orai1  (Alomone Labs)


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    Structured Review

    Alomone Labs orai1
    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: <t>ORAI1,</t> H (1, 47) = 9.1, p
    Orai1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/orai1/product/Alomone Labs
    Average 94 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    orai1 - by Bioz Stars, 2022-12
    94/100 stars

    Images

    1) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

    2) Product Images from "Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes"

    Article Title: Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0208981

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-pregnant controls and pregnant women. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Ca V 1.1 subunit mRNA was not detected in any sample. Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: ORAI1, df = 48, p = 0.003; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI2, H (1, 46) = 28.5, p

    Techniques Used: Expressing

    Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p
    Figure Legend Snippet: Altered mRNA expression of specific calcium release activated calcium (CRACs) channel and voltage-gated calcium channel (Ca v ) subunits in PBMCs from non-diabetic controls and T1D patients. Data from each group is presented as scatter dot plot (°) or box and whiskers plot with median and whiskers plotted by Tukey method to determine outliers (• - above or below the whiskers). Statistical analysis was performed by excluding outliers depending on normality distribution of the data and only the subunits with statistically significant differences are mentioned below. One-Way ANOVA with Bonferroni post-hoc test: STIM2, df = 45, p = 0.002; Kruskal–Wallis ANOVA on ranks with Dunn’s post hoc test: ORAI1, H (1, 47) = 9.1, p

    Techniques Used: Expressing

    Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.
    Figure Legend Snippet: Western blot analysis. Western blot analysis showing the expression of Ca 2+ sensor protein stromal interaction molecules (STIM) 2, voltage-gated Ca 2+ (Ca V ) channel subunits Cav1.3 and Cav2.3, and Ca 2+ release-activated Ca 2+ (CRAC) channel proteins ORAI1 and 2 in PBMCs from ND (n = 3 or 4) and T1D (n = 4) individuals. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) served as loading controls.

    Techniques Used: Western Blot, Expressing

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    Alomone Labs anti human orai1 extracellular antibody
    <t>ORAI1</t> , STIM1 and STIM2 expression in human eMSCs. ( A ) RT-PCR analysis of ORAI , STIM1 and STIM2 expression in eMSCs. M—size marker. Line 1—primers specific for ORAI1 , STIM1 and STIM2 amplified the PCR products of the expected sizes (78, 124 and 247 bp, correspondingly). Line 2—RT-PCR negative control in which reverse transcriptase was omitted. Shown are cropped gels with enhanced contrast. Original gel is shown in Supplementary Figure S5 . The distribution of ORAI1 ( B ), STIM1 ( C ) and STIM2 ( D ). Immunoreactivity in eMSCs was examined with confocal microscopy. No staining of the cells was observed after incubation of the cells with only fluorescent secondary antibodies ( Supplementary Figure S2 ). Scale bar is 50 µm.
    Anti Human Orai1 Extracellular Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti human orai1 extracellular antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti human orai1 extracellular antibody - by Bioz Stars, 2022-12
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    ORAI1 , STIM1 and STIM2 expression in human eMSCs. ( A ) RT-PCR analysis of ORAI , STIM1 and STIM2 expression in eMSCs. M—size marker. Line 1—primers specific for ORAI1 , STIM1 and STIM2 amplified the PCR products of the expected sizes (78, 124 and 247 bp, correspondingly). Line 2—RT-PCR negative control in which reverse transcriptase was omitted. Shown are cropped gels with enhanced contrast. Original gel is shown in Supplementary Figure S5 . The distribution of ORAI1 ( B ), STIM1 ( C ) and STIM2 ( D ). Immunoreactivity in eMSCs was examined with confocal microscopy. No staining of the cells was observed after incubation of the cells with only fluorescent secondary antibodies ( Supplementary Figure S2 ). Scale bar is 50 µm.

    Journal: International Journal of Molecular Sciences

    Article Title: Store-Operated Ca2+ Entry Contributes to Piezo1-Induced Ca2+ Increase in Human Endometrial Stem Cells

    doi: 10.3390/ijms23073763

    Figure Lengend Snippet: ORAI1 , STIM1 and STIM2 expression in human eMSCs. ( A ) RT-PCR analysis of ORAI , STIM1 and STIM2 expression in eMSCs. M—size marker. Line 1—primers specific for ORAI1 , STIM1 and STIM2 amplified the PCR products of the expected sizes (78, 124 and 247 bp, correspondingly). Line 2—RT-PCR negative control in which reverse transcriptase was omitted. Shown are cropped gels with enhanced contrast. Original gel is shown in Supplementary Figure S5 . The distribution of ORAI1 ( B ), STIM1 ( C ) and STIM2 ( D ). Immunoreactivity in eMSCs was examined with confocal microscopy. No staining of the cells was observed after incubation of the cells with only fluorescent secondary antibodies ( Supplementary Figure S2 ). Scale bar is 50 µm.

