive CD4+ T cells were transfected with ORAI3 siRNA smart pool or control siRNA; after 48 h, Ca2+ influx was monitored over time (210 s) by flow cytometry of Fura Red with 0.1 µM AA added after the first 30 s (left). Right, knockdown efficiency. b CD4+ naive T cells transduced with lentivirus expressing ORAI3 shRNA #1, ORAI3 shRNA #2, or control shRNA were assayed for p-ERK (Thr202/Tyr204) at 0.5, 1, or 4 h after 0.3 µM AA stimulation. Uncropped Western blots in Supplementary Fig. 9. c Representative histograms of constitutive CD69 expression in Jurkat cells stably transduced with ORAI3 shRNA or control shRNA (left). Results of CD69 expression in control and ORAI3-silenced Jurkat cells from five experiments (middle, *** = p < 0.0001). The efficiency of ORAI3 silencing was confirmed by immunoblotting (right). d Immunoblot analysis of NUR77 expression in Jurkat T cell transduced with ORAI3 shRNA #1, ORAI3 shRNA #2, or control shRNA. Data represent one of two independent experiments. e Flow cytometry of AA-induced Ca2+ influx as determined from the ratio of shifts in Fura Red (at 406 and 532 nm) fluorescence in Jurkat T cells stably overexpressing ORAI3. Representative tracing of Fura Red ratios (left) and the peak calcium influxes from three experiments are shown (right). f Immunoblot of ERK phosphorylated at Thr202/Tyr204 from ORAI3-overexpressing Jurkat cells at indicated time points (0, 0.5, 1, and 4 h) after 0.3 µM AA stimulation (left) and peak p-ERK responses from three experiments are shown (right). g Flow cytometry of constitutive CD69 expression by ORAI3-overexpressing and vector control Jurkat T cells (left). CD69 expression summarized from five experiments (middle). Immunoblots documenting ORAI3 overexpression (right). Uncropped Western blots of c, d, f, g in Supplementary Fig. 10. Data are presented as mean ± SEM (a, c, e, f, g). Results in a, b, e, and f are each representative of three experiments. Data were analyzed by paired two-tailed Student’s t test (a), by one-way ANOVA followed by Tukey’s multiple comparison test (c), or by unpaired two-tailed Student’s t test (e–g). Source data are provided as a Source Data file.
Fig 4: Increased expression of the Ca2+ channel component ORAI3 in RA CD4+ T cells.a Transcriptional profiling of Ca2+ channel and Ca2+ sensor gene transcripts in CD4+ naive T cells from HC (n = 6 white bars and circles) and RA (n = 6 gray bars and squares) patients. Gene expression was normalized to β-actin. Data are presented as mean ± SEM. b ORAI3 transcripts quantified by qPCR and normalized to 18S rRNA in CD4+ naive T cells from HC (n = 22) and RA (n = 21). Data are shown as dot plots with mean ± SEM. c ORAI3 transcript levels in RA patients with CDAI activity scores <10 (n = 9) or >20 (n = 5). d, e ORAI3 transcripts in CD4+ naive T cells from RA patients dichotomized for whether they were treated (n = 5) or not treated with methotrexate (MTX) (n = 16) (d) or whether they were (n = 10) or were not (n = 11) on TNF inhibitors (TNFi, etanercept or adalimumab) (e). f ORAI3 protein levels are shown as a representative immunoblot (left) and as dot plots of densities of ORAI3 relative to β-actin of purified naive CD4+ T cells from HC (n = 10) and RA (n = 9) patients. Uncropped Western blots in Supplementary Fig. 9. g ORAI3 transcripts quantified by qPCR and normalized to 18S rRNA in CD4+ naive T cells from HC (n = 22) and gout (n = 6), systemic lupus erythematosus (SLE) (n = 7), and PsA (n = 10) patients (HC vs. gout p = 0.80; HC vs. SLE p = 0.85). Statistical analyses were performed with one-way ANOVA followed by Tukey’s multiple comparison test (Fig. 2g) or with unpaired two-tailed Student’s t test (a–f). n.s. not significant. Source data are provided as a Source Data file.
Fig 5: Enrichment of Orai3 in CSC populations. (A) Expression of Orai3 was assessed in tumor spheres (Sph.) and their corresponding adherent monolayer cells (Mono.) derived from multiple OSCC cell lines by qPCR. Data are means ± SD of three independent assays. * p < 0.05 and ** p < 0.01 compared to controls. On the right, a representative image of spheres derived from OSCC cell lines was displayed. (B) ALDH1HIGH (High) and ALDH1low (Low) cell populations were sorted from SCC4 cells by flow cytometry, and their Orai3 expression level was assessed by qPCR (upper) and Western blot (lower). Relative numeric protein levels were indicated under the corresponding bands. * p < 0.05. (C) Expression of Orai3 was assessed in cisplatin-sensitive (Sens) and cisplatin-resistant (Resist) SCC4 cells by qPCR. Orai3 levels were obtained from three independent qPCRs. ** p < 0.01. (D) Expression of Orai3 was assessed in CSC populations and their corresponding non-CSC populations by Western blot. Relative numeric protein levels were indicated under the corresponding bands.
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