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  • 99
    Thermo Fisher bapta am
    A persistent increase in intracellular Ca 2+ leads to MT depolymerization, causing proteosomal GEF-H1 degradation (A) (i) Representative fluorescent images of EB1-GFP-expressing HUVECs cultured on a nano-grooved patterned substratum following treatment by DMSO, <t>BAPTA</t> (1 μM), <t>ionomycin</t> (3 μM) and thapsigargin (1 μM) for 15 hours. The white arrow indicates the direction of the grooves and ridges in the pattern. Scale bar: 40 μm. Results of an automated tracking analysis representing (ii) speed and (iii) angle of trajectories of EB1-GFP clusters from images taken every 2 seconds for 2 minutes. (B) Comparison of the length of persistent MT growth over 2 minutes for the conditions of treatment by DMSO, BAPTA (1 μM) (* P = 0.0401), ionomycin (3 μM) (* P = 0.0113) and thapsigargin (1 μM) (* P = 0.0447). Data were analyzed by unpaired two-tailed t test with error bar representing s.e.m. (C) Comparison of axial ratios of HUVECs under the conditions of treatment with DMSO (n = 106 cells), paclitaxel (0.5 μM) (n = 140 cells), colchicine (0.5 μM) (n = 132 cells), BAPTA (1 μM) (n = 135 cells), ionomycin (3 μM) (n = 130 cells) and thapsigargin (1 μM) (n = 113 cells). Data were analyzed by one-way ANOVA with error bar representing s.e.m. (* P = 0.0116, **** P
    Bapta Am, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bapta am
    IXD extract increases intracellular calcium release in the HSG cells Notes: ( A ) Fura-2-loaded HSG cells were promptly treated with IXD extract (0.02 mg/mL) and changes in F340/F380 were monitored. ( B ) The effect of 10 μM BAPTA-AM in IXD induced Ca 2+ release. The figures represent the typical Ca 2+ transients from more than three experiments. Abbreviations: BAPTA-AM, 1, 2-Bis (2-aminophenoxy) ethane-N, N, N′, <t>N′-tetraacetic</t> acid tetrakis (acetoxymethyl ester); Ca 2+ , calcium; HSG, human salivary gland; IXD, Ixeris dentata .
    Bapta Am, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1896 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris bapta am
    Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM <t>Ionomycin</t> and 50 μM <t>Bapta-AM</t> was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P
    Bapta Am, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Dojindo Labs bapta am
    Ca 2+ transients are required for LEM migration. ( a ) Ca 2+ transients in DMZ explants treated with <t>BAPTA-AM</t> for 3 hours. Red bars indicate average values. <t>DMSO:</t> n = 7 embryos and BAPTA-AM (50 μM): n = 8 embryos. Mann–Whitney U-test, **P
    Bapta Am, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 92/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Selleck Chemicals bapta am
    Ca 2+ transients are required for LEM migration. ( a ) Ca 2+ transients in DMZ explants treated with <t>BAPTA-AM</t> for 3 hours. Red bars indicate average values. <t>DMSO:</t> n = 7 embryos and BAPTA-AM (50 μM): n = 8 embryos. Mann–Whitney U-test, **P
    Bapta Am, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 98/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam bapta am
    Ca 2+ transients are required for LEM migration. ( a ) Ca 2+ transients in DMZ explants treated with <t>BAPTA-AM</t> for 3 hours. Red bars indicate average values. <t>DMSO:</t> n = 7 embryos and BAPTA-AM (50 μM): n = 8 embryos. Mann–Whitney U-test, **P
    Bapta Am, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Cayman Chemical bapta am
    HRS is not required for ESCRT-III endomembrane damage response. (A and B) HRS does not assemble with ESCRT machinery on <t>LLOME-disrupted</t> endolysosomes. U20S cells were treated with LLOME or the solvent control for 10 min and then stained for HRS and EEA1 (A) or for HRS and CHMP4A (B). Boxed areas are magnified at right. The middle two columns show each indicated stain in grayscale; overlap of both stains in leftmost and rightmost columns appears white. Individual cells are outlined by white dashed lines; scale bars equal 10 μm (2 μm in magnified views). (C) RAW cells were treated with siRNA to deplete HRS or TSG101 for 2 days. Macrophages were then treated with LLOME for 15 min, and ubiquitin (FK2 antibody [red]) and CHMP4B (green) were examined. FK2-positive cells are outlined by white dashed lines. (D and E) Automated image analysis was used to quantify the mean fluorescent intensity (MFI) of FK2 (D) or the number of CHMP4B (E) punctae on 50 macrophages per sample. The number of CHMP4B punctae was compared in cells with FK2 MFI greater than and less than 700. Data are means ± SEM from one representative experiment from at least two independent experiments. *** * , P ≤ 0.0001, Student’s t test. ns, not significant. (F) HeLa cells were transfected with M. tuberculosis (Mtb) EsxG-EsxH or vector control, preincubated for 1 h with <t>BAPTA-AM,</t> and then treated with LLOME for 15 min and stained for CHMP4A (green) and EsxG-EsxH (red). EsxG-EsxH was detected with anti-V5 antibody. Nuclei were stained with DAPI. Images are maximum-intensity projections. Scale bar, 10 μm.
    Bapta Am, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 98/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore calcium chelator bapta am
    HRS is not required for ESCRT-III endomembrane damage response. (A and B) HRS does not assemble with ESCRT machinery on <t>LLOME-disrupted</t> endolysosomes. U20S cells were treated with LLOME or the solvent control for 10 min and then stained for HRS and EEA1 (A) or for HRS and CHMP4A (B). Boxed areas are magnified at right. The middle two columns show each indicated stain in grayscale; overlap of both stains in leftmost and rightmost columns appears white. Individual cells are outlined by white dashed lines; scale bars equal 10 μm (2 μm in magnified views). (C) RAW cells were treated with siRNA to deplete HRS or TSG101 for 2 days. Macrophages were then treated with LLOME for 15 min, and ubiquitin (FK2 antibody [red]) and CHMP4B (green) were examined. FK2-positive cells are outlined by white dashed lines. (D and E) Automated image analysis was used to quantify the mean fluorescent intensity (MFI) of FK2 (D) or the number of CHMP4B (E) punctae on 50 macrophages per sample. The number of CHMP4B punctae was compared in cells with FK2 MFI greater than and less than 700. Data are means ± SEM from one representative experiment from at least two independent experiments. *** * , P ≤ 0.0001, Student’s t test. ns, not significant. (F) HeLa cells were transfected with M. tuberculosis (Mtb) EsxG-EsxH or vector control, preincubated for 1 h with <t>BAPTA-AM,</t> and then treated with LLOME for 15 min and stained for CHMP4A (green) and EsxG-EsxH (red). EsxG-EsxH was detected with anti-V5 antibody. Nuclei were stained with DAPI. Images are maximum-intensity projections. Scale bar, 10 μm.
    Calcium Chelator Bapta Am, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH bapta am
    Involvement of PKC-α in BK-induced ROS generation and MMP-9 expression in RBA-1. ( A ) Cells were pretreated without or with <t>GF109203X</t> (GF, 30 μM) or Gö6976 (Gö, 1 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. ( B ) Cells were pretreated with or without GF109203X (GF, 30 μM) or Gö6976 (Gö, 1 μM) for 1 h before exposure to 10 nM BK for 16 h. The total RNA was collected and analyzed by RT-PCR. ( C ) Cells were pretreated with or without <t>BAPTA</t> (BAP, 30 μM) or Gö (1 μM) for 1 h and then treated with 10 nM BK for the indicated time intervals (upper part) or 5 min (lower part). The membrane and cytosol fractions were prepared and analyzed by Western blotting. ( D ) Cells were pretreated without or with GF or Gö for 1 h before exposure to 10 nM BK for 5 min. The Nox activity and ROS generation were analyzed. ( E ) Cells were transfected with scramble (scra) or PKC-α siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography or Western blotting. ( F ) Cells were pretreated without or with Gö (1 μM), BAPTA (30 μM), or TG (1 μM) for 1 h before exposure to 10 nM BK for 5 min. The cell lysates were collected and analyzed by Western blotting using an anti-phospho-serine (p-Ser), p47 phox , or GAPDH antibody. Data are expressed as the mean ± SEM (n = 3). * P
    Bapta Am, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA bapta am
    PDD-induced TRPV4 activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of <t>4α-PDD</t> or DMSO. Luc -DMSO=cells transfected with Luc control siRNA and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by <t>BAPTA-AM)</t> and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P
    Bapta Am, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Enzo Biochem bapta am
    PDD-induced TRPV4 activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of <t>4α-PDD</t> or DMSO. Luc -DMSO=cells transfected with Luc control siRNA and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by <t>BAPTA-AM)</t> and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P
    Bapta Am, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 95/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Nacalai bapta am
    <t>ALG-2</t> stabilizes the localization of Sec31A at ER exit sites. (A and A′) HeLa cells were transfected with the ALG-2 siRNA vector (A-siRNA1), and double stained with anti-Sec31A (A) and anti-ALG-2 (A′) antibodies. Asterisks and arrowheads indicate ALG-2–depleted and undepleted cells, respectively. (B) HeLa cells transfected with the mock or A-siRNA1 vector were homogenized and fractionated into nuclear (N), cytoplasmic (C), and membrane (M) fractions. Proteins in each fraction were immunoblotted with antibodies against Sec31A (top) and ALG-2 (bottom). (C) The experiment shown in B was repeated four times, and the intensity of the Sec31A band in each fraction was quantified. The mean ± SD of the Sec31A ratio (%) in each fraction is shown. (D–E″) HeLa cells were treated without (D–D″) or with (E–E″) <t>BAPTA-AM</t> and double stained with anti-ALG-2 (D and E) and anti-Sec31A (D′ and E′) antibodies. D″ and E″ are merged images. N indicates nuclei. Bars, 20 μm.
    Bapta Am, supplied by Nacalai, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Merck & Co bapta am
    Cytoskeleton of neurites of rat hippocampal neurons after treatment with <t>BAPTA/AM</t> and <t>LPA</t> (Confocal Microscopy). In the magnified images, the red microfilaments in protrusion terminals showed increased fluorescence intensity, and the spinous protrusions
    Bapta Am, supplied by Merck & Co, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Unimed bapta am
    Monocytes and <t>PMN</t> phagocytosis—Ca 2+ chelation. Whole blood samples were pre-treated with 100 μM of the calcium chelator <t>BAPTA-AM</t> for 30 min and incubated in a water bath for 20 min at 37°C with opsonized E . coli (1-2x10 9 /ml). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the monocytes population. A regular phagocytosis was observed on monocytes and PMN from HD and XLA even after calcium chelation demonstrating that the process is Ca 2+ independent. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean valu es and bars standard deviation. In (A and B) are shown monocytes and in (C and D) PMN phagocytosis of representative XLA patient: black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).
    Bapta Am, supplied by Unimed, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher oregon green 488 bapta am
    Monocytes and <t>PMN</t> phagocytosis—Ca 2+ chelation. Whole blood samples were pre-treated with 100 μM of the calcium chelator <t>BAPTA-AM</t> for 30 min and incubated in a water bath for 20 min at 37°C with opsonized E . coli (1-2x10 9 /ml). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the monocytes population. A regular phagocytosis was observed on monocytes and PMN from HD and XLA even after calcium chelation demonstrating that the process is Ca 2+ independent. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean valu es and bars standard deviation. In (A and B) are shown monocytes and in (C and D) PMN phagocytosis of representative XLA patient: black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).
    Oregon Green 488 Bapta Am, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    BAPTA AM 1 2 Bis 2 aminophenoxy ethane N N N N tetraacetic acid tetrakis acetoxymethyl ester is a non fluorescent membrane permeable form of BAPTA Whereas the BAPTA Free
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    N/A
    BAPTA Item No 11706 is membrane impermeable calcium chelator that binds extracellular calcium ions K 0 11 μM with selectivity over magnesium ions or protons BAPTA and its derivatives can
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    N/A
    A cell permeable selective chelator of intracellular calcium stores BAPTA is105 fold greater affinity for Ca2 than for Mg2 Inhibits thapsigargin induced apoptosis in rat thymocytes
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    Image Search Results