    Article Snippet: Then cells were incubated with goat anti-rabbit polyclonal Abs against Piezo1 (Novus Bio, Centennial, CO, USA, cat. no. NBP1-78537) or rabbit polyclonal Abs against ORAI1 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-060) or Abs against STIM1 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-063) or STIM2 (Alomone Labs, Jerusalem, Israel, cat. no. ACC-064) at 4 °C overnight.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Amplification, Polymerase Chain Reaction, Negative Control, Confocal Microscopy, Staining, Incubation

    ORAI1 knockout (KO) reduces store-operated calcium entry (SOCE). ( A ) Representative traces of a single Ca 2+ imaging experiment. For both panels, all recorded cells are represented with different colored lanes, the mean value is presented in black, and the 95% confidence interval (CI) is shown in grey. Each experiment was repeated at least three times. ORAI1 KO cells exhibit a greatly reduced SOCE compared with wild-type (WT) cells. ( B ) The integrated area of the curve following Ca 2+ addition representing SOCE. Values from each individual cells from three independent experiments are displayed in semitransparent colors (replicate N°1 yellow circles, replicate N°2 blue squares, and replicate N°3 green diamonds). For each experiment, the median values are displayed in plain shapes (replicate N°1 yellow circles, replicate N°2 blue squares, and replicate N°3 green diamonds). Data generated for each experiment were considered nonpaired. Black bars represent the mean and the 95% CI of the three independent experiments. The difference in SOCE between WT HEK-293 cells (mean = 1511 with a 95% CI of 906–2117) compared with ORAI1 KO HEK-293 cells (mean = 282 with a 95% CI of 154–411) was assessed by Welch’s t -test and was significant ( p = 0.007).

    Journal: Cells

    Article Title: Impact of SOCE Abolition by ORAI1 Knockout on the Proliferation, Adhesion, and Migration of HEK-293 Cells

    doi: 10.3390/cells10113016

    Figure Lengend Snippet: ORAI1 knockout (KO) reduces store-operated calcium entry (SOCE). ( A ) Representative traces of a single Ca 2+ imaging experiment. For both panels, all recorded cells are represented with different colored lanes, the mean value is presented in black, and the 95% confidence interval (CI) is shown in grey. Each experiment was repeated at least three times. ORAI1 KO cells exhibit a greatly reduced SOCE compared with wild-type (WT) cells. ( B ) The integrated area of the curve following Ca 2+ addition representing SOCE. Values from each individual cells from three independent experiments are displayed in semitransparent colors (replicate N°1 yellow circles, replicate N°2 blue squares, and replicate N°3 green diamonds). For each experiment, the median values are displayed in plain shapes (replicate N°1 yellow circles, replicate N°2 blue squares, and replicate N°3 green diamonds). Data generated for each experiment were considered nonpaired. Black bars represent the mean and the 95% CI of the three independent experiments. The difference in SOCE between WT HEK-293 cells (mean = 1511 with a 95% CI of 906–2117) compared with ORAI1 KO HEK-293 cells (mean = 282 with a 95% CI of 154–411) was assessed by Welch’s t -test and was significant ( p = 0.007).

    Article Snippet: The primary antibodies were anti-ORAI1 (Alomone, Jerusalemn, Israel: Acc-060), used at a 1/200 dilution, and anti-β-actin Sigma-Aldrich, St. Quentin Fallavier, France: A5441), used at a 1/5000 dilution.

    Techniques: Knock-Out, Imaging, Generated

    Genomic validation of complete ORAI1 knockout (KO) generation. ( A ) Polymerase chain reaction (PCR) performed on genomic DNA (gDNA) of nonclonal cells corresponding to non-transfected cells and cells transfected with two guide RNAs (gRNAs). Cells transfected with two gRNA present a smaller band (384 bp) indicating the existence of recombination events. ( B ) PCR performed on gDNA of single-cell-derived clones. Clones 1, 3, and 5 presenting a single band around the expected size (384 bp) were selected for further experiments. ( C ) PCR performed on gDNA of wild type (WT) and ORAI1 KO HEK-293 cells. The ORAI1 KO clone presents a lower band indicating successful ORAI1 gene deletion. ( D ) Western blot analysis of WT and ORAI1 KO HEK-293 cells. WT cells exhibit an ORAI1 band around 37 kDa, while ORAI1 KO cells lack this band.

    Journal: Cells

    Article Title: Impact of SOCE Abolition by ORAI1 Knockout on the Proliferation, Adhesion, and Migration of HEK-293 Cells

    doi: 10.3390/cells10113016

    Figure Lengend Snippet: Genomic validation of complete ORAI1 knockout (KO) generation. ( A ) Polymerase chain reaction (PCR) performed on genomic DNA (gDNA) of nonclonal cells corresponding to non-transfected cells and cells transfected with two guide RNAs (gRNAs). Cells transfected with two gRNA present a smaller band (384 bp) indicating the existence of recombination events. ( B ) PCR performed on gDNA of single-cell-derived clones. Clones 1, 3, and 5 presenting a single band around the expected size (384 bp) were selected for further experiments. ( C ) PCR performed on gDNA of wild type (WT) and ORAI1 KO HEK-293 cells. The ORAI1 KO clone presents a lower band indicating successful ORAI1 gene deletion. ( D ) Western blot analysis of WT and ORAI1 KO HEK-293 cells. WT cells exhibit an ORAI1 band around 37 kDa, while ORAI1 KO cells lack this band.

    Article Snippet: The primary antibodies were anti-ORAI1 (Alomone, Jerusalemn, Israel: Acc-060), used at a 1/200 dilution, and anti-β-actin Sigma-Aldrich, St. Quentin Fallavier, France: A5441), used at a 1/5000 dilution.

