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  • 96
    Millipore xestospongin c
    Communicating Ca 2+ spikes in AMs ( a ) Fluorescent LPS (green), AM (red) and DC (blue) in interstitium (Int) and alveolar lumen (Alv). n = 3 ( b ) YFP-expressing AMs (yellow; topmost panel), pseudocolored sequential images show increased fluo-4 fluorescence (arrowheads), 24 h after LPS. Dashes sketch spike path between AMs. Tracings show synchronous AM responses (arrows). n = 4 ( c ) Ca 2+ spikes normalized for 10 AMs per imaging field. Total, all spikes; Sync, Synchronous spikes; BL, baseline (n = 8); 1h, n = 6; 4h, n = 4; 24h, n = 12 ( d) Ca 2+ oscillations and spikes (arrows) in AMs and adjoining alveolar epithelium (AE) 24 h after LPS or PBS. GAP , GAP26/27. n = 4 ( e ) Ca 2+ spikes are 24h after LPS, normalized for 10 AMs per imaging field. ; Ctl , LPS alone (n = 12); PP , PPADS (n = 4); XeC, <t>xestospongin</t> C (n = 4); Ca 0 , Ca 2+ depletion (n = 3). GAP, n = 5. Scale bars, 20 μm. Bars, mean±SEM. *P
    Xestospongin C, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cayman Chemical xestospongin c
    Communicating Ca 2+ spikes in AMs ( a ) Fluorescent LPS (green), AM (red) and DC (blue) in interstitium (Int) and alveolar lumen (Alv). n = 3 ( b ) YFP-expressing AMs (yellow; topmost panel), pseudocolored sequential images show increased fluo-4 fluorescence (arrowheads), 24 h after LPS. Dashes sketch spike path between AMs. Tracings show synchronous AM responses (arrows). n = 4 ( c ) Ca 2+ spikes normalized for 10 AMs per imaging field. Total, all spikes; Sync, Synchronous spikes; BL, baseline (n = 8); 1h, n = 6; 4h, n = 4; 24h, n = 12 ( d) Ca 2+ oscillations and spikes (arrows) in AMs and adjoining alveolar epithelium (AE) 24 h after LPS or PBS. GAP , GAP26/27. n = 4 ( e ) Ca 2+ spikes are 24h after LPS, normalized for 10 AMs per imaging field. ; Ctl , LPS alone (n = 12); PP , PPADS (n = 4); XeC, <t>xestospongin</t> C (n = 4); Ca 0 , Ca 2+ depletion (n = 3). GAP, n = 5. Scale bars, 20 μm. Bars, mean±SEM. *P
    Xestospongin C, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 95/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris xestospongin c
    Communicating Ca 2+ spikes in AMs ( a ) Fluorescent LPS (green), AM (red) and DC (blue) in interstitium (Int) and alveolar lumen (Alv). n = 3 ( b ) YFP-expressing AMs (yellow; topmost panel), pseudocolored sequential images show increased fluo-4 fluorescence (arrowheads), 24 h after LPS. Dashes sketch spike path between AMs. Tracings show synchronous AM responses (arrows). n = 4 ( c ) Ca 2+ spikes normalized for 10 AMs per imaging field. Total, all spikes; Sync, Synchronous spikes; BL, baseline (n = 8); 1h, n = 6; 4h, n = 4; 24h, n = 12 ( d) Ca 2+ oscillations and spikes (arrows) in AMs and adjoining alveolar epithelium (AE) 24 h after LPS or PBS. GAP , GAP26/27. n = 4 ( e ) Ca 2+ spikes are 24h after LPS, normalized for 10 AMs per imaging field. ; Ctl , LPS alone (n = 12); PP , PPADS (n = 4); XeC, <t>xestospongin</t> C (n = 4); Ca 0 , Ca 2+ depletion (n = 3). GAP, n = 5. Scale bars, 20 μm. Bars, mean±SEM. *P
    Xestospongin C, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam xestospongin c
    Cytosolic Ca 2+ and PKC regulate CLSs containing EGFR but not those harboring TfR. RPE cells stably expressing clathrin light chain fused to eGFP (eGFP-CLCa), treated with various inhibitors as in Figures 3 and 4: 3 μM <t>XeC</t> for 30 min, 10 μM BAPTA-AM (BA) for 15 min, 1 μM <t>BIM</t> for 30 min, or left untreated (control), and then treated with A555-EGF (A) or A555-Tfn (B) for 5 min. Shown are representative micrographs obtained by TIRF-M. Scale bar: 5 μm. Full image panels are shown in Supplemental Figure 7. (C–F) TIRF-M images were subjected to automated detection of CLSs, followed by quantification of mean A555-conjugated ligand fluorescence intensity therein (C, E). CLSs were sorted into A555-EGF-enriched or A555-Tfn-enriched cohorts, followed by quantification of the mean eGFP-CLC within each CLS cohort (D, F). For C–F, the overall median of the cellular means (bar) and 25th/75th percentiles (boxes) are shown. The number of CLSs and cells analyzed, respectively, for each condition are as follows: EGF control: 14,114 and 114; EGF XeC: 11,322 and 98; EGF BAPTA-AM: 15,786 and 104; EGF BIM: 3860 and 46, Tfn control: 16,983 and 117; Tfn XeC: 10,838 and 98; Tfn BAPTA-AM: 8868 and 64; Tfn BIM: 3718 and 44; from a minimum of three independent experiments in each condition.
    Xestospongin C, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    FUJIFILM xestospongin c
    Effects of <t>xestospongin</t> C on progesterone‐enhanced hyperactivation. After exposure to xestospongin C for 5 min, spermatozoa were exposed to progesterone. The percentages of motile spermatozoa ( a ) and hyperactivated spermatozoa ( b ) are shown when 500 nM or 1 μM xestospongin C were added to the mTALP medium. The percentages of motile spermatozoa ( c ) and hyperactivated spermatozoa ( d ) are shown when 500 nM or 1 μM xestospongin C and 20 ng/ml progesterone were added to the mTALP medium. Data are expressed as mean ± SD. In a and b (Vehicle), mTALP + 0.1 % (v/v) DMSO; (500 nM Xestospongin C), mTALP + 500 nM xestospongin C + 0.1 % (v/v) DMSO; (1 μM xestospongin C), mTALP + 1 μM xestospongin C + 0.1 % (v/v) DMSO. In c and d (Vehicle), mTALP + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P), mTALP + 20 ng/ml progesterone + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; ( P + 500 nM Xestospongin C), mTALP + 20 ng/ml progesterone + 500 nM xestospongin C + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; ( P + 1 μM Xestospongin C), mTALP + 20 ng/ml progesterone + 1 μM xestospongin C + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO. a Significant difference compared with “Vehicle” and “ P + 1 μM Xestospongin C” ( P
