Structured Review

Adipogen ionomycin
Lung injury, bacterial counts, and survival in NETosis-impaired mice. ( A – C ) Bone marrow neutrophils were isolated from WT or PAD4 –/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with <t>ionomycin</t> (4 μM) for 4 hours. ( B ) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA ( n = 4) and ( C ) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. ( D – K ) WT, PAD4 –/– , and PAD4 +/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). ( E ) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. ( F ) NETs (NE-DNA ELISA), ( G ) CitH3-DNA complexes, and ( H ) protein content were quantified in BAL. ( I ) Bacterial counts in the lung. ( J and K ) IL-6 and IL-1β concentration in BAL. ( B , F – K ) Data were analyzed using 1-way ANOVA ( n = 11–19). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ( L ) Survival curves for WT, PAD4 –/– , and PAD4 +/– littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test ( n = 10–20). ns, not significant.
Ionomycin, supplied by Adipogen, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury"

Article Title: Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury

Journal: JCI Insight

doi: 10.1172/jci.insight.98178

Lung injury, bacterial counts, and survival in NETosis-impaired mice. ( A – C ) Bone marrow neutrophils were isolated from WT or PAD4 –/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with ionomycin (4 μM) for 4 hours. ( B ) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA ( n = 4) and ( C ) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. ( D – K ) WT, PAD4 –/– , and PAD4 +/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). ( E ) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. ( F ) NETs (NE-DNA ELISA), ( G ) CitH3-DNA complexes, and ( H ) protein content were quantified in BAL. ( I ) Bacterial counts in the lung. ( J and K ) IL-6 and IL-1β concentration in BAL. ( B , F – K ) Data were analyzed using 1-way ANOVA ( n = 11–19). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ( L ) Survival curves for WT, PAD4 –/– , and PAD4 +/– littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test ( n = 10–20). ns, not significant.
Figure Legend Snippet: Lung injury, bacterial counts, and survival in NETosis-impaired mice. ( A – C ) Bone marrow neutrophils were isolated from WT or PAD4 –/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with ionomycin (4 μM) for 4 hours. ( B ) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA ( n = 4) and ( C ) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. ( D – K ) WT, PAD4 –/– , and PAD4 +/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). ( E ) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. ( F ) NETs (NE-DNA ELISA), ( G ) CitH3-DNA complexes, and ( H ) protein content were quantified in BAL. ( I ) Bacterial counts in the lung. ( J and K ) IL-6 and IL-1β concentration in BAL. ( B , F – K ) Data were analyzed using 1-way ANOVA ( n = 11–19). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ( L ) Survival curves for WT, PAD4 –/– , and PAD4 +/– littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test ( n = 10–20). ns, not significant.

Techniques Used: Mouse Assay, Isolation, In Vitro, Enzyme-linked Immunosorbent Assay, Immunofluorescence, In Vivo, Ex Vivo, Concentration Assay

2) Product Images from "CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A"

Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A

Journal: PLoS ONE

doi: 10.1371/journal.pone.0196512

CLCA2 enhances I ClCa in TMEM16A-transduced HEK293 cells. Representative whole-cell chloride currents from cells stably expressing vector (A), CLCA2 (B), TMEM16A plus vector (D) or CLCA2 plus TMEM16A (E) are shown. I CaCC was induced by application of 2 micromolar ionomycin in the presence of extracellular calcium. When the current peaked, a voltage ramp from -60 to +60mV was applied and the current response was recorded. In A and B, current was blocked by DIDS, 100 micromolar. In both D and E, current was fully inhibited by substitution of sodium gluconate (S.G.) for chloride. C, F, relative current amplitudes at +60mV +/- S.E.M. C, n = 9 for CLCA2; n = 7 for vector control; **, p
Figure Legend Snippet: CLCA2 enhances I ClCa in TMEM16A-transduced HEK293 cells. Representative whole-cell chloride currents from cells stably expressing vector (A), CLCA2 (B), TMEM16A plus vector (D) or CLCA2 plus TMEM16A (E) are shown. I CaCC was induced by application of 2 micromolar ionomycin in the presence of extracellular calcium. When the current peaked, a voltage ramp from -60 to +60mV was applied and the current response was recorded. In A and B, current was blocked by DIDS, 100 micromolar. In both D and E, current was fully inhibited by substitution of sodium gluconate (S.G.) for chloride. C, F, relative current amplitudes at +60mV +/- S.E.M. C, n = 9 for CLCA2; n = 7 for vector control; **, p