    A persistent increase in intracellular Ca 2+ leads to MT depolymerization, causing proteosomal GEF-H1 degradation (A) (i) Representative fluorescent images of EB1-GFP-expressing HUVECs cultured on a nano-grooved patterned substratum following treatment by DMSO, BAPTA (1 μM), ionomycin (3 μM) and thapsigargin (1 μM) for 15 hours. The white arrow indicates the direction of the grooves and ridges in the pattern. Scale bar: 40 μm. Results of an automated tracking analysis representing (ii) speed and (iii) angle of trajectories of EB1-GFP clusters from images taken every 2 seconds for 2 minutes. (B) Comparison of the length of persistent MT growth over 2 minutes for the conditions of treatment by DMSO, BAPTA (1 μM) (* P = 0.0401), ionomycin (3 μM) (* P = 0.0113) and thapsigargin (1 μM) (* P = 0.0447). Data were analyzed by unpaired two-tailed t test with error bar representing s.e.m. (C) Comparison of axial ratios of HUVECs under the conditions of treatment with DMSO (n = 106 cells), paclitaxel (0.5 μM) (n = 140 cells), colchicine (0.5 μM) (n = 132 cells), BAPTA (1 μM) (n = 135 cells), ionomycin (3 μM) (n = 130 cells) and thapsigargin (1 μM) (n = 113 cells). Data were analyzed by one-way ANOVA with error bar representing s.e.m. (* P = 0.0116, **** P

