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  • 99
    Thermo Fisher bapta am
    Electron microscopy of mitochondria and cytoplasm of curcumin-treated Huh-7 cells. ( a ) Electron micrographs illustrating time-dependent mitochondrial swelling in cells incubated with 25 μ M curcumin for 1, 3 or 24 h. ( b and c ) Western blots of cytochrome c released in cells treated with different concentrations of curcumin, in the presence or absence of cyclosporine A. Triton X-100 treatment results in the release of the total cytochrome c available in cells. ( c ) Quantification of cytochrome c released in cells treated with different concentrations of curcumin and inhibition of this release by <t>EGTA-AM</t> or <t>BAPTA-AM,</t> which control intracellular calcium levels after curcumin addition. ( d ) Electron microscopy picture of autophagic vacuoles in cells treated with 25 μ M curcumin for 24 h. AV, autophagic vacuoles; SM, swollen mitochondria; V, vacuole.
    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
    β1-Integrin coclusters with IgE-FcεRI on patterned DNP-SLB in an active process that depends on actin polymerization and intracellular Ca 2+ . (A) Sensitized RBL cells were labeled with anti-β1 mAb (green) after incubation at 37°C on patterned features of DNP-SLB (red, left pair) or <t>DNP-BSA</t> (red, right pair). (B) Same as left pair in A, except that cells were pretreated with 2 µM CytoD. (C) Same as left pair in A, except that cells were incubated on patterned DNP-SLB surfaces in the presence of 1.5 µM RGD peptide. (D) Sensitized cells incubated at 4°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (E) Sensitized cells pretreated with 5 µM <t>BAPTA-AM,</t> incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (F) Sensitized cells pretreated with 10 µg/ml anti-α4 mAb, incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (G) Radial analysis for samples represented in A–C (carried out as described in the legend to Figure 1 ) quantify β1-integrin recruitment to DNP-SLB features. N = 30 cells from three independent experiments for each sample type. Error bars represent SEM; statistical significance: **, p ≤ 0.01. Scale bars: 10 µm.
    Bapta Am, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris bapta am
    A , immunoblotting for RhoG on platelet whole cell lysates demonstrated RhoG expression in human platelets. GST-ELMO fusion protein pulldown assays were then used to assess activation of RhoG in human platelets. The fusion protein interacted specifically with the activated (GTP-bound) form of RhoG. Platelet whole cell lysates ( WCL ) were stimulated with cross-linked CRP or with bovine α-thrombin at the indicated final concentrations, and the time course of RhoG activation was assessed by immunoblotting for RhoG in the pulldown samples. B , the GST-ELMO activation assay was then used to evaluate the effect of various pharmacologic inhibitors on RhoG activation in CRP-stimulated human platelets. Inhibitors were incubated with washed platelet suspensions for 10 min prior to stimulation. Compared with the appropriate vehicle control dimethyl sulfoxide ( DMSO ) or Tyrode's HEPES buffer ( Tyr ), RhoG activation was reduced by Su6656 (20 μ m ) and piceatannol ( Piceat ; 60 μ m ) but not by U73122 (10 μ m ), wortmannin ( Wort ; 100 n m ), <t>BAPTA-AM</t> ( B-AM ; 10 μ m ), BIM1 (10 μ m ), Abciximab ( Abcix ; 10 μg/ml), or <t>AR-C66096</t> ( AR-C ; 1 μ m ). This suggests that RhoG activation in human platelets is dependent on Src family kinases and Syk activity and that RhoG activation is independent of PLC, PI3K, or PKC activity and does not require intracellular calcium or signaling downstream of α IIb β 3 or P2Y 12 receptors. All images are representative of three separate experiments. The white lines in the lower immunoblot panels denote gaps between different sections of the same membrane. C , the effects of these inhibitors on RhoG activation were quantified by densitometry using ImageJ. Bars represent the mean, and error bars represent S.E. from at least three membranes/group. The dotted gray line represents the mean level of the dimethyl sulfoxide controls.
    Bapta Am, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Dojindo Labs bapta am
    Ca 2+ -depletion causes defects in the localisation of CENP-F at the outer kinetochore. Fluorescence images of metaphase cells after control, <t>BAPTA-AM</t> and BAPTA (with ionomycin) treatments for the localisation of Aurora B ( a ), Mis12 ( b ), Hec1 ( c ) and CENP-F ( d ). Bar, 5 µm.
    Bapta Am, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 93/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Selleck Chemicals bapta am
    ACYP2 promotes STAT3 phosphorylation in glioma cells through regulating intracellular Ca 2+ homeostasis and calpain activity. Cells transfected with the indicated shRNAs were treated with 5 μM <t>BAPTA-AM</t> for 6 h ( a ), 10 μM <t>calpeptin</t> for 10 h ( b ), 10 μM Na3VO4 for 1 h ( c ), or the vehicle. Western blot analysis was then performed to test their effect on STAT3 phosphorylation. GAPDH was used as a loading control. d , Cells transfected with the indicated shRNAs were treated with the vehicle or 10 μM Na3VO4 for 1 h, and the MTT assay was then carried out to evaluate their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). Cells stably knocking down ACYP2 and control cells were transfected with siRNA targeting PTP1B or not. e , Western blot analysis was then performed to evaluate their effect on STAT3 phosphorylation. f , MTT assay was performed to assess their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). **, 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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    97