    Xestospongin C, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem xestospongin c
    Long-lasting increase of fIPSPs after DHPG application is mediated by IP 3 receptors activation. DHPG failed to increase fIPSPs in all CA1 (a) and specifically in its different sub-regions (b–g) in presence of the cell-permeable IP 3 receptors blockers 2-APB or <t>Xestospongin</t> C (XestoC). In all figures, left panel shows the time course, while right panel is showing mean over time. Statistical analysis in right panel figures is one-way ANOVA followed by Dunnet post-hoc test. Vehicles of both IP 3 receptors antagonists have been pooled together since they are not different. n = (slices, mice): Vehicle group = (11, 9), 2-APB group = (5, 5), Xestospongin C group (5, 3). Data are mean ± s.e.m. *p
    Xestospongin C, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AG Scientific xestospongin c
    Long-lasting increase of fIPSPs after DHPG application is mediated by IP 3 receptors activation. DHPG failed to increase fIPSPs in all CA1 (a) and specifically in its different sub-regions (b–g) in presence of the cell-permeable IP 3 receptors blockers 2-APB or <t>Xestospongin</t> C (XestoC). In all figures, left panel shows the time course, while right panel is showing mean over time. Statistical analysis in right panel figures is one-way ANOVA followed by Dunnet post-hoc test. Vehicles of both IP 3 receptors antagonists have been pooled together since they are not different. n = (slices, mice): Vehicle group = (11, 9), 2-APB group = (5, 5), Xestospongin C group (5, 3). Data are mean ± s.e.m. *p
    Xestospongin C, supplied by AG Scientific, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA xestospongin c
    After cells were treated with 1.2% isoflurane for 8 h, cytosolic calcium concentration ([Ca 2+ ] c ) was determined by flow cytometry. Briefly, the cells were harvested, stained with Fluo-3/AM and were then analyzed. (A-C) Representative results from one of six independent experiments on mutated β-amyloid precursor protein (APP)-transfected SH-SY5Y cells treated with (A) the control condition (Con), (B) <t>xestospongin</t> C plus isoflurane (Xes) and (C) isoflurane (Iso). (D-F) Representative results from vector-transfected SH-SY5Y cells treated with (D) Con, (E) Xes and (F) Iso. The positive rate of [Ca 2+ ] c loading with Fluo-3/AM was > 99%. (A-F) X-axis refers to the average value of calcium fluorescence intensity, and Y-axis refers to cell count. (G) Data are presented as the mean ± standard deviation of at least three separate experiments. *P
    Xestospongin C, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Focus Biomolecules xestospongin c
    After cells were treated with 1.2% isoflurane for 8 h, cytosolic calcium concentration ([Ca 2+ ] c ) was determined by flow cytometry. Briefly, the cells were harvested, stained with Fluo-3/AM and were then analyzed. (A-C) Representative results from one of six independent experiments on mutated β-amyloid precursor protein (APP)-transfected SH-SY5Y cells treated with (A) the control condition (Con), (B) <t>xestospongin</t> C plus isoflurane (Xes) and (C) isoflurane (Iso). (D-F) Representative results from vector-transfected SH-SY5Y cells treated with (D) Con, (E) Xes and (F) Iso. The positive rate of [Ca 2+ ] c loading with Fluo-3/AM was > 99%. (A-F) X-axis refers to the average value of calcium fluorescence intensity, and Y-axis refers to cell count. (G) Data are presented as the mean ± standard deviation of at least three separate experiments. *P
    Xestospongin C, supplied by Focus Biomolecules, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co xestospongin c
    After cells were treated with 1.2% isoflurane for 8 h, cytosolic calcium concentration ([Ca 2+ ] c ) was determined by flow cytometry. Briefly, the cells were harvested, stained with Fluo-3/AM and were then analyzed. (A-C) Representative results from one of six independent experiments on mutated β-amyloid precursor protein (APP)-transfected SH-SY5Y cells treated with (A) the control condition (Con), (B) <t>xestospongin</t> C plus isoflurane (Xes) and (C) isoflurane (Iso). (D-F) Representative results from vector-transfected SH-SY5Y cells treated with (D) Con, (E) Xes and (F) Iso. The positive rate of [Ca 2+ ] c loading with Fluo-3/AM was > 99%. (A-F) X-axis refers to the average value of calcium fluorescence intensity, and Y-axis refers to cell count. (G) Data are presented as the mean ± standard deviation of at least three separate experiments. *P
    Xestospongin C, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    Xestospongin C is a marine natural product isolated from Pacific basin sponges it antagonizes the calcium releasing action of inositol 1 4 5 trisphosphate IP3 at the receptor level Xestospongin
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    N/A
    Xestospongin C is a marine natural product which was first isolated from Pacific basin sponges and noted to have vasodilatory properties Xestospongin C antagonizes the calcium releasing action of inositol
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    N/A
    Xestospongin C is a Xestospongin which represents a class of potent membrane permeable IP3 receptor blockers exhibiting a high selectivity over ryanodine receptors Xestospongin C is a marine alkaloid isolated
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    Image Search Results