    Journal: bioRxiv

    Article Title: A molecular clock controls periodically driven cell migration in confined spaces

    doi: 10.1101/2020.12.29.424673

    Figure Lengend Snippet: A persistent increase in intracellular Ca 2+ leads to MT depolymerization, causing proteosomal GEF-H1 degradation (A) (i) Representative fluorescent images of EB1-GFP-expressing HUVECs cultured on a nano-grooved patterned substratum following treatment by DMSO, BAPTA (1 μM), ionomycin (3 μM) and thapsigargin (1 μM) for 15 hours. The white arrow indicates the direction of the grooves and ridges in the pattern. Scale bar: 40 μm. Results of an automated tracking analysis representing (ii) speed and (iii) angle of trajectories of EB1-GFP clusters from images taken every 2 seconds for 2 minutes. (B) Comparison of the length of persistent MT growth over 2 minutes for the conditions of treatment by DMSO, BAPTA (1 μM) (* P = 0.0401), ionomycin (3 μM) (* P = 0.0113) and thapsigargin (1 μM) (* P = 0.0447). Data were analyzed by unpaired two-tailed t test with error bar representing s.e.m. (C) Comparison of axial ratios of HUVECs under the conditions of treatment with DMSO (n = 106 cells), paclitaxel (0.5 μM) (n = 140 cells), colchicine (0.5 μM) (n = 132 cells), BAPTA (1 μM) (n = 135 cells), ionomycin (3 μM) (n = 130 cells) and thapsigargin (1 μM) (n = 113 cells). Data were analyzed by one-way ANOVA with error bar representing s.e.m. (* P = 0.0116, **** P

    Article Snippet: Chemicals and antibodiesThe following chemicals were used: Ionomycin (Cayman, 10004974), BAPTA (Invitrogen, B6769), Thapsigargin (Invitrogen, T7459), Paclitaxel (VWR, T104-0005), Colchicine (Sigma, C9754), Nocodazole (Sigma, M1404), C646 (Cayman, 328968-36-1), VIVIT (Cayman, 249537-733), BML 210 (Cayman, 537034-17-6), Fluo-4 AM (Introgen, F14201), SMIFH2 (Tocris, 4401), MG- 132 (Sellekchem, S2619) and Chloroquine (Sigma, C6628).

    Techniques: Expressing, Cell Culture, Two Tailed Test

    Persistently elevated intracellular Ca 2+ enhances CREB-dependent GEF-H1 transcription (A) (i) Immunoblot of GEF-H1 abundance in HUVECs following treatment with DMSO, BAPTA (1 μM), ionomycin (3 μM) and thapsigardin (1 μM) for 15 hours. GAPDH was used as a loading control. Analysis is based on 3 independent experiments. (ii) Fold-change quantification of GEF-H1 abundance normalized over GAPDH (** P = 0.0034, **** P

    Journal: bioRxiv

    Article Title: A molecular clock controls periodically driven cell migration in confined spaces

    doi: 10.1101/2020.12.29.424673

    Figure Lengend Snippet: Persistently elevated intracellular Ca 2+ enhances CREB-dependent GEF-H1 transcription (A) (i) Immunoblot of GEF-H1 abundance in HUVECs following treatment with DMSO, BAPTA (1 μM), ionomycin (3 μM) and thapsigardin (1 μM) for 15 hours. GAPDH was used as a loading control. Analysis is based on 3 independent experiments. (ii) Fold-change quantification of GEF-H1 abundance normalized over GAPDH (** P = 0.0034, **** P

    Article Snippet: Chemicals and antibodiesThe following chemicals were used: Ionomycin (Cayman, 10004974), BAPTA (Invitrogen, B6769), Thapsigargin (Invitrogen, T7459), Paclitaxel (VWR, T104-0005), Colchicine (Sigma, C9754), Nocodazole (Sigma, M1404), C646 (Cayman, 328968-36-1), VIVIT (Cayman, 249537-733), BML 210 (Cayman, 537034-17-6), Fluo-4 AM (Introgen, F14201), SMIFH2 (Tocris, 4401), MG- 132 (Sellekchem, S2619) and Chloroquine (Sigma, C6628).

    Techniques:

    IXD extract increases intracellular calcium release in the HSG cells Notes: ( A ) Fura-2-loaded HSG cells were promptly treated with IXD extract (0.02 mg/mL) and changes in F340/F380 were monitored. ( B ) The effect of 10 μM BAPTA-AM in IXD induced Ca 2+ release. The figures represent the typical Ca 2+ transients from more than three experiments. Abbreviations: BAPTA-AM, 1, 2-Bis (2-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid tetrakis (acetoxymethyl ester); Ca 2+ , calcium; HSG, human salivary gland; IXD, Ixeris dentata .

    Journal: Journal of Experimental Pharmacology

    Article Title: Ixeris dentata extract regulates salivary secretion through the activation of aquaporin-5 and prevents diabetes-induced xerostomia

    doi: 10.2147/JEP.S141807

    Figure Lengend Snippet: IXD extract increases intracellular calcium release in the HSG cells Notes: ( A ) Fura-2-loaded HSG cells were promptly treated with IXD extract (0.02 mg/mL) and changes in F340/F380 were monitored. ( B ) The effect of 10 μM BAPTA-AM in IXD induced Ca 2+ release. The figures represent the typical Ca 2+ transients from more than three experiments. Abbreviations: BAPTA-AM, 1, 2-Bis (2-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid tetrakis (acetoxymethyl ester); Ca 2+ , calcium; HSG, human salivary gland; IXD, Ixeris dentata .

    Article Snippet: Chemicals and reagents Chemicals, including streptozotocin (STZ), citric acid, pilocarpine hydrochloride, and 1, 2-Bis (2-aminophenoxy) ethane-N, N, N′, N′- tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).

    Techniques:

    Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P

    Journal: Biology Open

    Article Title: Plasma membrane damage caused by listeriolysin O is not repaired through endocytosis of the membrane pore

    doi: 10.1242/bio.035287

    Figure Lengend Snippet: Pore formation by LLO represses GRAF1 assembly but does not influence the number of caveolin1- or SNX9-positive structures. (A) Graphs showing the number of endocytic spots detected by live-cell TIRF microscopy of HeLa Flp-In TRex cells expressing different endocytic proteins treated with LLO. The number of endocytic structures present in the TIRF field was quantified every 3 s for 5 min before and after LLO addition and plotted as the relative number of spots where the first frame of respective movie was set as 1. Error bars represent the s.d. Only movies with cells showing ANXA6-mCherry recruitment to the plasma membrane after addition of LLO were quantified. Time 0 s represent the acquisition start time in all subfigures. The data represent quantifications derived from several cells and independent movies from three independent experiments n =3 except for GRAF1, where n =1 on 7 cells. (B) Representative micrographs of maximum-projected confocal z-stacks showing fixed Flp-In TRex cells expressing GFP-GRAF1. Vehicle DMSO (control), 10 μM Ionomycin and 50 μM Bapta-AM was added to the cells for 30 min followed fixation and imaging, respectively, as indicated. Scale bars 10 μm. (C) Bar plot of the number of GFP-GRAF1 structures quantified after 30 min of drug treatment. P -values from one-way ANOVA: control versus Ionomycin P

    Article Snippet: FM4-64FX and Fluo-3 were from Molecular Probes, Ionomycin from LC Laboratories and Bapta-AM from Tocris Bioscience.