    Techniques: Knock-Out, Polymerase Chain Reaction, Transfection, Derivative Assay, Clone Assay, Western Blot

    Experimental design of the strategy used to create total knockout of ORAI1 protein (ORAI1 KO). ( A ) Structure of ORAI1 messenger RNA (mRNA) including the noncoding region (light grey), the coding sequence (dark grey), the original start codon (ATG, full-length ORAI1 origin), and the alternative start codon (ATG, ORAI1β isoform). ( B ) Structure of ORAI1 protein including the transmembrane domain (white boxes) and the stromal interaction molecule (STIM) interaction domain (hatched boxes). ( C ) Guide RNA (gRNA) strategy used to generate complete ORAI1 KO. The green dotted lines represent the localization of the two gRNAs used. Exact coordinates of gRNA double-strand break (DSB) induction sites (relative to the original ORAI1 ATG) are indicated next to the name of the corresponding gRNA (with nt standing for nucleotide and aa standing for amino acid). The light green insert displays the genomic (g), coding sequence (c), and protein (p) coordinates of the induced deletion. The dark green insert presents the sequences surrounding the deletion on genomic DNA level (g) for both alleles (underlined signs indicate the position of the original ORAI1 start codon).

    Journal: Cells

    Article Title: Impact of SOCE Abolition by ORAI1 Knockout on the Proliferation, Adhesion, and Migration of HEK-293 Cells

    doi: 10.3390/cells10113016

    Figure Lengend Snippet: Experimental design of the strategy used to create total knockout of ORAI1 protein (ORAI1 KO). ( A ) Structure of ORAI1 messenger RNA (mRNA) including the noncoding region (light grey), the coding sequence (dark grey), the original start codon (ATG, full-length ORAI1 origin), and the alternative start codon (ATG, ORAI1β isoform). ( B ) Structure of ORAI1 protein including the transmembrane domain (white boxes) and the stromal interaction molecule (STIM) interaction domain (hatched boxes). ( C ) Guide RNA (gRNA) strategy used to generate complete ORAI1 KO. The green dotted lines represent the localization of the two gRNAs used. Exact coordinates of gRNA double-strand break (DSB) induction sites (relative to the original ORAI1 ATG) are indicated next to the name of the corresponding gRNA (with nt standing for nucleotide and aa standing for amino acid). The light green insert displays the genomic (g), coding sequence (c), and protein (p) coordinates of the induced deletion. The dark green insert presents the sequences surrounding the deletion on genomic DNA level (g) for both alleles (underlined signs indicate the position of the original ORAI1 start codon).

    Article Snippet: The primary antibodies were anti-ORAI1 (Alomone, Jerusalemn, Israel: Acc-060), used at a 1/200 dilution, and anti-β-actin Sigma-Aldrich, St. Quentin Fallavier, France: A5441), used at a 1/5000 dilution.

    Techniques: Knock-Out, Sequencing

    ORAI1 knockout (KO) modifies HEK-293 cell migration properties. ( A ) Comparison of the adhesive properties of wild type (WT) and ORAI1 KO cells. Data points represent the percentage of adhered cells after an incubation for 1 h at 37 °C. Plain circles represent the mean values of individual replicates (R1, R2, R3, and R4); semitransparent circles display individual data points within each replicate. The mean values of the four replicates are indicated as black bars ± standard deviation (CI). The difference in adhesion between WT cells (25.56% ± standard deviation [SD] 6.90%) and KO ORAI1 cells (22.49% ± 8.61%) was assessed with Welch’s t -test for nonpaired data and was not significant ( p = 0.6; mean difference between conditions = −3.07% with a 95% CI of −16.73 to 10.59). ( B ) Middle panel: relative wound area as a function of time (WT in blue, ORAI1 KO in green). The curves represent the means of five independent experiments with their 95% CI (semitransparent colors). Right panels: Relative wound area as a function of time with each repeat presented individually (down panel: WT in blue; top panel: KO in green). Black lines represent the mean value of the experiments with the 95% CI in semitransparent color; the thin colored lanes represent repeats of every single experiment performed. Wound closure area analysis performed for each time point (i.e., every hour) indicates that ORAI1 KO cells migrated significantly faster than WT cells during hours 1–9 of the experiment (* p

    Journal: Cells

    Article Title: Impact of SOCE Abolition by ORAI1 Knockout on the Proliferation, Adhesion, and Migration of HEK-293 Cells

    doi: 10.3390/cells10113016

    Figure Lengend Snippet: ORAI1 knockout (KO) modifies HEK-293 cell migration properties. ( A ) Comparison of the adhesive properties of wild type (WT) and ORAI1 KO cells. Data points represent the percentage of adhered cells after an incubation for 1 h at 37 °C. Plain circles represent the mean values of individual replicates (R1, R2, R3, and R4); semitransparent circles display individual data points within each replicate. The mean values of the four replicates are indicated as black bars ± standard deviation (CI). The difference in adhesion between WT cells (25.56% ± standard deviation [SD] 6.90%) and KO ORAI1 cells (22.49% ± 8.61%) was assessed with Welch’s t -test for nonpaired data and was not significant ( p = 0.6; mean difference between conditions = −3.07% with a 95% CI of −16.73 to 10.59). ( B ) Middle panel: relative wound area as a function of time (WT in blue, ORAI1 KO in green). The curves represent the means of five independent experiments with their 95% CI (semitransparent colors). Right panels: Relative wound area as a function of time with each repeat presented individually (down panel: WT in blue; top panel: KO in green). Black lines represent the mean value of the experiments with the 95% CI in semitransparent color; the thin colored lanes represent repeats of every single experiment performed. Wound closure area analysis performed for each time point (i.e., every hour) indicates that ORAI1 KO cells migrated significantly faster than WT cells during hours 1–9 of the experiment (* p

    Article Snippet: The primary antibodies were anti-ORAI1 (Alomone, Jerusalemn, Israel: Acc-060), used at a 1/200 dilution, and anti-β-actin Sigma-Aldrich, St. Quentin Fallavier, France: A5441), used at a 1/5000 dilution.