    Communicating Ca 2+ spikes in AMs ( a ) Fluorescent LPS (green), AM (red) and DC (blue) in interstitium (Int) and alveolar lumen (Alv). n = 3 ( b ) YFP-expressing AMs (yellow; topmost panel), pseudocolored sequential images show increased fluo-4 fluorescence (arrowheads), 24 h after LPS. Dashes sketch spike path between AMs. Tracings show synchronous AM responses (arrows). n = 4 ( c ) Ca 2+ spikes normalized for 10 AMs per imaging field. Total, all spikes; Sync, Synchronous spikes; BL, baseline (n = 8); 1h, n = 6; 4h, n = 4; 24h, n = 12 ( d) Ca 2+ oscillations and spikes (arrows) in AMs and adjoining alveolar epithelium (AE) 24 h after LPS or PBS. GAP , GAP26/27. n = 4 ( e ) Ca 2+ spikes are 24h after LPS, normalized for 10 AMs per imaging field. ; Ctl , LPS alone (n = 12); PP , PPADS (n = 4); XeC, xestospongin C (n = 4); Ca 0 , Ca 2+ depletion (n = 3). GAP, n = 5. Scale bars, 20 μm. Bars, mean±SEM. *P

    Journal: Nature

    Article Title: Sessile alveolar macrophages modulate immunity through connexin 43-based epithelial communication

    doi: 10.1038/nature12902

    Figure Lengend Snippet: Communicating Ca 2+ spikes in AMs ( a ) Fluorescent LPS (green), AM (red) and DC (blue) in interstitium (Int) and alveolar lumen (Alv). n = 3 ( b ) YFP-expressing AMs (yellow; topmost panel), pseudocolored sequential images show increased fluo-4 fluorescence (arrowheads), 24 h after LPS. Dashes sketch spike path between AMs. Tracings show synchronous AM responses (arrows). n = 4 ( c ) Ca 2+ spikes normalized for 10 AMs per imaging field. Total, all spikes; Sync, Synchronous spikes; BL, baseline (n = 8); 1h, n = 6; 4h, n = 4; 24h, n = 12 ( d) Ca 2+ oscillations and spikes (arrows) in AMs and adjoining alveolar epithelium (AE) 24 h after LPS or PBS. GAP , GAP26/27. n = 4 ( e ) Ca 2+ spikes are 24h after LPS, normalized for 10 AMs per imaging field. ; Ctl , LPS alone (n = 12); PP , PPADS (n = 4); XeC, xestospongin C (n = 4); Ca 0 , Ca 2+ depletion (n = 3). GAP, n = 5. Scale bars, 20 μm. Bars, mean±SEM. *P

    Article Snippet: Reagents We purchased o -Nitrophenyl EGTA (NP-EGTA) (100 μM) and BAPTA (100 μM) from Invitrogen, Gap 27 (500 μM) from Tocris, Gap 26 (200 μM) from Alpha Diagnostics, xestospongin C (25 μM) from Calbiochem, Saponin (0.01 %) from ICN Biomedicals, Tween 20 (0.5%) from BIO-RAD and LPS (E.coli 0111:B4), fluorescent LPS (E.coli 0111:B4), Triton X-100 (0.2%), PPADS (100 μM), and doxycycline from Sigma.