    Abcam bapta am
    Effect of seawater exposure on the average fluorescence intensity in A549 cells. (A) Seawater exposure evoked a rapid fluorescence intensity increase that represented [Ca 2+ ]c elevation. Left panel, A549 cells without stimulation; right panel, A549 cells exposed to seawater. (B) Quantification of fluorescence intensity following seawater treatment. (C) Pretreatment with <t>BAPTA-AM</t> may reverse the elevation of [Ca 2+ ]c. (D) Chelation of calcium with <t>EGTA</t> reduced the rise of [Ca 2+ ]c triggered by seawater. (E) Average fluorescence intensities for all groups. * P
    Bapta Am, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical bapta am
    Effect of <t>BAPTA-AM</t> on <t>RuV</t> infection. (A) Endocytic infection. RuV or SFV was prebound to Vero cells on ice for 1.5 h in binding medium, shifted to 37°C for 20 min in medium containing 2 mM CaCl 2 and the indicated concentration of BAPTA-AM, and
    Bapta Am, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology bapta am
    Gene expression regulated by <t>celastrol-mediated</t> Ca 2+ mobilization analysis in RASF. RT-qPCR independent validation from RASF cells untreated (control), or treated with 1 μM Celastrol (Cel), and 10 μM <t>BAPTA/AM</t> (BM) for 24 h. Gene expressions were normalized to GAPDH, relative to control, and analyzed using the 2 −ΔΔCT method. The data is represented as the mean ± SD. ** P ≤ 0.01; *** P ≤ 0.001 compared with control. # P ≤ 0.05; ## P ≤ 0.01; ### P ≤ 0.001 compared with Celastrol.
    Bapta Am, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH bapta am
    Ca 2+ /CaMKII-dependent LTA-mediated activation of JNK and c-Jun in RBA-1 cells . (A) Cells were pretreated with <t>BAPTA</t> (upper part) or TG (lower part) for 1 h and then incubated with LTA for the indicated time intervals. Cell lysates were assayed by western blot using anti-phospho-CaMKII antibody. (B) Cells were pretreated <t>CaMI</t> or KN-62 for 1 h and then incubated with LTA for the indicated time intervals. Cell lysates were assayed by western blot using an anti-phospho-JNK or phospho-c-Jun antibody. The membranes were stripped and re-probed with anti-GAPDH antibody as a control. Data are expressed as mean of three independent experiments (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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    93
    Merck KGaA bapta am
    Peroxynitrite negatively regulates mitochondrial degradation. (a) and (b) SH-SY5Y cells overexpressing parkin were treated with <t>BAPTA-AM</t> (10 μM) (a) or <t>FeTPPS</t> (5 μM) (b) with CCCP (10 μM) for 3 hr, and lysates were analysed by SNO-RAC. The quantity of S-nitrosylated parkin, as measured by scanning densitometry, is expressed as a percentage of control, normalized with respect to total parkin. Data shown are mean ± SE (n = 3); *p
    Bapta Am, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem bapta am
    Effect of AD2900 on proliferation is dependent on <t>cAMP</t> reduction and calcium signal transduction The effect of Edelfosine (Et-18-OCH 3 ) (Et), <t>BAPTA-AM</t> (BAPTA), or 8-bromo-cAMP (8-b-CAMP) on the inhibition of PBMC proliferation was tested in a 5-day MLR experiment. The cells were incubated with 1 μM Edelfosine, 0.2 μM BAPTA-AM, or 5 μM 8-bromo-cAMP for 1 hour before the addition of sphingolipid analogues, 2 μM AD2900 (AD) (A) , FTY720 (FTY) (B) , or S1P (C) . Significances are compared to AD2900/FTY/S1P without preincubation, respectively. Graphs summarize the results of at least four independent experiments. Results of Student’s t -test: *(P
    Bapta Am, supplied by Enzo Biochem, 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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    91
    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: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    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: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Electron microscopy of mitochondria and cytoplasm of curcumin-treated Huh-7 cells. ( a ) Electron micrographs illustrating time-dependent mitochondrial swelling in cells incubated with 25 μ M curcumin for 1, 3 or 24 h. ( b and c ) Western blots of cytochrome c released in cells treated with different concentrations of curcumin, in the presence or absence of cyclosporine A. Triton X-100 treatment results in the release of the total cytochrome c available in cells. ( c ) Quantification of cytochrome c released in cells treated with different concentrations of curcumin and inhibition of this release by EGTA-AM or BAPTA-AM, which control intracellular calcium levels after curcumin addition. ( d ) Electron microscopy picture of autophagic vacuoles in cells treated with 25 μ M curcumin for 24 h. AV, autophagic vacuoles; SM, swollen mitochondria; V, vacuole.