Techniques Used: Stable Transfection, Expressing, Plasmid Preparation

CLCA2 and TMEM16A do not directly interact. (A) Immunoblots of cells transfected with CLCA2, TMEM16A, or both. Proteins were immunoprecipitated and detected with antibodies specific for either protein. (B) Cells stably expressing vector or CLCA2 were transiently transfected with TMEM16A (TMEM) and treated 48h later with the protein cross-linker DSS, followed by immunoprecipitation and immunoblot. TMEM16A was detected with anti-Flag tag antibody, and CLCA2 was detected using TVE20 antibody. In indicated experiments, cells were treated with 1 micromolar ionomycin 5min before adding cross-linker. 1, position of protein monomer. For CLCA2, this includes the 130 kDa precursor and the 100 kDa N-terminal product. 2, apparent multimers. Right, size marker positions are indicated.
Figure Legend Snippet: CLCA2 and TMEM16A do not directly interact. (A) Immunoblots of cells transfected with CLCA2, TMEM16A, or both. Proteins were immunoprecipitated and detected with antibodies specific for either protein. (B) Cells stably expressing vector or CLCA2 were transiently transfected with TMEM16A (TMEM) and treated 48h later with the protein cross-linker DSS, followed by immunoprecipitation and immunoblot. TMEM16A was detected with anti-Flag tag antibody, and CLCA2 was detected using TVE20 antibody. In indicated experiments, cells were treated with 1 micromolar ionomycin 5min before adding cross-linker. 1, position of protein monomer. For CLCA2, this includes the 130 kDa precursor and the 100 kDa N-terminal product. 2, apparent multimers. Right, size marker positions are indicated.

Techniques Used: Western Blot, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Plasmid Preparation, FLAG-tag, Marker

CLCA2 enhances ER calcium stores and SOCE. Calcium imaging studies using HEK293 cells loaded with calcium fluorophore Fluo-4. (A) Cells were treated with 2 micromolar ionomycin in the absence of extracellular calcium to allow ER depletion, then calcium was added to allow SOCE. For CLCA2, n = 52; for control, n = 49. (B) CLCA2-expressing cells were treated with ionomycin, and the SOCE inhibitor BTP-2 (10 micromolar) was added after extracellular calcium (n = 121). Control cells expressed CLCA2 but were untreated with BTP-2 (n = 124). The temporal displacement in the SOCE peaks was due to a minor difference in timing of calcium addition. (C) The same cell lines were treated with SERCA inhibitor CPA (15 micromolar). For CLCA2, n = 246; for control, n = 240. (D) Bar graph showing enhancement by CLCA2 of cytosolic calcium from the ER (Peak 1) and SOCE (Peak 2) in A and C. The height of each peak minus background is plotted. P-values were determined for pairwise comparisons between CLCA2 and control peaks. For ionomycin, Peak 1 p = 0.001, and Peak 2 p = 4.58x10 -11 . For CPA, Peak 1, p = 2.63 x10 -21 , and Peak 2 p = 9.23 x10 -10 . For A and C, traces represent the mean of three independent experiments.
Figure Legend Snippet: CLCA2 enhances ER calcium stores and SOCE. Calcium imaging studies using HEK293 cells loaded with calcium fluorophore Fluo-4. (A) Cells were treated with 2 micromolar ionomycin in the absence of extracellular calcium to allow ER depletion, then calcium was added to allow SOCE. For CLCA2, n = 52; for control, n = 49. (B) CLCA2-expressing cells were treated with ionomycin, and the SOCE inhibitor BTP-2 (10 micromolar) was added after extracellular calcium (n = 121). Control cells expressed CLCA2 but were untreated with BTP-2 (n = 124). The temporal displacement in the SOCE peaks was due to a minor difference in timing of calcium addition. (C) The same cell lines were treated with SERCA inhibitor CPA (15 micromolar). For CLCA2, n = 246; for control, n = 240. (D) Bar graph showing enhancement by CLCA2 of cytosolic calcium from the ER (Peak 1) and SOCE (Peak 2) in A and C. The height of each peak minus background is plotted. P-values were determined for pairwise comparisons between CLCA2 and control peaks. For ionomycin, Peak 1 p = 0.001, and Peak 2 p = 4.58x10 -11 . For CPA, Peak 1, p = 2.63 x10 -21 , and Peak 2 p = 9.23 x10 -10 . For A and C, traces represent the mean of three independent experiments.