    Techniques: Affinity Magnetic Separation, Expressing, Fluorescence, Imaging, CTL Assay

    Inhibition of IP3R by xestospongin C (XeC) or MCU by ruthenium red (RuR) reverses the opening of mPTP in Cdk5 −/− MEFs. a Wt and Cdk5 −/− MEFs pretreated with or without XeC (3 µM) or RuR (3 µM) were loaded with calcein-AM, then treated with or without CoCl 2 . Cell were then analysed by flow cytometry. Cells treated with the potent mPTP desensitizers, cyclosporin A (CsA, 1 µM) or sanglifehrin A (SFA, 2 µM), were used as positive controls. b Quantitative analysis of the relative calcein fluorescence in wt and Cdk5 −/− MEFs in the presence or absence of the treatments indicated above. Untreated cells and cells treated with ionomycin served as controls. Values are means ± SEM from three ( n = 3) independent experiments. * p

    Journal: Oncogene

    Article Title: mPTP opening caused by Cdk5 loss is due to increased mitochondrial Ca2+ uptake

    doi: 10.1038/s41388-020-1188-5

    Figure Lengend Snippet: Inhibition of IP3R by xestospongin C (XeC) or MCU by ruthenium red (RuR) reverses the opening of mPTP in Cdk5 −/− MEFs. a Wt and Cdk5 −/− MEFs pretreated with or without XeC (3 µM) or RuR (3 µM) were loaded with calcein-AM, then treated with or without CoCl 2 . Cell were then analysed by flow cytometry. Cells treated with the potent mPTP desensitizers, cyclosporin A (CsA, 1 µM) or sanglifehrin A (SFA, 2 µM), were used as positive controls. b Quantitative analysis of the relative calcein fluorescence in wt and Cdk5 −/− MEFs in the presence or absence of the treatments indicated above. Untreated cells and cells treated with ionomycin served as controls. Values are means ± SEM from three ( n = 3) independent experiments. * p

    Article Snippet: The protease inhibitor cocktail, Cyclosporine A (CsA), Ruthenium Red (RuR), ATP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and Xestospongin C (XeC) were from Sigma.

    Techniques: Inhibition, Flow Cytometry, Fluorescence

    ORP4L sustains Ca 2+ -dependent bioenergetics in T-ALL cells. ( a , b ) Western blot analysis of PDH activation in Jurkat, Molt-4 and primary T-ALL cells with ORP4L knockdown ( a ) and overexpression ( b ). p-PDH/PDH expressed as fold change over control. ( c ) PDH activation (left), OCR (middle) and ATP levels (right) in Jurkat T-cells with ORP4L knockdown alone or in combination with shPLCβ3. ( d ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without U73122 (5 μM for 1 h) or XeC (2 μM for 1 h). ( e ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L knockdown Jurkat T-cells treated with or without ionomycin (2 mg l −1 for 1 h). ( f ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without BAPTA-AM (50 μM for 1 h). ( g ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L knockdown Jurkat T-cells treated with or without MCU agonist, kaempferol (2 μM, 30 min). ( h ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without MCU inhibitor, RU360 (5 μM, 30 min). The data represent mean±s.d. value from an experiment performed in triplicate. * P

    Journal: Nature Communications

    Article Title: ORP4L is essential for T-cell acute lymphoblastic leukemia cell survival

    doi: 10.1038/ncomms12702

    Figure Lengend Snippet: ORP4L sustains Ca 2+ -dependent bioenergetics in T-ALL cells. ( a , b ) Western blot analysis of PDH activation in Jurkat, Molt-4 and primary T-ALL cells with ORP4L knockdown ( a ) and overexpression ( b ). p-PDH/PDH expressed as fold change over control. ( c ) PDH activation (left), OCR (middle) and ATP levels (right) in Jurkat T-cells with ORP4L knockdown alone or in combination with shPLCβ3. ( d ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without U73122 (5 μM for 1 h) or XeC (2 μM for 1 h). ( e ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L knockdown Jurkat T-cells treated with or without ionomycin (2 mg l −1 for 1 h). ( f ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without BAPTA-AM (50 μM for 1 h). ( g ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L knockdown Jurkat T-cells treated with or without MCU agonist, kaempferol (2 μM, 30 min). ( h ) PDH activation (left), OCR (middle) and ATP levels (right) in control or ORP4L overexpressing Jurkat T-cells treated with or without MCU inhibitor, RU360 (5 μM, 30 min). The data represent mean±s.d. value from an experiment performed in triplicate. * P

    Article Snippet: Hoechst 33342, U73122, XeC, thapsigargin and JC-1 were from Sigma-Aldrich (St Louis, MO, USA).