    Techniques: Knock-Out, Migration, Incubation, Standard Deviation

    ORAI1 knockout (KO) leads to the downregulation of ORAI2 and ORAI3 messenger RNA (mRNA) expression. Dotted boxes represent the mean expression level of ORAI2 and ORAI3 mRNA measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) from four independent experiments. The levels of ORAI2 and ORAI3 mRNA in wild-type (WT) HEK-293 cells were arbitrarily set at 1. In ORAI1 KO cells, ORAI2 (low-density dots, left box) and ORAI3 (high-density dots, right box) are downregulated ( ORAI2 relative mean expression level 0.42 with a 95% confidence interval [CI] of 0.19–0.87; ORAI3 relative mean expression level 0.20 with a 95% CI of 0.07–0.60 compared with WT. The significance of the results was assessed by unpaired t -test (* p = 0.02 for ORAI2 and * p = 0.01 for ORAI3 ).

    Journal: Cells

    Article Title: Impact of SOCE Abolition by ORAI1 Knockout on the Proliferation, Adhesion, and Migration of HEK-293 Cells

    doi: 10.3390/cells10113016

    Figure Lengend Snippet: ORAI1 knockout (KO) leads to the downregulation of ORAI2 and ORAI3 messenger RNA (mRNA) expression. Dotted boxes represent the mean expression level of ORAI2 and ORAI3 mRNA measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) from four independent experiments. The levels of ORAI2 and ORAI3 mRNA in wild-type (WT) HEK-293 cells were arbitrarily set at 1. In ORAI1 KO cells, ORAI2 (low-density dots, left box) and ORAI3 (high-density dots, right box) are downregulated ( ORAI2 relative mean expression level 0.42 with a 95% confidence interval [CI] of 0.19–0.87; ORAI3 relative mean expression level 0.20 with a 95% CI of 0.07–0.60 compared with WT. The significance of the results was assessed by unpaired t -test (* p = 0.02 for ORAI2 and * p = 0.01 for ORAI3 ).

    Article Snippet: The primary antibodies were anti-ORAI1 (Alomone, Jerusalemn, Israel: Acc-060), used at a 1/200 dilution, and anti-β-actin Sigma-Aldrich, St. Quentin Fallavier, France: A5441), used at a 1/5000 dilution.

    Techniques: Knock-Out, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    ORAI1 knockout (KO) does not affect proliferation of HEK-293 cells. ( A ) The doubling time of wild type (WT) and ORAI1 KO HEK-293 cells was measured by cell counting over a 5–6 day period. Semitransparent dots represent each doubling time calculated from one experiment (6–14 points per experiment). Plain colored circles display the mean of seven independent experiments. Black lanes represent the median value of each individual experiment and their 95% confidence interval (CI). The difference in the mean doubling time between condition was calculated by using Welch’s t -test and was not significant (WT doubling time of 25.77 h with a 95% CI of 23.75–27.79 h; ORAI1 KO doubling time of 26.81 h with a 95% CI of 24.18–29.43 h; difference between the conditions = 1.04 h; Welch’s t -test p = 0.426). ( B ) The normalized cell proliferation rate assessed by the SRB assay 5 days after seeding WT and ORAI1 KO HEK-293 cells. The semitransparent dots represent single values obtained within each experiment (eight measurements). The median values of three independent experiments are displayed in plain colored circles. The black lanes indicate the mean value of the three individual experiments and their 95% CI (WT = 9.29 with a 95% CI of 6.44–12.14; ORAI1 KO = 9.58 with a 95% CI of 6.73–12.44). The significance of the difference between conditions (−0.29 with a 95% CI of 1.83–2.42) was calculated with Welch’s t -test ( p = 0.721). ( C ) Cell-cycle analysis of the DNA content determined by propidium iodide staining for WT (blue) and ORAI1 KO (green) HEK-293 cells. The histograms represent the percentage of cells in each phase of the cell cycle (exact value indicated within the histogram bars) with error bars indicating the standard deviation. Experiments were repeated six times, and no significant differences between conditions were identified by using Welch’s t test.

    Journal: Cells

    Article Title: Impact of SOCE Abolition by ORAI1 Knockout on the Proliferation, Adhesion, and Migration of HEK-293 Cells