Techniques Used: Imaging, Expressing

3) Product Images from "VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation"

Article Title: VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation

Journal: FEBS Open Bio

doi: 10.1002/2211-5463.12918

Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of Slc17a9 in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 exposed to compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m ionomycin, 10 μ m brefeldin A, or 10 μ m clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2 N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n = 3). Statistical analysis was performed with unpaired t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P
Figure Legend Snippet: Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of Slc17a9 in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 exposed to compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m ionomycin, 10 μ m brefeldin A, or 10 μ m clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2 N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n = 3). Statistical analysis was performed with unpaired t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P

Techniques Used: Inhibition, Stable Transfection, Expressing, shRNA, Marker, Real-time Polymerase Chain Reaction, Activity Assay, Centrifugation

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Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A
Article Snippet: Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.

Cell Culture:

Article Title: Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury
Article Snippet: .. Neutrophils were cultured at 37°C and 5% CO2 and stimulated with either 4 μM ionomycin (Adipogen) for 4 hours, 100 nM PMA (Sigma-Aldrich) for 4 hours, or MRSA (108 CFU/ml) for 20 minutes. .. In selected experiments, neutrophils were pretreated for 1 hour with Cl-amidine (200 μM) or lipoxin A4 (EMD Millipore, 300 nM).

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    Adipogen reagents ionomycin
    CLCA2 enhances I ClCa in TMEM16A-transduced HEK293 cells. Representative whole-cell chloride currents from cells stably expressing vector (A), CLCA2 (B), TMEM16A plus vector (D) or CLCA2 plus TMEM16A (E) are shown. I CaCC was induced by application of 2 micromolar <t>ionomycin</t> in the presence of extracellular calcium. When the current peaked, a voltage ramp from -60 to +60mV was applied and the current response was recorded. In A and B, current was blocked by DIDS, 100 micromolar. In both D and E, current was fully inhibited by substitution of sodium gluconate (S.G.) for chloride. C, F, relative current amplitudes at +60mV +/- S.E.M. C, n = 9 for CLCA2; n = 7 for vector control; **, p
    Reagents Ionomycin, supplied by Adipogen, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reagents ionomycin/product/Adipogen
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    reagents ionomycin - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    92
    Adipogen ionomycin
    Lung injury, bacterial counts, and survival in NETosis-impaired mice. ( A – C ) Bone marrow neutrophils were isolated from WT or PAD4 –/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with <t>ionomycin</t> (4 μM) for 4 hours. ( B ) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA ( n = 4) and ( C ) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. ( D – K ) WT, PAD4 –/– , and PAD4 +/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). ( E ) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. ( F ) NETs (NE-DNA ELISA), ( G ) CitH3-DNA complexes, and ( H ) protein content were quantified in BAL. ( I ) Bacterial counts in the lung. ( J and K ) IL-6 and IL-1β concentration in BAL. ( B , F – K ) Data were analyzed using 1-way ANOVA ( n = 11–19). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ( L ) Survival curves for WT, PAD4 –/– , and PAD4 +/– littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test ( n = 10–20). ns, not significant.
    Ionomycin, supplied by Adipogen, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/Adipogen
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ionomycin - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    CLCA2 enhances I ClCa in TMEM16A-transduced HEK293 cells. Representative whole-cell chloride currents from cells stably expressing vector (A), CLCA2 (B), TMEM16A plus vector (D) or CLCA2 plus TMEM16A (E) are shown. I CaCC was induced by application of 2 micromolar ionomycin in the presence of extracellular calcium. When the current peaked, a voltage ramp from -60 to +60mV was applied and the current response was recorded. In A and B, current was blocked by DIDS, 100 micromolar. In both D and E, current was fully inhibited by substitution of sodium gluconate (S.G.) for chloride. C, F, relative current amplitudes at +60mV +/- S.E.M. C, n = 9 for CLCA2; n = 7 for vector control; **, p

    Journal: PLoS ONE

    Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A

    doi: 10.1371/journal.pone.0196512

    Figure Lengend Snippet: CLCA2 enhances I ClCa in TMEM16A-transduced HEK293 cells. Representative whole-cell chloride currents from cells stably expressing vector (A), CLCA2 (B), TMEM16A plus vector (D) or CLCA2 plus TMEM16A (E) are shown. I CaCC was induced by application of 2 micromolar ionomycin in the presence of extracellular calcium. When the current peaked, a voltage ramp from -60 to +60mV was applied and the current response was recorded. In A and B, current was blocked by DIDS, 100 micromolar. In both D and E, current was fully inhibited by substitution of sodium gluconate (S.G.) for chloride. C, F, relative current amplitudes at +60mV +/- S.E.M. C, n = 9 for CLCA2; n = 7 for vector control; **, p

    Article Snippet: 2.1 Reagents Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.