    Techniques: Western Blot, Activation Assay, Over Expression

    CnAα is activated downstream of the IP3R. A: Amylase secretion from WT salivary gland suspensions pretreated for 30 minutes with cyclosporine (100 μmol/L) or xestospongin followed by acetylcholine (100 μmol/L) for 15 minutes. *

    Journal: The American Journal of Pathology

    Article Title: Rescue of Calcineurin A?−/− Mice Reveals a Novel Role for the ? Isoform in the Salivary Gland

    doi: 10.1016/j.ajpath.2010.12.054

    Figure Lengend Snippet: CnAα is activated downstream of the IP3R. A: Amylase secretion from WT salivary gland suspensions pretreated for 30 minutes with cyclosporine (100 μmol/L) or xestospongin followed by acetylcholine (100 μmol/L) for 15 minutes. *

    Article Snippet: Cyclosporine, brefeldin A, acetylcholine, and xestospongin were purchased from Sigma-Aldrich (St. Louis, MO).

    Techniques:

    Functional contributions of IP 3 Rs to intracellular Ca 2+ release in primary cultured rat ONHAs In order to determine the contribution of IP 3 Rs to Ca 2+ release from intracellular stores, we quantified the rise in intracellular Ca 2+ concentration upon stimulation with the IP 3 R-selective agonist, IP 3 , using optical imaging and the Ca 2+ fluorophore, Fura-2. For delivery, we used the membrane-permeable molecules IP 3 -AM (10 µM) and Fura-2-AM (1 µM). Representative cellular responses ( A ) and traces ( B ) are shown. Warmer colors indicate higher fluorescence intensity. IP 3 stimulation resulted in a mean 19.0 ± 1.3% increase of normalized fluorescence intensity over baseline (F/F 0 max; n=47; C ). This corresponded to an experimentally determined AUC of 168 ± 18 iRFU (n=45; D ). In our experiments, 81 ± 13% of cells responded to IP 3 stimulation (n=3 separate experiments, with at least 15 cells per experiment; E ). In order to determine the specificity of the response, we pre-incubated cell with the IP 3 R blocker, Xestospongin D (Xes D), for 30 min, and experiments performed with Xes D co-application. Xes D resulted in a 15.4 ± 2.5% reduction of F/F 0 max (n=18, P

    Journal: Experimental neurology

    Article Title: Differential subcellular Ca2+ signaling in a highly specialized subpopulation of astrocytes

    doi: 10.1016/j.expneurol.2014.12.014

    Figure Lengend Snippet: Functional contributions of IP 3 Rs to intracellular Ca 2+ release in primary cultured rat ONHAs In order to determine the contribution of IP 3 Rs to Ca 2+ release from intracellular stores, we quantified the rise in intracellular Ca 2+ concentration upon stimulation with the IP 3 R-selective agonist, IP 3 , using optical imaging and the Ca 2+ fluorophore, Fura-2. For delivery, we used the membrane-permeable molecules IP 3 -AM (10 µM) and Fura-2-AM (1 µM). Representative cellular responses ( A ) and traces ( B ) are shown. Warmer colors indicate higher fluorescence intensity. IP 3 stimulation resulted in a mean 19.0 ± 1.3% increase of normalized fluorescence intensity over baseline (F/F 0 max; n=47; C ). This corresponded to an experimentally determined AUC of 168 ± 18 iRFU (n=45; D ). In our experiments, 81 ± 13% of cells responded to IP 3 stimulation (n=3 separate experiments, with at least 15 cells per experiment; E ). In order to determine the specificity of the response, we pre-incubated cell with the IP 3 R blocker, Xestospongin D (Xes D), for 30 min, and experiments performed with Xes D co-application. Xes D resulted in a 15.4 ± 2.5% reduction of F/F 0 max (n=18, P

    Article Snippet: For pharmacological experiments, dantrolene (DAN; 20 µM; Enzo, Farmingdale, NY) and Xestospongin D (Xes D; 10 µM; EMD Millipore, Gibbstown, NJ), inhibitors of RyR and IP3 R, respectively, were applied 30 min prior to application of the respective agonists, and the concentration maintained during the experiment.