    Techniques: Microscopy, Expressing, Derivative Assay, Imaging

    TBS-induced PP → CA1 tuft LTP is blocked by intracellular calcium buffering with a high concentration of BAPTA, but not a low concentration of BAPTA or low or high EGTA. ( A – D ) Representative time course of EPSP amplitude before and after TBSx3+Current was delivered (arrows) from cells buffered with 0.5 mM EGTA ( A ), 0.5 mM BAPTA ( B ), 10 mM EGTA ( C ), or 10 mM BAPTA ( D ). CaCl 2 was added to maintain basal calcium level (∼50 nM; see ‘Materials and methods’). Calcium buffer was included in the intracellular solution. Top, representative traces (single trials) of EPSP before (1) and 25 min after (2) TBSx3+Current was delivered. The scale bar in A applies to all panels. ( E ) Summary of the LTP experiments with different calcium buffering. EPSP amplitude is normalized to the average EPSP amplitude before LTP induction. Solid lines and shaded areas represent mean and S.E.M., respectively. ( F ) Potentiation ratio in different experimental conditions (0.5 mM EGTA, n = 8; 0.5 mM BAPTA, n = 5; 10 mM EGTA, n = 5; 10 mM BAPTA, n = 8). ##p

    Journal: eLife

    Article Title: Dendritic sodium spikes are required for long-term potentiation at distal synapses on hippocampal pyramidal neurons

    doi: 10.7554/eLife.06414

    Figure Lengend Snippet: TBS-induced PP → CA1 tuft LTP is blocked by intracellular calcium buffering with a high concentration of BAPTA, but not a low concentration of BAPTA or low or high EGTA. ( A – D ) Representative time course of EPSP amplitude before and after TBSx3+Current was delivered (arrows) from cells buffered with 0.5 mM EGTA ( A ), 0.5 mM BAPTA ( B ), 10 mM EGTA ( C ), or 10 mM BAPTA ( D ). CaCl 2 was added to maintain basal calcium level (∼50 nM; see ‘Materials and methods’). Calcium buffer was included in the intracellular solution. Top, representative traces (single trials) of EPSP before (1) and 25 min after (2) TBSx3+Current was delivered. The scale bar in A applies to all panels. ( E ) Summary of the LTP experiments with different calcium buffering. EPSP amplitude is normalized to the average EPSP amplitude before LTP induction. Solid lines and shaded areas represent mean and S.E.M., respectively. ( F ) Potentiation ratio in different experimental conditions (0.5 mM EGTA, n = 8; 0.5 mM BAPTA, n = 5; 10 mM EGTA, n = 5; 10 mM BAPTA, n = 8). ##p

    Article Snippet: D-(−)-2-amino-5-phosphonopentanoic acid (D-AP5), tetrodotoxin citrate (TTX), nimodipine, CGP 52432, EGTA, and BAPTA were from Tocris Bioscience (Minneapolis, MN).

    Techniques: Concentration Assay

    Ca 2+ transients are required for LEM migration. ( a ) Ca 2+ transients in DMZ explants treated with BAPTA-AM for 3 hours. Red bars indicate average values. DMSO: n = 7 embryos and BAPTA-AM (50 μM): n = 8 embryos. Mann–Whitney U-test, **P

    Journal: Scientific Reports

    Article Title: Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation

    doi: 10.1038/s41598-018-20747-w

    Figure Lengend Snippet: Ca 2+ transients are required for LEM migration. ( a ) Ca 2+ transients in DMZ explants treated with BAPTA-AM for 3 hours. Red bars indicate average values. DMSO: n = 7 embryos and BAPTA-AM (50 μM): n = 8 embryos. Mann–Whitney U-test, **P

    Article Snippet: BAPTA-AM (B035, Dojindo) in DMSO was added to the medium 30 minutes before starting the live imaging.

    Techniques: Migration, MANN-WHITNEY

    Suppression of Ca 2+ transients reduce the protrusive activity in LEM cells. ( a ) Snapshots of BAPTA-AM- and DMSO-treated DMZ explants. Scale bar: 50 μm. ( b ) Procedure for measuring the protrusion activity in LEM cells. ( c ) Protrusion size in DMSO- and BAPTA-AM-treated LEM cells. DMSO: n = 63 cells from 14 embryos, BAPTA-AM: n = 79 cells from 13 embryos. Error bars indicate s.e. ± Mann–Whitney U-test, **P

    Journal: Scientific Reports

    Article Title: Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation

    doi: 10.1038/s41598-018-20747-w

    Figure Lengend Snippet: Suppression of Ca 2+ transients reduce the protrusive activity in LEM cells. ( a ) Snapshots of BAPTA-AM- and DMSO-treated DMZ explants. Scale bar: 50 μm. ( b ) Procedure for measuring the protrusion activity in LEM cells. ( c ) Protrusion size in DMSO- and BAPTA-AM-treated LEM cells. DMSO: n = 63 cells from 14 embryos, BAPTA-AM: n = 79 cells from 13 embryos. Error bars indicate s.e. ± Mann–Whitney U-test, **P

    Article Snippet: BAPTA-AM (B035, Dojindo) in DMSO was added to the medium 30 minutes before starting the live imaging.

    Techniques: Activity Assay, MANN-WHITNEY

    HRS is not required for ESCRT-III endomembrane damage response. (A and B) HRS does not assemble with ESCRT machinery on LLOME-disrupted endolysosomes. U20S cells were treated with LLOME or the solvent control for 10 min and then stained for HRS and EEA1 (A) or for HRS and CHMP4A (B). Boxed areas are magnified at right. The middle two columns show each indicated stain in grayscale; overlap of both stains in leftmost and rightmost columns appears white. Individual cells are outlined by white dashed lines; scale bars equal 10 μm (2 μm in magnified views). (C) RAW cells were treated with siRNA to deplete HRS or TSG101 for 2 days. Macrophages were then treated with LLOME for 15 min, and ubiquitin (FK2 antibody [red]) and CHMP4B (green) were examined. FK2-positive cells are outlined by white dashed lines. (D and E) Automated image analysis was used to quantify the mean fluorescent intensity (MFI) of FK2 (D) or the number of CHMP4B (E) punctae on 50 macrophages per sample. The number of CHMP4B punctae was compared in cells with FK2 MFI greater than and less than 700. Data are means ± SEM from one representative experiment from at least two independent experiments. *** * , P ≤ 0.0001, Student’s t test. ns, not significant. (F) HeLa cells were transfected with M. tuberculosis (Mtb) EsxG-EsxH or vector control, preincubated for 1 h with BAPTA-AM, and then treated with LLOME for 15 min and stained for CHMP4A (green) and EsxG-EsxH (red). EsxG-EsxH was detected with anti-V5 antibody. Nuclei were stained with DAPI. Images are maximum-intensity projections. Scale bar, 10 μm.