    doi: 10.3390/cells10113016

    Figure Lengend Snippet: ORAI1 knockout (KO) does not affect proliferation of HEK-293 cells. ( A ) The doubling time of wild type (WT) and ORAI1 KO HEK-293 cells was measured by cell counting over a 5–6 day period. Semitransparent dots represent each doubling time calculated from one experiment (6–14 points per experiment). Plain colored circles display the mean of seven independent experiments. Black lanes represent the median value of each individual experiment and their 95% confidence interval (CI). The difference in the mean doubling time between condition was calculated by using Welch’s t -test and was not significant (WT doubling time of 25.77 h with a 95% CI of 23.75–27.79 h; ORAI1 KO doubling time of 26.81 h with a 95% CI of 24.18–29.43 h; difference between the conditions = 1.04 h; Welch’s t -test p = 0.426). ( B ) The normalized cell proliferation rate assessed by the SRB assay 5 days after seeding WT and ORAI1 KO HEK-293 cells. The semitransparent dots represent single values obtained within each experiment (eight measurements). The median values of three independent experiments are displayed in plain colored circles. The black lanes indicate the mean value of the three individual experiments and their 95% CI (WT = 9.29 with a 95% CI of 6.44–12.14; ORAI1 KO = 9.58 with a 95% CI of 6.73–12.44). The significance of the difference between conditions (−0.29 with a 95% CI of 1.83–2.42) was calculated with Welch’s t -test ( p = 0.721). ( C ) Cell-cycle analysis of the DNA content determined by propidium iodide staining for WT (blue) and ORAI1 KO (green) HEK-293 cells. The histograms represent the percentage of cells in each phase of the cell cycle (exact value indicated within the histogram bars) with error bars indicating the standard deviation. Experiments were repeated six times, and no significant differences between conditions were identified by using Welch’s t test.

    Article Snippet: The primary antibodies were anti-ORAI1 (Alomone, Jerusalemn, Israel: Acc-060), used at a 1/200 dilution, and anti-β-actin Sigma-Aldrich, St. Quentin Fallavier, France: A5441), used at a 1/5000 dilution.

    Techniques: Knock-Out, Cell Counting, Sulforhodamine B Assay, Cell Cycle Assay, Staining, Standard Deviation

    Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p

    Journal: Frontiers in Pharmacology

    Article Title: Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

    doi: 10.3389/fphar.2021.742862

    Figure Lengend Snippet: Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p

    Article Snippet: For determination of ion channel expression, cells were fixed with 4% paraformaldehyde, washed with PBS followed by overnight incubation at 4°C with the following primary antibodies, mouse anti-human KCa3.1 (6C1/ATTO-488), guinea pig anti-human Kv1.3, rabbit anti-human Orai1 (all from Alomone labs) followed by anti-guinea pig (Dy350 goat anti-guinea pig IgG/Thermo Fisher) and anti-rabbit (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher) secondary antibodies.

    Techniques: Inhibition, Patch Clamp, Transferring, Fluorescence, Chick Chorioallantoic Membrane Assay, Flow Cytometry

    αPD-1 treatment increases K + channel activity in HNSCC T cells. (A) Representative current traces of KCa3.1 and Kv1.3 channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs cells from a HNSCC patient in absence or presence of αPD-1 (10 μg/ml, for 6 h). Data are normalized to maximum current at +50 mV recorded using a ramp pulse protocol from −120 mV to +50 mV for 200 ms every 15 s. The holding potential used was −70 mV. (B,C) KCa3.1 (B) and Kv1.3 (C) conductance (G) measured in the absence or presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 68 cells without pembrolizumab and n = 55 cells with pembrolizumab from 14 patients). (D) Representative current traces of divalent free current (DVF) through CRAC channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs from a HNSCC patient. Data were recorded using a ramp pulse protocol from −100 to +100 mV with at holding potential of +30 mV every 1.5 s. Cells were perfused with 0 mM Ca 2+ solution (1 min) followed by 20 mM Ca 2+ (1 min) and DVF solutions (2 min, see methods) to amplify currents during recordings. (E) Peak DVF current values measured in absence and presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 34 cells without αPD-1 and n = 31 cells with αPD-1 from 8 patients). The values in panels (B,C) and (E) are represented as box plots: the horizontal line indicates the median; the lower box is the 25 th percentile; the upper box is the 75 th percentile; and the whiskers represent the 10 th and 90 th percentiles. (F) Ion channel expression (KCa3.1, Kv1.3, Orai1 and STIM1) in HNSCC patient T cells after treatment with αPD-1 (10 μg/ml for 6 h). Effect of αPD-1 treatment is shown as ratio of mean fluorescence intensity (MFI, fold change) values of treatment versus control group. Data are represented as scatter plot where each symbol represents an individual patient ( n = 4–5). Horizontal line represents mean values for each group. Data in panels (B,C,E) were analyzed by Mann-Whitney rank sum test.

    Journal: Frontiers in Pharmacology

    Article Title: Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

    doi: 10.3389/fphar.2021.742862

    Figure Lengend Snippet: αPD-1 treatment increases K + channel activity in HNSCC T cells. (A) Representative current traces of KCa3.1 and Kv1.3 channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs cells from a HNSCC patient in absence or presence of αPD-1 (10 μg/ml, for 6 h). Data are normalized to maximum current at +50 mV recorded using a ramp pulse protocol from −120 mV to +50 mV for 200 ms every 15 s. The holding potential used was −70 mV. (B,C) KCa3.1 (B) and Kv1.3 (C) conductance (G) measured in the absence or presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 68 cells without pembrolizumab and n = 55 cells with pembrolizumab from 14 patients). (D) Representative current traces of divalent free current (DVF) through CRAC channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs from a HNSCC patient. Data were recorded using a ramp pulse protocol from −100 to +100 mV with at holding potential of +30 mV every 1.5 s. Cells were perfused with 0 mM Ca 2+ solution (1 min) followed by 20 mM Ca 2+ (1 min) and DVF solutions (2 min, see methods) to amplify currents during recordings. (E) Peak DVF current values measured in absence and presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 34 cells without αPD-1 and n = 31 cells with αPD-1 from 8 patients). The values in panels (B,C) and (E) are represented as box plots: the horizontal line indicates the median; the lower box is the 25 th percentile; the upper box is the 75 th percentile; and the whiskers represent the 10 th and 90 th percentiles. (F) Ion channel expression (KCa3.1, Kv1.3, Orai1 and STIM1) in HNSCC patient T cells after treatment with αPD-1 (10 μg/ml for 6 h). Effect of αPD-1 treatment is shown as ratio of mean fluorescence intensity (MFI, fold change) values of treatment versus control group. Data are represented as scatter plot where each symbol represents an individual patient ( n = 4–5). Horizontal line represents mean values for each group. Data in panels (B,C,E) were analyzed by Mann-Whitney rank sum test.