    Techniques: Functional Assay, Cell Culture, Concentration Assay, Optical Imaging, Fluorescence, Incubation

    Cytosolic Ca 2+ and PKC regulate CLSs containing EGFR but not those harboring TfR. RPE cells stably expressing clathrin light chain fused to eGFP (eGFP-CLCa), treated with various inhibitors as in Figures 3 and 4: 3 μM XeC for 30 min, 10 μM BAPTA-AM (BA) for 15 min, 1 μM BIM for 30 min, or left untreated (control), and then treated with A555-EGF (A) or A555-Tfn (B) for 5 min. Shown are representative micrographs obtained by TIRF-M. Scale bar: 5 μm. Full image panels are shown in Supplemental Figure 7. (C–F) TIRF-M images were subjected to automated detection of CLSs, followed by quantification of mean A555-conjugated ligand fluorescence intensity therein (C, E). CLSs were sorted into A555-EGF-enriched or A555-Tfn-enriched cohorts, followed by quantification of the mean eGFP-CLC within each CLS cohort (D, F). For C–F, the overall median of the cellular means (bar) and 25th/75th percentiles (boxes) are shown. The number of CLSs and cells analyzed, respectively, for each condition are as follows: EGF control: 14,114 and 114; EGF XeC: 11,322 and 98; EGF BAPTA-AM: 15,786 and 104; EGF BIM: 3860 and 46, Tfn control: 16,983 and 117; Tfn XeC: 10,838 and 98; Tfn BAPTA-AM: 8868 and 64; Tfn BIM: 3718 and 44; from a minimum of three independent experiments in each condition.

    Journal: Molecular Biology of the Cell

    Article Title: Selective regulation of clathrin-mediated epidermal growth factor receptor signaling and endocytosis by phospholipase C and calcium

    doi: 10.1091/mbc.E16-12-0871

    Figure Lengend Snippet: Cytosolic Ca 2+ and PKC regulate CLSs containing EGFR but not those harboring TfR. RPE cells stably expressing clathrin light chain fused to eGFP (eGFP-CLCa), treated with various inhibitors as in Figures 3 and 4: 3 μM XeC for 30 min, 10 μM BAPTA-AM (BA) for 15 min, 1 μM BIM for 30 min, or left untreated (control), and then treated with A555-EGF (A) or A555-Tfn (B) for 5 min. Shown are representative micrographs obtained by TIRF-M. Scale bar: 5 μm. Full image panels are shown in Supplemental Figure 7. (C–F) TIRF-M images were subjected to automated detection of CLSs, followed by quantification of mean A555-conjugated ligand fluorescence intensity therein (C, E). CLSs were sorted into A555-EGF-enriched or A555-Tfn-enriched cohorts, followed by quantification of the mean eGFP-CLC within each CLS cohort (D, F). For C–F, the overall median of the cellular means (bar) and 25th/75th percentiles (boxes) are shown. The number of CLSs and cells analyzed, respectively, for each condition are as follows: EGF control: 14,114 and 114; EGF XeC: 11,322 and 98; EGF BAPTA-AM: 15,786 and 104; EGF BIM: 3860 and 46, Tfn control: 16,983 and 117; Tfn XeC: 10,838 and 98; Tfn BAPTA-AM: 8868 and 64; Tfn BIM: 3718 and 44; from a minimum of three independent experiments in each condition.

    Article Snippet: BIM and CsA were obtained from Cell Signaling (Danvers, MA), XeC was obtained from Abcam (Cambridge, MA), and BAPTA-AM was obtained from Santa Cruz Biotechnology (Dallas, TX).

    Techniques: Stable Transfection, Expressing, Fluorescence

    Effects of xestospongin C on progesterone‐enhanced hyperactivation. After exposure to xestospongin C for 5 min, spermatozoa were exposed to progesterone. The percentages of motile spermatozoa ( a ) and hyperactivated spermatozoa ( b ) are shown when 500 nM or 1 μM xestospongin C were added to the mTALP medium. The percentages of motile spermatozoa ( c ) and hyperactivated spermatozoa ( d ) are shown when 500 nM or 1 μM xestospongin C and 20 ng/ml progesterone were added to the mTALP medium. Data are expressed as mean ± SD. In a and b (Vehicle), mTALP + 0.1 % (v/v) DMSO; (500 nM Xestospongin C), mTALP + 500 nM xestospongin C + 0.1 % (v/v) DMSO; (1 μM xestospongin C), mTALP + 1 μM xestospongin C + 0.1 % (v/v) DMSO. In c and d (Vehicle), mTALP + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P), mTALP + 20 ng/ml progesterone + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; ( P + 500 nM Xestospongin C), mTALP + 20 ng/ml progesterone + 500 nM xestospongin C + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; ( P + 1 μM Xestospongin C), mTALP + 20 ng/ml progesterone + 1 μM xestospongin C + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO. a Significant difference compared with “Vehicle” and “ P + 1 μM Xestospongin C” ( P