    Journal: mBio

    Article Title: Mycobacterium tuberculosis Type VII Secretion System Effectors Differentially Impact the ESCRT Endomembrane Damage Response

    doi: 10.1128/mBio.01765-18

    Figure Lengend Snippet: HRS is not required for ESCRT-III endomembrane damage response. (A and B) HRS does not assemble with ESCRT machinery on LLOME-disrupted endolysosomes. U20S cells were treated with LLOME or the solvent control for 10 min and then stained for HRS and EEA1 (A) or for HRS and CHMP4A (B). Boxed areas are magnified at right. The middle two columns show each indicated stain in grayscale; overlap of both stains in leftmost and rightmost columns appears white. Individual cells are outlined by white dashed lines; scale bars equal 10 μm (2 μm in magnified views). (C) RAW cells were treated with siRNA to deplete HRS or TSG101 for 2 days. Macrophages were then treated with LLOME for 15 min, and ubiquitin (FK2 antibody [red]) and CHMP4B (green) were examined. FK2-positive cells are outlined by white dashed lines. (D and E) Automated image analysis was used to quantify the mean fluorescent intensity (MFI) of FK2 (D) or the number of CHMP4B (E) punctae on 50 macrophages per sample. The number of CHMP4B punctae was compared in cells with FK2 MFI greater than and less than 700. Data are means ± SEM from one representative experiment from at least two independent experiments. *** * , P ≤ 0.0001, Student’s t test. ns, not significant. (F) HeLa cells were transfected with M. tuberculosis (Mtb) EsxG-EsxH or vector control, preincubated for 1 h with BAPTA-AM, and then treated with LLOME for 15 min and stained for CHMP4A (green) and EsxG-EsxH (red). EsxG-EsxH was detected with anti-V5 antibody. Nuclei were stained with DAPI. Images are maximum-intensity projections. Scale bar, 10 μm.

    Article Snippet: LLOME (no. L7393; Sigma-Aldrich) and BAPTA-AM (no. 15551; Cayman Chemical) were prepared in dimethyl sulfoxide (DMSO) and stored at −80°C in single-use aliquots.

    Techniques: Staining, Transfection, Plasmid Preparation

    Involvement of PKC-α in BK-induced ROS generation and MMP-9 expression in RBA-1. ( A ) Cells were pretreated without or with GF109203X (GF, 30 μM) or Gö6976 (Gö, 1 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. ( B ) Cells were pretreated with or without GF109203X (GF, 30 μM) or Gö6976 (Gö, 1 μM) for 1 h before exposure to 10 nM BK for 16 h. The total RNA was collected and analyzed by RT-PCR. ( C ) Cells were pretreated with or without BAPTA (BAP, 30 μM) or Gö (1 μM) for 1 h and then treated with 10 nM BK for the indicated time intervals (upper part) or 5 min (lower part). The membrane and cytosol fractions were prepared and analyzed by Western blotting. ( D ) Cells were pretreated without or with GF or Gö for 1 h before exposure to 10 nM BK for 5 min. The Nox activity and ROS generation were analyzed. ( E ) Cells were transfected with scramble (scra) or PKC-α siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography or Western blotting. ( F ) Cells were pretreated without or with Gö (1 μM), BAPTA (30 μM), or TG (1 μM) for 1 h before exposure to 10 nM BK for 5 min. The cell lysates were collected and analyzed by Western blotting using an anti-phospho-serine (p-Ser), p47 phox , or GAPDH antibody. Data are expressed as the mean ± SEM (n = 3). * P

    Journal: Cell Communication and Signaling : CCS

    Article Title: NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin-induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes

    doi: 10.1186/1478-811X-10-35

    Figure Lengend Snippet: Involvement of PKC-α in BK-induced ROS generation and MMP-9 expression in RBA-1. ( A ) Cells were pretreated without or with GF109203X (GF, 30 μM) or Gö6976 (Gö, 1 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. ( B ) Cells were pretreated with or without GF109203X (GF, 30 μM) or Gö6976 (Gö, 1 μM) for 1 h before exposure to 10 nM BK for 16 h. The total RNA was collected and analyzed by RT-PCR. ( C ) Cells were pretreated with or without BAPTA (BAP, 30 μM) or Gö (1 μM) for 1 h and then treated with 10 nM BK for the indicated time intervals (upper part) or 5 min (lower part). The membrane and cytosol fractions were prepared and analyzed by Western blotting. ( D ) Cells were pretreated without or with GF or Gö for 1 h before exposure to 10 nM BK for 5 min. The Nox activity and ROS generation were analyzed. ( E ) Cells were transfected with scramble (scra) or PKC-α siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography or Western blotting. ( F ) Cells were pretreated without or with Gö (1 μM), BAPTA (30 μM), or TG (1 μM) for 1 h before exposure to 10 nM BK for 5 min. The cell lysates were collected and analyzed by Western blotting using an anti-phospho-serine (p-Ser), p47 phox , or GAPDH antibody. Data are expressed as the mean ± SEM (n = 3). * P

    Article Snippet: BAPTA/AM, thapsigargin, GF109203X, Gö6976, apocynin, diphenyleneiodonium chloride (DPI), and tanshinone IIA (TSIIA) were from Biomol (Plymouth Meeting, PA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Transfection, Incubation, Zymography

    BK induces astrocytic migration through Nox/ROS-dependent AP-1 increasing MMP-9 gene expression. ( A ) Cells were incubated with BK (10 nM) for the indicated times (upper part), or pretreated with BAPTA (BAP, 30 μM), Gö6976 (Gö, 1 μM), Apo (10 μM), DPI (1 μM), or NAC (10 mM) for 1 h and then incubated with 10 nM BK for 30 min. The c-Fos and c-Jun AP-1 binding activity were analyzed by chromatin-IP (ChIP)-PCR assay. ( B ) Cells were transiently cotransfected with pGL-MMP9-Luc and pGal for 24 h, pretreated with BAPTA (BAP), Gö, Apo, DPI, or transfection with c-Fos or c-Jun siRNA for 24 h and then incubated with BK for 16 h. ( C ) Schematic representation of the different MMP-9-luciferase constructs, either wild-type (WT) or modified by single-point mutation of the AP-1 binding site (upper part). After cotransfection, luciferase activities of different MMP-9-promoter constructs after stimulation with or without BK (10 nM) for 16 h, were measured as relative MMP-9 promoter activity to that of β-galactosidase. ( D ) Cells were plated on 6-well culture plates, grew to confluence and starved with serum-free DMEM/F-12 medium for 24 h. Cells were pretreated with TG, Gö, Apo, DPI, NAC, TSIIA, or MMP-9i (9i) for 1 h and the monolayer cells were manually scratched with a blue tip as described in Methods, and then incubated with BK (10 nM) for 48 h. Phase contrast images of cells were taken at 48 h and the number of cell migration was counted as described in Methods. ( E ) Rat primary cultured astrocytes were pretreated with BAPTA (BAP), Gö, Apo, DPI, NAC, or TSIIA before exposure to BK for 16 h. The conditioned media and total RNA were collected and analyzed by zymography and RT-PCR. Data are expressed as the mean ± SEM (n = 3). # P