    Techniques: Stable Transfection, Expressing, Plasmid Preparation

    CLCA2 and TMEM16A do not directly interact. (A) Immunoblots of cells transfected with CLCA2, TMEM16A, or both. Proteins were immunoprecipitated and detected with antibodies specific for either protein. (B) Cells stably expressing vector or CLCA2 were transiently transfected with TMEM16A (TMEM) and treated 48h later with the protein cross-linker DSS, followed by immunoprecipitation and immunoblot. TMEM16A was detected with anti-Flag tag antibody, and CLCA2 was detected using TVE20 antibody. In indicated experiments, cells were treated with 1 micromolar ionomycin 5min before adding cross-linker. 1, position of protein monomer. For CLCA2, this includes the 130 kDa precursor and the 100 kDa N-terminal product. 2, apparent multimers. Right, size marker positions are indicated.

    Journal: PLoS ONE

    Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A

    doi: 10.1371/journal.pone.0196512

    Figure Lengend Snippet: CLCA2 and TMEM16A do not directly interact. (A) Immunoblots of cells transfected with CLCA2, TMEM16A, or both. Proteins were immunoprecipitated and detected with antibodies specific for either protein. (B) Cells stably expressing vector or CLCA2 were transiently transfected with TMEM16A (TMEM) and treated 48h later with the protein cross-linker DSS, followed by immunoprecipitation and immunoblot. TMEM16A was detected with anti-Flag tag antibody, and CLCA2 was detected using TVE20 antibody. In indicated experiments, cells were treated with 1 micromolar ionomycin 5min before adding cross-linker. 1, position of protein monomer. For CLCA2, this includes the 130 kDa precursor and the 100 kDa N-terminal product. 2, apparent multimers. Right, size marker positions are indicated.

    Article Snippet: 2.1 Reagents Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.

    Techniques: Western Blot, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Plasmid Preparation, FLAG-tag, Marker

    Effect of CLCA2 knockdown on calcium trafficking. (A) Calcium imaging in MCF10A mammary epithelial cells with CLCA2 knockdown (shRNA; n = 134) or vector (control; n = 196). Cells were treated with ionomycin as in Fig 1 except that calcium was withdrawn after appearance of Peak 2 as indicated by bars at top. (B) The height of each peak minus background is plotted. Peak 1, p = 1.77 x10 -9 . Peak 2, p = 6.10 x10 -18 .

    Journal: PLoS ONE

    Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A

    doi: 10.1371/journal.pone.0196512

    Figure Lengend Snippet: Effect of CLCA2 knockdown on calcium trafficking. (A) Calcium imaging in MCF10A mammary epithelial cells with CLCA2 knockdown (shRNA; n = 134) or vector (control; n = 196). Cells were treated with ionomycin as in Fig 1 except that calcium was withdrawn after appearance of Peak 2 as indicated by bars at top. (B) The height of each peak minus background is plotted. Peak 1, p = 1.77 x10 -9 . Peak 2, p = 6.10 x10 -18 .

    Article Snippet: 2.1 Reagents Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.

    Techniques: Imaging, shRNA, Plasmid Preparation

    CLCA2 enhances ER calcium stores and SOCE. Calcium imaging studies using HEK293 cells loaded with calcium fluorophore Fluo-4. (A) Cells were treated with 2 micromolar ionomycin in the absence of extracellular calcium to allow ER depletion, then calcium was added to allow SOCE. For CLCA2, n = 52; for control, n = 49. (B) CLCA2-expressing cells were treated with ionomycin, and the SOCE inhibitor BTP-2 (10 micromolar) was added after extracellular calcium (n = 121). Control cells expressed CLCA2 but were untreated with BTP-2 (n = 124). The temporal displacement in the SOCE peaks was due to a minor difference in timing of calcium addition. (C) The same cell lines were treated with SERCA inhibitor CPA (15 micromolar). For CLCA2, n = 246; for control, n = 240. (D) Bar graph showing enhancement by CLCA2 of cytosolic calcium from the ER (Peak 1) and SOCE (Peak 2) in A and C. The height of each peak minus background is plotted. P-values were determined for pairwise comparisons between CLCA2 and control peaks. For ionomycin, Peak 1 p = 0.001, and Peak 2 p = 4.58x10 -11 . For CPA, Peak 1, p = 2.63 x10 -21 , and Peak 2 p = 9.23 x10 -10 . For A and C, traces represent the mean of three independent experiments.