    Journal: Reproductive Medicine and Biology

    Article Title: Progesterone‐enhanced sperm hyperactivation through IP3–PKC and PKA signals

    doi: 10.1007/s12522-012-0137-6

    Figure Lengend Snippet: Effects of xestospongin C on progesterone‐enhanced hyperactivation. After exposure to xestospongin C for 5 min, spermatozoa were exposed to progesterone. The percentages of motile spermatozoa ( a ) and hyperactivated spermatozoa ( b ) are shown when 500 nM or 1 μM xestospongin C were added to the mTALP medium. The percentages of motile spermatozoa ( c ) and hyperactivated spermatozoa ( d ) are shown when 500 nM or 1 μM xestospongin C and 20 ng/ml progesterone were added to the mTALP medium. Data are expressed as mean ± SD. In a and b (Vehicle), mTALP + 0.1 % (v/v) DMSO; (500 nM Xestospongin C), mTALP + 500 nM xestospongin C + 0.1 % (v/v) DMSO; (1 μM xestospongin C), mTALP + 1 μM xestospongin C + 0.1 % (v/v) DMSO. In c and d (Vehicle), mTALP + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; (P), mTALP + 20 ng/ml progesterone + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; ( P + 500 nM Xestospongin C), mTALP + 20 ng/ml progesterone + 500 nM xestospongin C + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO; ( P + 1 μM Xestospongin C), mTALP + 20 ng/ml progesterone + 1 μM xestospongin C + 0.1 % (v/v) MeOH + 0.1 % (v/v) DMSO. a Significant difference compared with “Vehicle” and “ P + 1 μM Xestospongin C” ( P

    Article Snippet: Xestospongin C and other reagent‐grade chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).

    Techniques:

    Long-lasting increase of fIPSPs after DHPG application is mediated by IP 3 receptors activation. DHPG failed to increase fIPSPs in all CA1 (a) and specifically in its different sub-regions (b–g) in presence of the cell-permeable IP 3 receptors blockers 2-APB or Xestospongin C (XestoC). In all figures, left panel shows the time course, while right panel is showing mean over time. Statistical analysis in right panel figures is one-way ANOVA followed by Dunnet post-hoc test. Vehicles of both IP 3 receptors antagonists have been pooled together since they are not different. n = (slices, mice): Vehicle group = (11, 9), 2-APB group = (5, 5), Xestospongin C group (5, 3). Data are mean ± s.e.m. *p

    Journal: Scientific Reports

    Article Title: Layer-specific potentiation of network GABAergic inhibition in the CA1 area of the hippocampus

    doi: 10.1038/srep28454

    Figure Lengend Snippet: Long-lasting increase of fIPSPs after DHPG application is mediated by IP 3 receptors activation. DHPG failed to increase fIPSPs in all CA1 (a) and specifically in its different sub-regions (b–g) in presence of the cell-permeable IP 3 receptors blockers 2-APB or Xestospongin C (XestoC). In all figures, left panel shows the time course, while right panel is showing mean over time. Statistical analysis in right panel figures is one-way ANOVA followed by Dunnet post-hoc test. Vehicles of both IP 3 receptors antagonists have been pooled together since they are not different. n = (slices, mice): Vehicle group = (11, 9), 2-APB group = (5, 5), Xestospongin C group (5, 3). Data are mean ± s.e.m. *p

    Article Snippet: Xestospongin C was from Enzo Life Sciences (France) and dissolved in DMSO (DMSO < 0.1%).

    Techniques: Activation Assay, Mouse Assay

    After cells were treated with 1.2% isoflurane for 8 h, cytosolic calcium concentration ([Ca 2+ ] c ) was determined by flow cytometry. Briefly, the cells were harvested, stained with Fluo-3/AM and were then analyzed. (A-C) Representative results from one of six independent experiments on mutated β-amyloid precursor protein (APP)-transfected SH-SY5Y cells treated with (A) the control condition (Con), (B) xestospongin C plus isoflurane (Xes) and (C) isoflurane (Iso). (D-F) Representative results from vector-transfected SH-SY5Y cells treated with (D) Con, (E) Xes and (F) Iso. The positive rate of [Ca 2+ ] c loading with Fluo-3/AM was > 99%. (A-F) X-axis refers to the average value of calcium fluorescence intensity, and Y-axis refers to cell count. (G) Data are presented as the mean ± standard deviation of at least three separate experiments. *P

    Journal: Molecular Medicine Reports

    Article Title: A mutation in β-amyloid precursor protein renders SH-SY5Y cells vulnerable to isoflurane toxicity: The role of inositol 1,4,5-trisphosphate receptors

    doi: 10.3892/mmr.2016.5930

    Figure Lengend Snippet: After cells were treated with 1.2% isoflurane for 8 h, cytosolic calcium concentration ([Ca 2+ ] c ) was determined by flow cytometry. Briefly, the cells were harvested, stained with Fluo-3/AM and were then analyzed. (A-C) Representative results from one of six independent experiments on mutated β-amyloid precursor protein (APP)-transfected SH-SY5Y cells treated with (A) the control condition (Con), (B) xestospongin C plus isoflurane (Xes) and (C) isoflurane (Iso). (D-F) Representative results from vector-transfected SH-SY5Y cells treated with (D) Con, (E) Xes and (F) Iso. The positive rate of [Ca 2+ ] c loading with Fluo-3/AM was > 99%. (A-F) X-axis refers to the average value of calcium fluorescence intensity, and Y-axis refers to cell count. (G) Data are presented as the mean ± standard deviation of at least three separate experiments. *P

    Article Snippet: A group of cells were pretreated with xestospongin C (100 nM; Merck Millipore) for 30 in at room temperature prior to isofluarne exposure.