    Article Snippet: For determination of ion channel expression, cells were fixed with 4% paraformaldehyde, washed with PBS followed by overnight incubation at 4°C with the following primary antibodies, mouse anti-human KCa3.1 (6C1/ATTO-488), guinea pig anti-human Kv1.3, rabbit anti-human Orai1 (all from Alomone labs) followed by anti-guinea pig (Dy350 goat anti-guinea pig IgG/Thermo Fisher) and anti-rabbit (Alexa Fluor 594 goat anti-rabbit IgG/Thermo Fisher) secondary antibodies.

    Techniques: Activity Assay, Incubation, Expressing, Fluorescence, MANN-WHITNEY

    Loss of ORAI1 or STIM1 affects cellular susceptibility to SARS-CoV-2 infection ( A ) Representative immunoblot showing expression of SARS-CoV-2 proteins in indicated HEK293-ACE2 cells under mock conditions or after infection with SARS-CoV-2 at indicated multiplicity of infection (MOIs). β-actin – loading control. Bar graph below shows densitometry analysis of normalized ratio of SARS-N-CoV-2 to β-actin (± s.e.m.) from three independent experiments. ( B ) Representative epifluorescence images showing expression of spike protein (green) in control, ORAI1 -/- , or STIM1 -/- HEK293-ACE2 cells after infection with SARS-CoV-2 at MOI 1.0. Cells were co-stained with DAPI for detection of nuclei. ORAI1 -/- HEK293-ACE2 cells were all detached due to high virus load. ( C ) Quantitative RT-PCR analysis of viral genome from indicated cell types under mock conditions or after infection with SARS-CoV-2 at indicated MOI for 5 (left graph) or 20 (right graph) hours. Shown is one representative triplicate from two independent experiments. * P

    Journal: bioRxiv

    Article Title: ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling

    doi: 10.1101/2021.05.04.442548

    Figure Lengend Snippet: Loss of ORAI1 or STIM1 affects cellular susceptibility to SARS-CoV-2 infection ( A ) Representative immunoblot showing expression of SARS-CoV-2 proteins in indicated HEK293-ACE2 cells under mock conditions or after infection with SARS-CoV-2 at indicated multiplicity of infection (MOIs). β-actin – loading control. Bar graph below shows densitometry analysis of normalized ratio of SARS-N-CoV-2 to β-actin (± s.e.m.) from three independent experiments. ( B ) Representative epifluorescence images showing expression of spike protein (green) in control, ORAI1 -/- , or STIM1 -/- HEK293-ACE2 cells after infection with SARS-CoV-2 at MOI 1.0. Cells were co-stained with DAPI for detection of nuclei. ORAI1 -/- HEK293-ACE2 cells were all detached due to high virus load. ( C ) Quantitative RT-PCR analysis of viral genome from indicated cell types under mock conditions or after infection with SARS-CoV-2 at indicated MOI for 5 (left graph) or 20 (right graph) hours. Shown is one representative triplicate from two independent experiments. * P

    Article Snippet: Antibodies for detection of human STIM1 (5668S) was purchased from Cell Signaling Technologies, that for detection of interferon alpha and beta receptor 1 (IFNAR-1) was purchased from Leinco Technologies, Inc. (I-400), that for detection of surface ORAI1 (ACC-060) was purchased from Alomone labs and that for detection of β-actin (sc-47778), was obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Expressing, Staining, Quantitative RT-PCR

    Transcriptome analysis of control and ORAI1 -/- HEK293-ACE2 cells. ( A ) Normalized read counts (log 2 ) of SARS-CoV-2 RNA products, showing transcriptional enrichment of viral genes in SARS-CoV-2 infected cells when compared to uninfected cells. ( B ) Bar graph shows proportion of total reads comprising SARS-CoV-2 transcripts in indicated cell types. The proportion of virus-aligned reads over total reads is shown for each sample. Error bars represent average (± s.d.m.) from three biological replicates. ( C ) Dot plot visualization of enriched pathways in ORAI1 -/- HEK293-ACE2 cells. Reactome pathway enrichment analysis was performed in PANTHER. The size of the dots represents the percentage of genes enriched in the total gene set, while its color represents the false discovery rate (FDR, p-adjusted) value for each enriched pathway. ( D ) Volcano plots with all DEGs from ORAI1 -/- and control mock (uninfected) samples in gray and the indicated gene sets of anti-viral ISG15 signaling (left) and MyD88 signaling (right) highlighted in blue (FDR