    Journal: Cell Death Discovery

    Article Title: Curcumin induces crosstalk between autophagy and apoptosis mediated by calcium release from the endoplasmic reticulum, lysosomal destabilization and mitochondrial events

    doi: 10.1038/cddiscovery.2015.17

    Figure Lengend Snippet: Electron microscopy of mitochondria and cytoplasm of curcumin-treated Huh-7 cells. ( a ) Electron micrographs illustrating time-dependent mitochondrial swelling in cells incubated with 25 μ M curcumin for 1, 3 or 24 h. ( b and c ) Western blots of cytochrome c released in cells treated with different concentrations of curcumin, in the presence or absence of cyclosporine A. Triton X-100 treatment results in the release of the total cytochrome c available in cells. ( c ) Quantification of cytochrome c released in cells treated with different concentrations of curcumin and inhibition of this release by EGTA-AM or BAPTA-AM, which control intracellular calcium levels after curcumin addition. ( d ) Electron microscopy picture of autophagic vacuoles in cells treated with 25 μ M curcumin for 24 h. AV, autophagic vacuoles; SM, swollen mitochondria; V, vacuole.

    Article Snippet: Skulachev (Lomonosov Moscow State University, Moscow, Russia), EGTA-AM (cell permeable) and BAPTA-AM (cell permeable) were from Life Technology, Invitrogen.