    Journal: PLoS ONE

    Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A

    doi: 10.1371/journal.pone.0196512

    Figure Lengend Snippet: CLCA2 enhances ER calcium stores and SOCE. Calcium imaging studies using HEK293 cells loaded with calcium fluorophore Fluo-4. (A) Cells were treated with 2 micromolar ionomycin in the absence of extracellular calcium to allow ER depletion, then calcium was added to allow SOCE. For CLCA2, n = 52; for control, n = 49. (B) CLCA2-expressing cells were treated with ionomycin, and the SOCE inhibitor BTP-2 (10 micromolar) was added after extracellular calcium (n = 121). Control cells expressed CLCA2 but were untreated with BTP-2 (n = 124). The temporal displacement in the SOCE peaks was due to a minor difference in timing of calcium addition. (C) The same cell lines were treated with SERCA inhibitor CPA (15 micromolar). For CLCA2, n = 246; for control, n = 240. (D) Bar graph showing enhancement by CLCA2 of cytosolic calcium from the ER (Peak 1) and SOCE (Peak 2) in A and C. The height of each peak minus background is plotted. P-values were determined for pairwise comparisons between CLCA2 and control peaks. For ionomycin, Peak 1 p = 0.001, and Peak 2 p = 4.58x10 -11 . For CPA, Peak 1, p = 2.63 x10 -21 , and Peak 2 p = 9.23 x10 -10 . For A and C, traces represent the mean of three independent experiments.

    Article Snippet: 2.1 Reagents Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.

    Techniques: Imaging, Expressing

    A second family member, CLCA1, also enhances ER calcium release and SOCE. (A) Cells stably expressing CLCA1 (n = 228 cells) or vector (n = 219) and loaded with Fluo-4 were treated with ionomycin and cytosolic calcium was measured over time as in Fig 2 . (B) Amplitudes of peaks 1 and 2 were plotted. For pairwise comparisons between CLCA1 and vector, the p-value of Peak 1 was 3.2 x10 -87 and the p-value of Peak 2 was 5.07 x10 -84 .

    Journal: PLoS ONE

    Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A

    doi: 10.1371/journal.pone.0196512

    Figure Lengend Snippet: A second family member, CLCA1, also enhances ER calcium release and SOCE. (A) Cells stably expressing CLCA1 (n = 228 cells) or vector (n = 219) and loaded with Fluo-4 were treated with ionomycin and cytosolic calcium was measured over time as in Fig 2 . (B) Amplitudes of peaks 1 and 2 were plotted. For pairwise comparisons between CLCA1 and vector, the p-value of Peak 1 was 3.2 x10 -87 and the p-value of Peak 2 was 5.07 x10 -84 .

    Article Snippet: 2.1 Reagents Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.

    Techniques: Stable Transfection, Expressing, Plasmid Preparation

    Lung injury, bacterial counts, and survival in NETosis-impaired mice. ( A – C ) Bone marrow neutrophils were isolated from WT or PAD4 –/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with ionomycin (4 μM) for 4 hours. ( B ) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA ( n = 4) and ( C ) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. ( D – K ) WT, PAD4 –/– , and PAD4 +/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). ( E ) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. ( F ) NETs (NE-DNA ELISA), ( G ) CitH3-DNA complexes, and ( H ) protein content were quantified in BAL. ( I ) Bacterial counts in the lung. ( J and K ) IL-6 and IL-1β concentration in BAL. ( B , F – K ) Data were analyzed using 1-way ANOVA ( n = 11–19). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ( L ) Survival curves for WT, PAD4 –/– , and PAD4 +/– littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test ( n = 10–20). ns, not significant.