    Journal: bioRxiv

    Article Title: ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling

    doi: 10.1101/2021.05.04.442548

    Figure Lengend Snippet: Transcriptome analysis of control and ORAI1 -/- HEK293-ACE2 cells. ( A ) Normalized read counts (log 2 ) of SARS-CoV-2 RNA products, showing transcriptional enrichment of viral genes in SARS-CoV-2 infected cells when compared to uninfected cells. ( B ) Bar graph shows proportion of total reads comprising SARS-CoV-2 transcripts in indicated cell types. The proportion of virus-aligned reads over total reads is shown for each sample. Error bars represent average (± s.d.m.) from three biological replicates. ( C ) Dot plot visualization of enriched pathways in ORAI1 -/- HEK293-ACE2 cells. Reactome pathway enrichment analysis was performed in PANTHER. The size of the dots represents the percentage of genes enriched in the total gene set, while its color represents the false discovery rate (FDR, p-adjusted) value for each enriched pathway. ( D ) Volcano plots with all DEGs from ORAI1 -/- and control mock (uninfected) samples in gray and the indicated gene sets of anti-viral ISG15 signaling (left) and MyD88 signaling (right) highlighted in blue (FDR

    Article Snippet: Antibodies for detection of human STIM1 (5668S) was purchased from Cell Signaling Technologies, that for detection of interferon alpha and beta receptor 1 (IFNAR-1) was purchased from Leinco Technologies, Inc. (I-400), that for detection of surface ORAI1 (ACC-060) was purchased from Alomone labs and that for detection of β-actin (sc-47778), was obtained from Santa Cruz Biotechnology.

    Techniques: Infection

    Loss of ORAI1 imparts susceptibility to SARS-CoV-2 infection by abrogating baseline interferon β levels. ( A ) Levels of IFNβ and IL-6 in culture supernatants from control or ORAI1 -/- HEK293-ACE2 cells under mock conditions or 20 hours after infection with SARS-CoV-2 at indicted MOIs. ( B ) Representative immunoblot showing expression of phosphorylated STAT1 (p-STAT1) or total STAT1 in lysates from control or ORAI1 -/- HEK293-ACE2 cells. β-actin – loading control. Data are representative of three independent experiments. ( C ) Quantitative RT-PCR analysis of viral genome from indicated cell types with or without pre-treatment with 100 U/ml of IFN-β (for 20 hours) under mock conditions or after infection with SARS-CoV-2 at MOI 0.1 for 20 h. ( D ) Representative immunoblot showing expression of SARS-CoV-2 proteins in indicated HEK293-ACE2 cells with or without pre-treatment with 100 U/ml of IFN-β (for 20 hours) under mock conditions or after infection with SARS-CoV-2 at MOI 0.1. β-actin – loading control. Data are representative of two independent infection experiments. ( E ) Levels of IFN-β in culture supernatants from control or ORAI1 -/- HEK293-ACE2 cells with or without pretreatment with 1μM cyclosporine A (CsA, 20 hours). P

    Journal: bioRxiv

    Article Title: ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling

    doi: 10.1101/2021.05.04.442548

    Figure Lengend Snippet: Loss of ORAI1 imparts susceptibility to SARS-CoV-2 infection by abrogating baseline interferon β levels. ( A ) Levels of IFNβ and IL-6 in culture supernatants from control or ORAI1 -/- HEK293-ACE2 cells under mock conditions or 20 hours after infection with SARS-CoV-2 at indicted MOIs. ( B ) Representative immunoblot showing expression of phosphorylated STAT1 (p-STAT1) or total STAT1 in lysates from control or ORAI1 -/- HEK293-ACE2 cells. β-actin – loading control. Data are representative of three independent experiments. ( C ) Quantitative RT-PCR analysis of viral genome from indicated cell types with or without pre-treatment with 100 U/ml of IFN-β (for 20 hours) under mock conditions or after infection with SARS-CoV-2 at MOI 0.1 for 20 h. ( D ) Representative immunoblot showing expression of SARS-CoV-2 proteins in indicated HEK293-ACE2 cells with or without pre-treatment with 100 U/ml of IFN-β (for 20 hours) under mock conditions or after infection with SARS-CoV-2 at MOI 0.1. β-actin – loading control. Data are representative of two independent infection experiments. ( E ) Levels of IFN-β in culture supernatants from control or ORAI1 -/- HEK293-ACE2 cells with or without pretreatment with 1μM cyclosporine A (CsA, 20 hours). P

    Article Snippet: Antibodies for detection of human STIM1 (5668S) was purchased from Cell Signaling Technologies, that for detection of interferon alpha and beta receptor 1 (IFNAR-1) was purchased from Leinco Technologies, Inc. (I-400), that for detection of surface ORAI1 (ACC-060) was purchased from Alomone labs and that for detection of β-actin (sc-47778), was obtained from Santa Cruz Biotechnology.