    Techniques: Electron Microscopy, Incubation, Western Blot, Inhibition

    Second messenger alterations and control of IL-6 release mediated by adenosine/A2bAR. Intracellular cAMP concentrations (A, B) were analyzed by ELISA. H69 cells were stimulated with 100 μM adenosine for 2 h in 96-well plates (5,000 cells per well) in H69 complete media in the presence of A2bAR antagonist (A) MRS-1754 (1 μM; n = 3, performed in triplicates) or (B) 72 h after transfection with an A2bAR-specific siRNA ( n = 4, performed in triplicate). #Significant when compared to the untreated cells; *significant when compared to vehicle (A) or nontarget siRNA (B). (C, D) Intracellular Ca 2+ mobilization was observed by confocal microscopy. Glass coverslip-plated, fluo-4/AM-loaded H69 cells were perifused with HEPES buffer alone or containing 500 μM adenosine. ATP (100 μM) was used as a positive control. Changes in fluorescence intensity were recorded with a Zeiss 510 confocal microscope. (C) Relative fluorescence intensity of individual cells; 29 individual cells were analyzed. (D) Representative image of the dye intensity variation during the course of the experiment. Scale bar: 50 μm. (E) H69 cells were stimulated by 100 μM of adenosine for 2 h in six-well plates (150,000 cells per well) in H69 complete media. RNA was extracted, and IL-6 mRNA expression was determined by qPCR. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation ( n = 3 for cAMPs-RP and n = 7 for BAPTA/AM). (F) H69 cells were stimulated by 100 μM adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate). #Significant when compared to unstimulated cells; *significant when compared to vehicle. (G) H69 cells were stimulated by 100 μM of 8-pCPT-2-O-Me-cAMP-AM (8-pCPT) or adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate).

    Journal: Gene Expression

    Article Title: The Cholangiocyte Adenosine–IL-6 Axis Regulates Survival During Biliary Cirrhosis

    doi: 10.3727/105221617X15042723767876

    Figure Lengend Snippet: Second messenger alterations and control of IL-6 release mediated by adenosine/A2bAR. Intracellular cAMP concentrations (A, B) were analyzed by ELISA. H69 cells were stimulated with 100 μM adenosine for 2 h in 96-well plates (5,000 cells per well) in H69 complete media in the presence of A2bAR antagonist (A) MRS-1754 (1 μM; n = 3, performed in triplicates) or (B) 72 h after transfection with an A2bAR-specific siRNA ( n = 4, performed in triplicate). #Significant when compared to the untreated cells; *significant when compared to vehicle (A) or nontarget siRNA (B). (C, D) Intracellular Ca 2+ mobilization was observed by confocal microscopy. Glass coverslip-plated, fluo-4/AM-loaded H69 cells were perifused with HEPES buffer alone or containing 500 μM adenosine. ATP (100 μM) was used as a positive control. Changes in fluorescence intensity were recorded with a Zeiss 510 confocal microscope. (C) Relative fluorescence intensity of individual cells; 29 individual cells were analyzed. (D) Representative image of the dye intensity variation during the course of the experiment. Scale bar: 50 μm. (E) H69 cells were stimulated by 100 μM of adenosine for 2 h in six-well plates (150,000 cells per well) in H69 complete media. RNA was extracted, and IL-6 mRNA expression was determined by qPCR. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation ( n = 3 for cAMPs-RP and n = 7 for BAPTA/AM). (F) H69 cells were stimulated by 100 μM adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Cells were preincubated with BAPTA/AM (50 μM) or cAMPs-RP (100 μM) 30 min prior to the stimulation. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate). #Significant when compared to unstimulated cells; *significant when compared to vehicle. (G) H69 cells were stimulated by 100 μM of 8-pCPT-2-O-Me-cAMP-AM (8-pCPT) or adenosine for 4 h in 48-well plates (30,000 cells per well) in H69 complete media. Supernatants were collected and centrifuged, and IL-6 content was analyzed by ELISA ( n = 3, performed in triplicate).

    Article Snippet: Transfected and nontransfected cells were stimulated with adenosine (Tocris Bioscience, Minneapolis, MN, USA) and 5′- N -ethylcarboxamidoadenosine (NECA ; Tocris Bioscience) for 2 h (qPCR and cAMP ELISA) or 4 h (IL-6 ELISA) +/−: selective A2bAR antagonist MRS-1754 (1 μM ; Tocris Bioscience), calcium chelator BAPTA/AM (50 μM; Life Technologies) cyclic AMP analog acting as an inhibitor of cAMP-dependent protein kinase, ( R )-adenosine, cyclic 3′,5′-(hydrogenphosphorothioate) triethylammonium (cAMPS-RP; 100 μM; Tocris Bioscience).

    Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Confocal Microscopy, Positive Control, Fluorescence, Microscopy, Expressing, Real-time Polymerase Chain Reaction

    Ca 2+ and CaMKII are implicated in TNF-induced renal vascular permeability. ( a–c) Male C57BL/6J mice were pre-injected i.v. with vehicle (6% DMSO in DPBS) or BAPTA-AM (2 mM in DPBS) 30 min prior to i.v. injection of TNF (LD 80 ); control condition is BAPTA-AM injected (i.v., 2 mM) without TNF; n = total number of mice per group. (a,b) Cumulative survival rates and body temperatures presented as a function of time. Pool of 3 independent experiments; * p = 0.0324. (c) Vascular permeability shown as RLU of FITC-dextran in colon, ileum, lungs and kidneys 6 hr after injection of control (BAPTA-AM) or TNF. Pool of 2 independent experiments. Colon: ** p = 0.0086, *** p = 0.0003; Lungs: * p = 0.0469; Kidney: **** p

    Journal: Scientific Reports

    Article Title: Blocking connexin43 hemichannels protects mice against tumour necrosis factor-induced inflammatory shock

    doi: 10.1038/s41598-019-52900-4

    Figure Lengend Snippet: Ca 2+ and CaMKII are implicated in TNF-induced renal vascular permeability. ( a–c) Male C57BL/6J mice were pre-injected i.v. with vehicle (6% DMSO in DPBS) or BAPTA-AM (2 mM in DPBS) 30 min prior to i.v. injection of TNF (LD 80 ); control condition is BAPTA-AM injected (i.v., 2 mM) without TNF; n = total number of mice per group. (a,b) Cumulative survival rates and body temperatures presented as a function of time. Pool of 3 independent experiments; * p = 0.0324. (c) Vascular permeability shown as RLU of FITC-dextran in colon, ileum, lungs and kidneys 6 hr after injection of control (BAPTA-AM) or TNF. Pool of 2 independent experiments. Colon: ** p = 0.0086, *** p = 0.0003; Lungs: * p = 0.0469; Kidney: **** p

    Article Snippet: BAPTA-AM (Invitrogen, B6769) was dissolved in dimethylsulfoxide (DMSO) (+0.01% pluronic® (Invitrogen)) and further diluted in endotoxin-free PBS for in vivo use.

    Techniques: Permeability, Mouse Assay, Injection

    β1-Integrin coclusters with IgE-FcεRI on patterned DNP-SLB in an active process that depends on actin polymerization and intracellular Ca 2+ . (A) Sensitized RBL cells were labeled with anti-β1 mAb (green) after incubation at 37°C on patterned features of DNP-SLB (red, left pair) or DNP-BSA (red, right pair). (B) Same as left pair in A, except that cells were pretreated with 2 µM CytoD. (C) Same as left pair in A, except that cells were incubated on patterned DNP-SLB surfaces in the presence of 1.5 µM RGD peptide. (D) Sensitized cells incubated at 4°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (E) Sensitized cells pretreated with 5 µM BAPTA-AM, incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (F) Sensitized cells pretreated with 10 µg/ml anti-α4 mAb, incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (G) Radial analysis for samples represented in A–C (carried out as described in the legend to Figure 1 ) quantify β1-integrin recruitment to DNP-SLB features. N = 30 cells from three independent experiments for each sample type. Error bars represent SEM; statistical significance: **, p ≤ 0.01. Scale bars: 10 µm.