    Journal: JCI Insight

    Article Title: Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury

    doi: 10.1172/jci.insight.98178

    Figure Lengend Snippet: Lung injury, bacterial counts, and survival in NETosis-impaired mice. ( A – C ) Bone marrow neutrophils were isolated from WT or PAD4 –/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with ionomycin (4 μM) for 4 hours. ( B ) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA ( n = 4) and ( C ) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. ( D – K ) WT, PAD4 –/– , and PAD4 +/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10 7 CFU, i.t.). ( E ) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. ( F ) NETs (NE-DNA ELISA), ( G ) CitH3-DNA complexes, and ( H ) protein content were quantified in BAL. ( I ) Bacterial counts in the lung. ( J and K ) IL-6 and IL-1β concentration in BAL. ( B , F – K ) Data were analyzed using 1-way ANOVA ( n = 11–19). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. ( L ) Survival curves for WT, PAD4 –/– , and PAD4 +/– littermates challenged with MRSA (1 × 10 8 CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test ( n = 10–20). ns, not significant.

    Article Snippet: Neutrophils were cultured at 37°C and 5% CO2 and stimulated with either 4 μM ionomycin (Adipogen) for 4 hours, 100 nM PMA (Sigma-Aldrich) for 4 hours, or MRSA (108 CFU/ml) for 20 minutes.

    Techniques: Mouse Assay, Isolation, In Vitro, Enzyme-linked Immunosorbent Assay, Immunofluorescence, In Vivo, Ex Vivo, Concentration Assay

    CLCA2 enhances I ClCa in TMEM16A-transduced HEK293 cells. Representative whole-cell chloride currents from cells stably expressing vector (A), CLCA2 (B), TMEM16A plus vector (D) or CLCA2 plus TMEM16A (E) are shown. I CaCC was induced by application of 2 micromolar ionomycin in the presence of extracellular calcium. When the current peaked, a voltage ramp from -60 to +60mV was applied and the current response was recorded. In A and B, current was blocked by DIDS, 100 micromolar. In both D and E, current was fully inhibited by substitution of sodium gluconate (S.G.) for chloride. C, F, relative current amplitudes at +60mV +/- S.E.M. C, n = 9 for CLCA2; n = 7 for vector control; **, p

    Journal: PLoS ONE

    Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A

    doi: 10.1371/journal.pone.0196512

    Figure Lengend Snippet: CLCA2 enhances I ClCa in TMEM16A-transduced HEK293 cells. Representative whole-cell chloride currents from cells stably expressing vector (A), CLCA2 (B), TMEM16A plus vector (D) or CLCA2 plus TMEM16A (E) are shown. I CaCC was induced by application of 2 micromolar ionomycin in the presence of extracellular calcium. When the current peaked, a voltage ramp from -60 to +60mV was applied and the current response was recorded. In A and B, current was blocked by DIDS, 100 micromolar. In both D and E, current was fully inhibited by substitution of sodium gluconate (S.G.) for chloride. C, F, relative current amplitudes at +60mV +/- S.E.M. C, n = 9 for CLCA2; n = 7 for vector control; **, p

    Article Snippet: Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.

    Techniques: Stable Transfection, Expressing, Plasmid Preparation

    CLCA2 and TMEM16A do not directly interact. (A) Immunoblots of cells transfected with CLCA2, TMEM16A, or both. Proteins were immunoprecipitated and detected with antibodies specific for either protein. (B) Cells stably expressing vector or CLCA2 were transiently transfected with TMEM16A (TMEM) and treated 48h later with the protein cross-linker DSS, followed by immunoprecipitation and immunoblot. TMEM16A was detected with anti-Flag tag antibody, and CLCA2 was detected using TVE20 antibody. In indicated experiments, cells were treated with 1 micromolar ionomycin 5min before adding cross-linker. 1, position of protein monomer. For CLCA2, this includes the 130 kDa precursor and the 100 kDa N-terminal product. 2, apparent multimers. Right, size marker positions are indicated.

    Journal: PLoS ONE

    Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A

    doi: 10.1371/journal.pone.0196512

    Figure Lengend Snippet: CLCA2 and TMEM16A do not directly interact. (A) Immunoblots of cells transfected with CLCA2, TMEM16A, or both. Proteins were immunoprecipitated and detected with antibodies specific for either protein. (B) Cells stably expressing vector or CLCA2 were transiently transfected with TMEM16A (TMEM) and treated 48h later with the protein cross-linker DSS, followed by immunoprecipitation and immunoblot. TMEM16A was detected with anti-Flag tag antibody, and CLCA2 was detected using TVE20 antibody. In indicated experiments, cells were treated with 1 micromolar ionomycin 5min before adding cross-linker. 1, position of protein monomer. For CLCA2, this includes the 130 kDa precursor and the 100 kDa N-terminal product. 2, apparent multimers. Right, size marker positions are indicated.