    Techniques: Concentration Assay, Flow Cytometry, Cytometry, Staining, Transfection, Plasmid Preparation, Fluorescence, Cell Counting, Standard Deviation

    After cells were treated with 1.2% isoflurane for 8 h, apoptosis was determined by flow cytometry. Briefly, the cells were harvested, stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), and were then analyzed. (A-C) Representative results from one of six independent experiments on mutated β-amyloid precursor protein (APP)-transfected SH-SY5Y cells treated with (A) the control condition (Con), (B) xestospongin C plus isoflurane (Xes) and (C) isoflurane (Iso). (D-F) Representative results from vector-transfected SH-SY5Y cells treated with (D) Con, (E) Xes and (F) Iso. The quadrants of each plot exhibit the following: Viable cells (Annexin − /PI − ; lower left), early apoptotic cells (Annexin + /PI − , lower right), necrotic cells (Annexin + /PI + , upper right), and late apoptotic cells (Annexin − /PI + , upper left). (G) Data are presented as a sum of the percentage of apoptotic cells in early apoptosis (annexin + /PI − ) and late apoptosis (annexin + /PI + ). Data are presented as the mean ± standard deviation of at least three separate experiments. ***P

    Journal: Molecular Medicine Reports

    Article Title: A mutation in β-amyloid precursor protein renders SH-SY5Y cells vulnerable to isoflurane toxicity: The role of inositol 1,4,5-trisphosphate receptors

    doi: 10.3892/mmr.2016.5930

    Figure Lengend Snippet: After cells were treated with 1.2% isoflurane for 8 h, apoptosis was determined by flow cytometry. Briefly, the cells were harvested, stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), and were then analyzed. (A-C) Representative results from one of six independent experiments on mutated β-amyloid precursor protein (APP)-transfected SH-SY5Y cells treated with (A) the control condition (Con), (B) xestospongin C plus isoflurane (Xes) and (C) isoflurane (Iso). (D-F) Representative results from vector-transfected SH-SY5Y cells treated with (D) Con, (E) Xes and (F) Iso. The quadrants of each plot exhibit the following: Viable cells (Annexin − /PI − ; lower left), early apoptotic cells (Annexin + /PI − , lower right), necrotic cells (Annexin + /PI + , upper right), and late apoptotic cells (Annexin − /PI + , upper left). (G) Data are presented as a sum of the percentage of apoptotic cells in early apoptosis (annexin + /PI − ) and late apoptosis (annexin + /PI + ). Data are presented as the mean ± standard deviation of at least three separate experiments. ***P

    Article Snippet: A group of cells were pretreated with xestospongin C (100 nM; Merck Millipore) for 30 in at room temperature prior to isofluarne exposure.

    Techniques: Flow Cytometry, Cytometry, Staining, Transfection, Plasmid Preparation, Standard Deviation

    Isoflurane induces inositol 1,4,5-trisphosphate receptor (IP3R) activation in mutated β-amyloid precursor protein (APP)-transfected SH-SY5Y cells. (A and B) Treatment with 1.2% isoflurane (Iso) inducesd IP3R cleavage compared with in the control (Con) and xestospongin C (Xes) groups in both cell types. There was no significant difference in β-actin expression between the groups. (C) Treatment with 1.2% isoflurane induced a higher increase in IP3R protein expression in the mutated APP-transfected SH-SY5Y cells compared with vector-transfected SH-SY5Y cells. Data are presented as the mean ± standard deviation of at least three separate experiments. *P

    Journal: Molecular Medicine Reports

    Article Title: A mutation in β-amyloid precursor protein renders SH-SY5Y cells vulnerable to isoflurane toxicity: The role of inositol 1,4,5-trisphosphate receptors

    doi: 10.3892/mmr.2016.5930

    Figure Lengend Snippet: Isoflurane induces inositol 1,4,5-trisphosphate receptor (IP3R) activation in mutated β-amyloid precursor protein (APP)-transfected SH-SY5Y cells. (A and B) Treatment with 1.2% isoflurane (Iso) inducesd IP3R cleavage compared with in the control (Con) and xestospongin C (Xes) groups in both cell types. There was no significant difference in β-actin expression between the groups. (C) Treatment with 1.2% isoflurane induced a higher increase in IP3R protein expression in the mutated APP-transfected SH-SY5Y cells compared with vector-transfected SH-SY5Y cells. Data are presented as the mean ± standard deviation of at least three separate experiments. *P

    Article Snippet: A group of cells were pretreated with xestospongin C (100 nM; Merck Millipore) for 30 in at room temperature prior to isofluarne exposure.

    Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Standard Deviation