    Journal: Molecular Biology of the Cell

    Article Title: The FcεRI signaling cascade and integrin trafficking converge at patterned ligand surfaces

    doi: 10.1091/mbc.E17-03-0208

    Figure Lengend Snippet: β1-Integrin coclusters with IgE-FcεRI on patterned DNP-SLB in an active process that depends on actin polymerization and intracellular Ca 2+ . (A) Sensitized RBL cells were labeled with anti-β1 mAb (green) after incubation at 37°C on patterned features of DNP-SLB (red, left pair) or DNP-BSA (red, right pair). (B) Same as left pair in A, except that cells were pretreated with 2 µM CytoD. (C) Same as left pair in A, except that cells were incubated on patterned DNP-SLB surfaces in the presence of 1.5 µM RGD peptide. (D) Sensitized cells incubated at 4°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (E) Sensitized cells pretreated with 5 µM BAPTA-AM, incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (F) Sensitized cells pretreated with 10 µg/ml anti-α4 mAb, incubated at 37°C on patterned DNP-SLB (red) surfaces and labeled with anti-β1 (green, left pair) or A488-IgE (green, right pair). (G) Radial analysis for samples represented in A–C (carried out as described in the legend to Figure 1 ) quantify β1-integrin recruitment to DNP-SLB features. N = 30 cells from three independent experiments for each sample type. Error bars represent SEM; statistical significance: **, p ≤ 0.01. Scale bars: 10 µm.

    Article Snippet: BSA, bovine FN, sulfinpyrazone, BAPTA-AM, (3-mercaptopropyl)trimethoxysilane (MPTS), and N-(γ-maleimidobutyryloxy)succinimide ester (GMBS) were purchased from Sigma-Aldrich.

    Techniques: Labeling, Incubation

    Circulating histone H2A in plasma from dengue-infected patients activates platelets. ( A-B ) Platelets isolated from healthy volunteers were stimulated with recombinant human histone H2A at the indicated concentrations. ( A ) Platelet surface P-selectin (CD62-P) was evaluated 1, 2 and 4 hours post stimulation by flow cytometry and ( B ) PF4/CXCL4 concentration was measured in supernatants 4 hours post stimulation. ( C-F ) Surface P-selectin and PF4/CXCL4 concentration in the supernatants of platelets stimulated with recombinant histone H2A for 2 hour in the presence of ( C-D ) the calcium chelator BAPTA-AM (20 μM) or vehicle (DMSO); or ( E-F ) blocking antibody against TLR4 (20 μg/mL) or isotype matched IgG. ( G-H ) P-selectin expression on platelets exposed to ( G ) plasma from six dengue-infected patients (dengue plasma) or four heterologous healthy volunteers (control plasma) for the indicated time-points; and ( H ) platelets exposed to dengue plasma or control plasma for 4 hours in the presence of anti-histone H2A (20 μg/mL) or isotype matched IgG. Bars represent mean ± standard error of the mean of 3 independent experiments ( A-F ) and of 4 to 6 independent plasma donors ( G-H ). * indicates p

    Journal: PLoS Pathogens

    Article Title: Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

    doi: 10.1371/journal.ppat.1006385

    Figure Lengend Snippet: Circulating histone H2A in plasma from dengue-infected patients activates platelets. ( A-B ) Platelets isolated from healthy volunteers were stimulated with recombinant human histone H2A at the indicated concentrations. ( A ) Platelet surface P-selectin (CD62-P) was evaluated 1, 2 and 4 hours post stimulation by flow cytometry and ( B ) PF4/CXCL4 concentration was measured in supernatants 4 hours post stimulation. ( C-F ) Surface P-selectin and PF4/CXCL4 concentration in the supernatants of platelets stimulated with recombinant histone H2A for 2 hour in the presence of ( C-D ) the calcium chelator BAPTA-AM (20 μM) or vehicle (DMSO); or ( E-F ) blocking antibody against TLR4 (20 μg/mL) or isotype matched IgG. ( G-H ) P-selectin expression on platelets exposed to ( G ) plasma from six dengue-infected patients (dengue plasma) or four heterologous healthy volunteers (control plasma) for the indicated time-points; and ( H ) platelets exposed to dengue plasma or control plasma for 4 hours in the presence of anti-histone H2A (20 μg/mL) or isotype matched IgG. Bars represent mean ± standard error of the mean of 3 independent experiments ( A-F ) and of 4 to 6 independent plasma donors ( G-H ). * indicates p

    Article Snippet: To characterize the mechanisms involved in platelet activation by cell free histone H2A, platelets were pretreated with the calcium chelator BAPTA-AM (Sigma) (20 μM) or anti-TLR4 neutralizing antibodies (eBioscience 169917–82) (20 μg/mL) for 30 min prior stimulation with histone H2A.