    Journal: Cell Communication and Signaling : CCS

    Article Title: NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin-induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes

    doi: 10.1186/1478-811X-10-35

    Figure Lengend Snippet: BK induces astrocytic migration through Nox/ROS-dependent AP-1 increasing MMP-9 gene expression. ( A ) Cells were incubated with BK (10 nM) for the indicated times (upper part), or pretreated with BAPTA (BAP, 30 μM), Gö6976 (Gö, 1 μM), Apo (10 μM), DPI (1 μM), or NAC (10 mM) for 1 h and then incubated with 10 nM BK for 30 min. The c-Fos and c-Jun AP-1 binding activity were analyzed by chromatin-IP (ChIP)-PCR assay. ( B ) Cells were transiently cotransfected with pGL-MMP9-Luc and pGal for 24 h, pretreated with BAPTA (BAP), Gö, Apo, DPI, or transfection with c-Fos or c-Jun siRNA for 24 h and then incubated with BK for 16 h. ( C ) Schematic representation of the different MMP-9-luciferase constructs, either wild-type (WT) or modified by single-point mutation of the AP-1 binding site (upper part). After cotransfection, luciferase activities of different MMP-9-promoter constructs after stimulation with or without BK (10 nM) for 16 h, were measured as relative MMP-9 promoter activity to that of β-galactosidase. ( D ) Cells were plated on 6-well culture plates, grew to confluence and starved with serum-free DMEM/F-12 medium for 24 h. Cells were pretreated with TG, Gö, Apo, DPI, NAC, TSIIA, or MMP-9i (9i) for 1 h and the monolayer cells were manually scratched with a blue tip as described in Methods, and then incubated with BK (10 nM) for 48 h. Phase contrast images of cells were taken at 48 h and the number of cell migration was counted as described in Methods. ( E ) Rat primary cultured astrocytes were pretreated with BAPTA (BAP), Gö, Apo, DPI, NAC, or TSIIA before exposure to BK for 16 h. The conditioned media and total RNA were collected and analyzed by zymography and RT-PCR. Data are expressed as the mean ± SEM (n = 3). # P

    Article Snippet: BAPTA/AM, thapsigargin, GF109203X, Gö6976, apocynin, diphenyleneiodonium chloride (DPI), and tanshinone IIA (TSIIA) were from Biomol (Plymouth Meeting, PA).

    Techniques: Migration, Expressing, Incubation, Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Transfection, Luciferase, Construct, Modification, Mutagenesis, Cotransfection, Cell Culture, Zymography, Reverse Transcription Polymerase Chain Reaction

    BK-induced Ca 2+ release from internal TG-sensitive Ca 2+ store plays a role in BK-induced MMP-9 expression. ( A ) Cells were pretreated without or with BAPTA/AM (30 μM) or TG (1 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. ( B ) Cells were pretreated with or without BAPTA/AM (30 μM) or TG (1 μM) for 1 h before exposure to 10 nM BK for 16 h. The total RNA was collected and analyzed by RT-PCR. ( C ) For Ca 2+ mobilization, confluent cultures of RBA-1 cells on glass coverslips were loaded with Fura-2/AM and fluorescent measurement of [Ca 2+ ] i was carried out in a dual excitation wavelength spectrofluorometer, with excitation at 340 nm and 380 nm. Cells were incubated in Ca 2+ -containing normal buffer (solid line) or Ca 2+ -free buffer (dot line) and then exposed to BK at 50 sec. In a Ca 2+ -free buffer, cells were pretreated with or without TG (1 μM), Apo (10 μM), or DPI (1 μM) for 30 min, exposed to BK at 50 sec, and then 2 mM Ca 2+ was added to the cells. ( D ) RBA-1 cells were pretreated without or with BAPTA/AM (30 μM) or TG (1 μM) for 1 h and then incubated with 10 nM BK for 5 min. The Nox activity and ROS generation were analyzed. Data are expressed as the mean ± SEM (n = 3). * P

    Journal: Cell Communication and Signaling : CCS

    Article Title: NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin-induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes

    doi: 10.1186/1478-811X-10-35

    Figure Lengend Snippet: BK-induced Ca 2+ release from internal TG-sensitive Ca 2+ store plays a role in BK-induced MMP-9 expression. ( A ) Cells were pretreated without or with BAPTA/AM (30 μM) or TG (1 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. ( B ) Cells were pretreated with or without BAPTA/AM (30 μM) or TG (1 μM) for 1 h before exposure to 10 nM BK for 16 h. The total RNA was collected and analyzed by RT-PCR. ( C ) For Ca 2+ mobilization, confluent cultures of RBA-1 cells on glass coverslips were loaded with Fura-2/AM and fluorescent measurement of [Ca 2+ ] i was carried out in a dual excitation wavelength spectrofluorometer, with excitation at 340 nm and 380 nm. Cells were incubated in Ca 2+ -containing normal buffer (solid line) or Ca 2+ -free buffer (dot line) and then exposed to BK at 50 sec. In a Ca 2+ -free buffer, cells were pretreated with or without TG (1 μM), Apo (10 μM), or DPI (1 μM) for 30 min, exposed to BK at 50 sec, and then 2 mM Ca 2+ was added to the cells. ( D ) RBA-1 cells were pretreated without or with BAPTA/AM (30 μM) or TG (1 μM) for 1 h and then incubated with 10 nM BK for 5 min. The Nox activity and ROS generation were analyzed. Data are expressed as the mean ± SEM (n = 3). * P

    Article Snippet: BAPTA/AM, thapsigargin, GF109203X, Gö6976, apocynin, diphenyleneiodonium chloride (DPI), and tanshinone IIA (TSIIA) were from Biomol (Plymouth Meeting, PA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Size-exclusion Chromatography, Activity Assay

    AP-1 (c-Fos/c-Jun) is essential for BK-induced MMP-9 expression through a Ca 2+ /PKC-α/Nox2/ROS cascade. ( A ) Cells were pretreated without or with tanshinone IIA (TSIIA, 10 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. ( B ) Cells were pretreated without or with BAPTA (BAP, 30 μM), Gö6976 (Gö, 1 μM), Apo (10 μM), DPI (1 μM), or NAC (10 mM) for 1 h before exposure to 10 nM BK for 30 min. The nuclear fraction was collected and analyzed by Western blotting using an anti-c-Fos, phospho-c-Jun, or c-Jun antibody. ( C ) Cells were transiently cotransfected with pAP1-Luc and pGal for 24 h, pretreated with BAPTA (BAP), TG, Gö, Apo, DPI, or NAC for 1 h and then incubated with BK for 6 h. The AP-1 promoter activity in the cell lysates was determined. ( D ) Cells were transfected with scramble (scra) or c-Fos/c-Jun siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography or Western blotting. Data are expressed as the mean ± SEM (n = 3). * P