    Article Snippet: Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.

    Techniques: Western Blot, Transfection, Immunoprecipitation, Stable Transfection, Expressing, Plasmid Preparation, FLAG-tag, Marker

    CLCA2 enhances ER calcium stores and SOCE. Calcium imaging studies using HEK293 cells loaded with calcium fluorophore Fluo-4. (A) Cells were treated with 2 micromolar ionomycin in the absence of extracellular calcium to allow ER depletion, then calcium was added to allow SOCE. For CLCA2, n = 52; for control, n = 49. (B) CLCA2-expressing cells were treated with ionomycin, and the SOCE inhibitor BTP-2 (10 micromolar) was added after extracellular calcium (n = 121). Control cells expressed CLCA2 but were untreated with BTP-2 (n = 124). The temporal displacement in the SOCE peaks was due to a minor difference in timing of calcium addition. (C) The same cell lines were treated with SERCA inhibitor CPA (15 micromolar). For CLCA2, n = 246; for control, n = 240. (D) Bar graph showing enhancement by CLCA2 of cytosolic calcium from the ER (Peak 1) and SOCE (Peak 2) in A and C. The height of each peak minus background is plotted. P-values were determined for pairwise comparisons between CLCA2 and control peaks. For ionomycin, Peak 1 p = 0.001, and Peak 2 p = 4.58x10 -11 . For CPA, Peak 1, p = 2.63 x10 -21 , and Peak 2 p = 9.23 x10 -10 . For A and C, traces represent the mean of three independent experiments.

    Journal: PLoS ONE

    Article Title: CLCA2 is a positive regulator of store-operated calcium entry and TMEM16A

    doi: 10.1371/journal.pone.0196512

    Figure Lengend Snippet: CLCA2 enhances ER calcium stores and SOCE. Calcium imaging studies using HEK293 cells loaded with calcium fluorophore Fluo-4. (A) Cells were treated with 2 micromolar ionomycin in the absence of extracellular calcium to allow ER depletion, then calcium was added to allow SOCE. For CLCA2, n = 52; for control, n = 49. (B) CLCA2-expressing cells were treated with ionomycin, and the SOCE inhibitor BTP-2 (10 micromolar) was added after extracellular calcium (n = 121). Control cells expressed CLCA2 but were untreated with BTP-2 (n = 124). The temporal displacement in the SOCE peaks was due to a minor difference in timing of calcium addition. (C) The same cell lines were treated with SERCA inhibitor CPA (15 micromolar). For CLCA2, n = 246; for control, n = 240. (D) Bar graph showing enhancement by CLCA2 of cytosolic calcium from the ER (Peak 1) and SOCE (Peak 2) in A and C. The height of each peak minus background is plotted. P-values were determined for pairwise comparisons between CLCA2 and control peaks. For ionomycin, Peak 1 p = 0.001, and Peak 2 p = 4.58x10 -11 . For CPA, Peak 1, p = 2.63 x10 -21 , and Peak 2 p = 9.23 x10 -10 . For A and C, traces represent the mean of three independent experiments.

    Article Snippet: Ionomycin was purchased from Adipogen; Cyclopiazonic Acid (CPA) was obtained from Tocris; Pluronic-F12 from Biotium and Life Technologies; Fluo4-AM from Life Technologies; BTP-2 from EMD-Millipore; 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) from Sigma.

    Techniques: Imaging, Expressing

    Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of Slc17a9 in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 exposed to compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m ionomycin, 10 μ m brefeldin A, or 10 μ m clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2 N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n = 3). Statistical analysis was performed with unpaired t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P

    Journal: FEBS Open Bio

    Article Title: VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation

    doi: 10.1002/2211-5463.12918

    Figure Lengend Snippet: Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of Slc17a9 in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 exposed to compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m ionomycin, 10 μ m brefeldin A, or 10 μ m clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2 N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n = 3). Statistical analysis was performed with unpaired t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P

    Article Snippet: Ionomycin (AdipoGen Life Sciences, San Diego, CA, USA) treatments were performed at 1 μm .

    Techniques: Inhibition, Stable Transfection, Expressing, shRNA, Marker, Real-time Polymerase Chain Reaction, Activity Assay, Centrifugation