    Techniques: Infection, Expressing, Quantitative RT-PCR

    Loss of ORAI1 reduces basal Ca 2+ concentration and abrogates SOCE in HEK293-ACE2 cells ( A ) Representative immunoblot showing expression of STIM1 in lysates from control, ORAI1 -/- , or STIM1 -/- HEK293-ACE2 cells. β-actin – loading control. Data are representative of two independent experiments. ( B ) Representative histograms showing levels of total ORAI1 protein in control, ORAI1 -/- , and STIM1 -/- HEK293-ACE2 cells after permeabilization and intracellular staining with anti-ORAI1 antibody. The bar graph shows average (± s.e.m.) from three independent experiments. ( C ) Representative pseudocoloured epifluorescence images of indicated cells under resting conditions or at the peak of SOCE. Below - Representative traces showing averaged SOCE from control (39 cells), ORAI1 -/- (35 cells) and STIM1 -/- (42 cells) HEK293-ACE2 cells after passive depletion of intracellular Ca 2+ stores with thapsigargin (TG – 1 μM) in Ca 2+ -free external solution. SOCE was measured after replacing external solution with that containing 2 mM CaCl 2 . Bar graph on the right shows averaged baseline subtracted ER Ca 2+ stores (TG) and SOCE (± s.e.m.) from four independent experiments. ( D ) Bar graph shows baseline Ca 2+ levels (as depicted by Fura-2 ratio) ± s.e.m. in indicated cell types upon perfusion with extracellular solution containing either 2 mM or 20 mM CaCl 2 . Each dot represents data obtained from an independent experiment. *** P

    Journal: bioRxiv

    Article Title: ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling

    doi: 10.1101/2021.05.04.442548

    Figure Lengend Snippet: Loss of ORAI1 reduces basal Ca 2+ concentration and abrogates SOCE in HEK293-ACE2 cells ( A ) Representative immunoblot showing expression of STIM1 in lysates from control, ORAI1 -/- , or STIM1 -/- HEK293-ACE2 cells. β-actin – loading control. Data are representative of two independent experiments. ( B ) Representative histograms showing levels of total ORAI1 protein in control, ORAI1 -/- , and STIM1 -/- HEK293-ACE2 cells after permeabilization and intracellular staining with anti-ORAI1 antibody. The bar graph shows average (± s.e.m.) from three independent experiments. ( C ) Representative pseudocoloured epifluorescence images of indicated cells under resting conditions or at the peak of SOCE. Below - Representative traces showing averaged SOCE from control (39 cells), ORAI1 -/- (35 cells) and STIM1 -/- (42 cells) HEK293-ACE2 cells after passive depletion of intracellular Ca 2+ stores with thapsigargin (TG – 1 μM) in Ca 2+ -free external solution. SOCE was measured after replacing external solution with that containing 2 mM CaCl 2 . Bar graph on the right shows averaged baseline subtracted ER Ca 2+ stores (TG) and SOCE (± s.e.m.) from four independent experiments. ( D ) Bar graph shows baseline Ca 2+ levels (as depicted by Fura-2 ratio) ± s.e.m. in indicated cell types upon perfusion with extracellular solution containing either 2 mM or 20 mM CaCl 2 . Each dot represents data obtained from an independent experiment. *** P

    Article Snippet: Antibodies for detection of human STIM1 (5668S) was purchased from Cell Signaling Technologies, that for detection of interferon alpha and beta receptor 1 (IFNAR-1) was purchased from Leinco Technologies, Inc. (I-400), that for detection of surface ORAI1 (ACC-060) was purchased from Alomone labs and that for detection of β-actin (sc-47778), was obtained from Santa Cruz Biotechnology.

    Techniques: Concentration Assay, Expressing, Staining

    Loss of ORAI1 or STIM1 influences host resistance to vesicular stomatitis virus. ( A ) Representative immunoblot showing expression of STIM1 in lysates from control and STIM1 -/- A549 cells. β-actin – loading control. Data are representative of two independent experiments. ( B ) Representative histograms showing levels of total ORAI1 protein in control, ORAI1 -/- , and STIM1 -/- A549 cells after permeabilization and intracellular staining with anti-ORAI1 antibody. The bar graph shows average (± s.e.m.) from three independent experiments. ( C ) Representative flow plots showing frequency of GFP-positive population in VSV-GFP-infected (at indicated MOIs for 20 hours) control, ORAI1 -/- , or STIM1 -/- A549 cells. Bar graph (right) shows averaged frequency of VSV-GFP-positive populations from three independent experiments. *** P

    Journal: bioRxiv

    Article Title: ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling

    doi: 10.1101/2021.05.04.442548

    Figure Lengend Snippet: Loss of ORAI1 or STIM1 influences host resistance to vesicular stomatitis virus. ( A ) Representative immunoblot showing expression of STIM1 in lysates from control and STIM1 -/- A549 cells. β-actin – loading control. Data are representative of two independent experiments. ( B ) Representative histograms showing levels of total ORAI1 protein in control, ORAI1 -/- , and STIM1 -/- A549 cells after permeabilization and intracellular staining with anti-ORAI1 antibody. The bar graph shows average (± s.e.m.) from three independent experiments. ( C ) Representative flow plots showing frequency of GFP-positive population in VSV-GFP-infected (at indicated MOIs for 20 hours) control, ORAI1 -/- , or STIM1 -/- A549 cells. Bar graph (right) shows averaged frequency of VSV-GFP-positive populations from three independent experiments. *** P

    Article Snippet: Antibodies for detection of human STIM1 (5668S) was purchased from Cell Signaling Technologies, that for detection of interferon alpha and beta receptor 1 (IFNAR-1) was purchased from Leinco Technologies, Inc. (I-400), that for detection of surface ORAI1 (ACC-060) was purchased from Alomone labs and that for detection of β-actin (sc-47778), was obtained from Santa Cruz Biotechnology.

    Techniques: Expressing, Staining, Infection