    Journal: Cell Communication and Signaling : CCS

    Article Title: NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin-induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes

    doi: 10.1186/1478-811X-10-35

    Figure Lengend Snippet: AP-1 (c-Fos/c-Jun) is essential for BK-induced MMP-9 expression through a Ca 2+ /PKC-α/Nox2/ROS cascade. ( A ) Cells were pretreated without or with tanshinone IIA (TSIIA, 10 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. ( B ) Cells were pretreated without or with BAPTA (BAP, 30 μM), Gö6976 (Gö, 1 μM), Apo (10 μM), DPI (1 μM), or NAC (10 mM) for 1 h before exposure to 10 nM BK for 30 min. The nuclear fraction was collected and analyzed by Western blotting using an anti-c-Fos, phospho-c-Jun, or c-Jun antibody. ( C ) Cells were transiently cotransfected with pAP1-Luc and pGal for 24 h, pretreated with BAPTA (BAP), TG, Gö, Apo, DPI, or NAC for 1 h and then incubated with BK for 6 h. The AP-1 promoter activity in the cell lysates was determined. ( D ) Cells were transfected with scramble (scra) or c-Fos/c-Jun siRNA for 24 h, followed by incubation with 10 nM BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography or Western blotting. Data are expressed as the mean ± SEM (n = 3). * P

    Article Snippet: BAPTA/AM, thapsigargin, GF109203X, Gö6976, apocynin, diphenyleneiodonium chloride (DPI), and tanshinone IIA (TSIIA) were from Biomol (Plymouth Meeting, PA).

    Techniques: Expressing, Western Blot, Incubation, Activity Assay, Transfection, Zymography

    PDD-induced TRPV4 activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of 4α-PDD or DMSO. Luc -DMSO=cells transfected with Luc control siRNA and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by BAPTA-AM) and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P

    Journal: Oncogenesis

    Article Title: TRPV4 plays a role in breast cancer cell migration via Ca2+-dependent activation of AKT and downregulation of E-cadherin cell cortex protein

    doi: 10.1038/oncsis.2017.39

    Figure Lengend Snippet: PDD-induced TRPV4 activation triggered Ca 2+ influx. ( a ) Intracellular Ca 2+ measurement in control or Trpv4 knocked-down 4T07 cells treated with 10 μ m of 4α-PDD or DMSO. Luc -DMSO=cells transfected with Luc control siRNA and treated with DMSO; Luc -PDD, cells transfected with Luc control siRNA and treated with 4α-PDD; S1-DMSO, cells transfected with Trpv4 siRNA (sequence 1) and treated with DMSO; S1-PDD, cells transfected with Trpv4 siRNA (sequence 1) and treated with 4α-PDD; S3-DMSO, cells transfected with Trpv4 siRNA (sequence 3) and treated with DMSO; S3-PDD, cells transfected with TRPV4 siRNA (sequence 3) and treated with 4α-PDD. ( b ) Effects of chelating intracellular (by BAPTA-AM) and extracellular (by EGTA) Ca 2+ on 4α-PDD-induced, TRPV4-mediated signal transduction. 4T07 cells were untreated or pre-treated with 20 μ m of BAPTA-AM or 2 m m of EGTA for 1 h before being stimulated with 4α-PDD for 15 min (to assay for AKT activation) or 16 h (to assay for FAK activation and effects on E-cadherin and β-catenin). Cell lysates were then subject to immunoblotting using the indicated antibodies and bands densitometry performed using the ‘Image J’ software. The fold induction (ratio) was then obtained by dividing the values at PDD 15 min or PDD16 h over DMSO. Data points represent mean±s.e.m. of three independent experiments, * P

    Article Snippet: Reagents 4α-alpha-phorbol 12,13-didecanoate, BAPTA-AM and AKT inhibitor IV were from Merck KGaA (Darmstadt, Germany).

    Techniques: Activation Assay, Transfection, Sequencing, Transduction, Software

    ALG-2 stabilizes the localization of Sec31A at ER exit sites. (A and A′) HeLa cells were transfected with the ALG-2 siRNA vector (A-siRNA1), and double stained with anti-Sec31A (A) and anti-ALG-2 (A′) antibodies. Asterisks and arrowheads indicate ALG-2–depleted and undepleted cells, respectively. (B) HeLa cells transfected with the mock or A-siRNA1 vector were homogenized and fractionated into nuclear (N), cytoplasmic (C), and membrane (M) fractions. Proteins in each fraction were immunoblotted with antibodies against Sec31A (top) and ALG-2 (bottom). (C) The experiment shown in B was repeated four times, and the intensity of the Sec31A band in each fraction was quantified. The mean ± SD of the Sec31A ratio (%) in each fraction is shown. (D–E″) HeLa cells were treated without (D–D″) or with (E–E″) BAPTA-AM and double stained with anti-ALG-2 (D and E) and anti-Sec31A (D′ and E′) antibodies. D″ and E″ are merged images. N indicates nuclei. Bars, 20 μm.

    Journal: Molecular Biology of the Cell

    Article Title: The Ca2+

    doi: 10.1091/mbc.E06-05-0444

    Figure Lengend Snippet: ALG-2 stabilizes the localization of Sec31A at ER exit sites. (A and A′) HeLa cells were transfected with the ALG-2 siRNA vector (A-siRNA1), and double stained with anti-Sec31A (A) and anti-ALG-2 (A′) antibodies. Asterisks and arrowheads indicate ALG-2–depleted and undepleted cells, respectively. (B) HeLa cells transfected with the mock or A-siRNA1 vector were homogenized and fractionated into nuclear (N), cytoplasmic (C), and membrane (M) fractions. Proteins in each fraction were immunoblotted with antibodies against Sec31A (top) and ALG-2 (bottom). (C) The experiment shown in B was repeated four times, and the intensity of the Sec31A band in each fraction was quantified. The mean ± SD of the Sec31A ratio (%) in each fraction is shown. (D–E″) HeLa cells were treated without (D–D″) or with (E–E″) BAPTA-AM and double stained with anti-ALG-2 (D and E) and anti-Sec31A (D′ and E′) antibodies. D″ and E″ are merged images. N indicates nuclei. Bars, 20 μm.

    Article Snippet: By contrast, the level of p125 at ER exit sites was unaffected when the mislocalization of ALG-2 was induced by BAPTA-AM (Supplemental Figures S2, B–B″ and C–C″).

    Techniques: Transfection, Plasmid Preparation, Staining

    Cytoskeleton of neurites of rat hippocampal neurons after treatment with BAPTA/AM and LPA (Confocal Microscopy). In the magnified images, the red microfilaments in protrusion terminals showed increased fluorescence intensity, and the spinous protrusions

    Journal: American Journal of Translational Research

    Article Title: Rho kinase regulates neurite outgrowth of hippocampal neurons via calcium dependent cytoskeleton regulation

    doi:

    Figure Lengend Snippet: Cytoskeleton of neurites of rat hippocampal neurons after treatment with BAPTA/AM and LPA (Confocal Microscopy). In the magnified images, the red microfilaments in protrusion terminals showed increased fluorescence intensity, and the spinous protrusions

    Article Snippet: DMEM/F12 (Invitrogen), Neurobasal medium, B27 supplement (Gibco), polylysine (Sigma), cytosine arabinoside (Ara-C) (Invitrogen), fetal bovine serum (Gibco), anti-rat α-tubulin antibody (Sigma), Rhodamine-Phalloidin (Invitrogen), FITC conjugated fluorescent secondary antibody (Amersham pharmacia), fluorescent secondary antibody (Jackson), LPA, Y27632 (Sigma), BAPTA/AM (Merck), and Fluo-3/AM (Molecular probes) were used in the present study.

    Techniques: Confocal Microscopy, Fluorescence

    Cytoskeleton of rat hippocampal neurons after treatment with BAPTA/AM and LPA (Confocal Microscopy). Neurite branches of neurons increased, the microfilaments and microtubules were observed in the neuronal body and protrusions of different levels, and

    Journal: American Journal of Translational Research

    Article Title: Rho kinase regulates neurite outgrowth of hippocampal neurons via calcium dependent cytoskeleton regulation

    doi:

    Figure Lengend Snippet: Cytoskeleton of rat hippocampal neurons after treatment with BAPTA/AM and LPA (Confocal Microscopy). Neurite branches of neurons increased, the microfilaments and microtubules were observed in the neuronal body and protrusions of different levels, and

    Article Snippet: DMEM/F12 (Invitrogen), Neurobasal medium, B27 supplement (Gibco), polylysine (Sigma), cytosine arabinoside (Ara-C) (Invitrogen), fetal bovine serum (Gibco), anti-rat α-tubulin antibody (Sigma), Rhodamine-Phalloidin (Invitrogen), FITC conjugated fluorescent secondary antibody (Amersham pharmacia), fluorescent secondary antibody (Jackson), LPA, Y27632 (Sigma), BAPTA/AM (Merck), and Fluo-3/AM (Molecular probes) were used in the present study.

    Techniques: Confocal Microscopy

    Monocytes and PMN phagocytosis—Ca 2+ chelation. Whole blood samples were pre-treated with 100 μM of the calcium chelator BAPTA-AM for 30 min and incubated in a water bath for 20 min at 37°C with opsonized E . coli (1-2x10 9 /ml). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the monocytes population. A regular phagocytosis was observed on monocytes and PMN from HD and XLA even after calcium chelation demonstrating that the process is Ca 2+ independent. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean valu es and bars standard deviation. In (A and B) are shown monocytes and in (C and D) PMN phagocytosis of representative XLA patient: black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).

    Journal: PLoS ONE

    Article Title: The lack of BTK does not impair monocytes and polymorphonuclear cells functions in X-linked agammaglobulinemia under treatment with intravenous immunoglobulin replacement

    doi: 10.1371/journal.pone.0175961

    Figure Lengend Snippet: Monocytes and PMN phagocytosis—Ca 2+ chelation. Whole blood samples were pre-treated with 100 μM of the calcium chelator BAPTA-AM for 30 min and incubated in a water bath for 20 min at 37°C with opsonized E . coli (1-2x10 9 /ml). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the monocytes population. A regular phagocytosis was observed on monocytes and PMN from HD and XLA even after calcium chelation demonstrating that the process is Ca 2+ independent. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean valu es and bars standard deviation. In (A and B) are shown monocytes and in (C and D) PMN phagocytosis of representative XLA patient: black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).

    Article Snippet: Also in PMN, BAPTA-AM induced a mild reduction of the E . coli -induced oxidative burst in XLA than HD ( ).

    Techniques: Incubation, Fluorescence, Standard Deviation

    PMN oxidative burst—Ca 2+ chelation. Whole blood samples were pre-treated with 100 μM of the calcium chelator BAPTA-AM for 30 min and incubated in a water bath for 20 min at 37°C with opsonized E . coli (1-2x10 9 /ml) and PMA (1.62 mM). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the PMN population. (A) BAPTA-AM treatment cause a stronger reduction of E . coli -induced oxidative burst in HD than XLA (▪▪▪p = 0.003). (A) The average reduction in HD was about 75% (▪p = 0.01) and it was about 50% in XLA patients (▪▪p = 0.02). (D) BAPTA-AM did not affect the oxidative burst induced by PMA both HD and XLA patients. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean values and bars standard deviation. Statistical significance, indicated as p value, was determined by the nonparametric Mann-Whitney test and Wilcoxon Signed Rank test. ( B and C) It shown PMN E . coli- induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+). (E and F) It shown PMN PMA-induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).

    Journal: PLoS ONE

    Article Title: The lack of BTK does not impair monocytes and polymorphonuclear cells functions in X-linked agammaglobulinemia under treatment with intravenous immunoglobulin replacement

    doi: 10.1371/journal.pone.0175961

    Figure Lengend Snippet: PMN oxidative burst—Ca 2+ chelation. Whole blood samples were pre-treated with 100 μM of the calcium chelator BAPTA-AM for 30 min and incubated in a water bath for 20 min at 37°C with opsonized E . coli (1-2x10 9 /ml) and PMA (1.62 mM). The conversion of DHR 123 to R 123 was used to evaluate the intracellular ROS production. FSC and SSC characteristics were used to identify the PMN population. (A) BAPTA-AM treatment cause a stronger reduction of E . coli -induced oxidative burst in HD than XLA (▪▪▪p = 0.003). (A) The average reduction in HD was about 75% (▪p = 0.01) and it was about 50% in XLA patients (▪▪p = 0.02). (D) BAPTA-AM did not affect the oxidative burst induced by PMA both HD and XLA patients. Results are expressed as Mean Fluorescence Intensity. Histograms denote mean values and bars standard deviation. Statistical significance, indicated as p value, was determined by the nonparametric Mann-Whitney test and Wilcoxon Signed Rank test. ( B and C) It shown PMN E . coli- induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+). (E and F) It shown PMN PMA-induced oxidative burst in a representative HD and XLA patient, black peak BAPTA-AM(-) and gray peak BAPTA-AM(+).

    Article Snippet: Also in PMN, BAPTA-AM induced a mild reduction of the E . coli -induced oxidative burst in XLA than HD ( ).

    Techniques: Incubation, Fluorescence, Standard Deviation, MANN-WHITNEY