    Techniques: Infection, Isolation, Recombinant, Flow Cytometry, Cytometry, Concentration Assay, Blocking Assay, Expressing

    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:

    Calcium as a second messenger for sperm chemotaxis. A , B , D , E , F , G and H : Percentage of oriented spermatozoa (OS) towards 10 pM P gradient after treating the cells with BAPTA-AM (1 min), sEBSS culture medium with and without extracellular calcium, Nifedipine (1 min), SKF96365 (1 min), TMB-8 (1 min), 2-APB (15 min) and Tetracaine (15 min), respectively. C : Ca 2+ signal (change in Oregon green 1 BAPTA florescence) in immobilized human spermatozoa, black line shows the mean of cells that responded to a step application of pM P and gray line shows the mean of those cells in which no response was detectable. Black arrow marks time of arrival of P in the imaging chamber. HAM was assayed as a negative control. Each bar shows mean ± SEM. a Significant differences vs. without inhibitor (p

    Journal: PLoS ONE

    Article Title: Molecular Mechanism for Human Sperm Chemotaxis Mediated by Progesterone

    doi: 10.1371/journal.pone.0008211

    Figure Lengend Snippet: Calcium as a second messenger for sperm chemotaxis. A , B , D , E , F , G and H : Percentage of oriented spermatozoa (OS) towards 10 pM P gradient after treating the cells with BAPTA-AM (1 min), sEBSS culture medium with and without extracellular calcium, Nifedipine (1 min), SKF96365 (1 min), TMB-8 (1 min), 2-APB (15 min) and Tetracaine (15 min), respectively. C : Ca 2+ signal (change in Oregon green 1 BAPTA florescence) in immobilized human spermatozoa, black line shows the mean of cells that responded to a step application of pM P and gray line shows the mean of those cells in which no response was detectable. Black arrow marks time of arrival of P in the imaging chamber. HAM was assayed as a negative control. Each bar shows mean ± SEM. a Significant differences vs. without inhibitor (p

    Article Snippet: Reagents HAM F-10 medium with L-glutamine and 25 mM Hepes (Invitrogen, USA); Human Serum Albumin (UNC, Argentina); Nifedipine, 2-APB (Calbiochem, Germany); KH7 (ChemDiv, USA); mouse monoclonal anti-phosphotyrosine, clone 4G10 (UPSTATE, USA); rabbit polyclonal anti-Rec30; fluorescent mounting medium (DAKO, Denmark); Poli-L-lysine (BIOCHROM AG, UK); Poli-D-lysine (BD Bioscience, UK); Streptavidin-Peroxidase (LAB-VISION, UK); cGMP and cAMP Enzymeimmunoassay Biotrak (EIA) System (Amersham Biosciences, UK); db-cAMP, db-cGMP, ddAdo, ODQ, KT5823, KT5720, SKF96365, LY-83.583, TMB-8, Tetracaine, BAPTA, BAPTA-AM, calcium ionophore A23187, Biotin-conjugated anti-mouse, TRITC-conjugated anti-rabbit, FITC-conjugated anti-mouse IgG, Percoll, DMSO, BSA, Triton X- 100, Formaldehyde, Progesterone and “AEC staining kit” were from SIGMA-Aldrich (USA).

    Techniques: Chemotaxis Assay, Imaging, Negative Control

    A , immunoblotting for RhoG on platelet whole cell lysates demonstrated RhoG expression in human platelets. GST-ELMO fusion protein pulldown assays were then used to assess activation of RhoG in human platelets. The fusion protein interacted specifically with the activated (GTP-bound) form of RhoG. Platelet whole cell lysates ( WCL ) were stimulated with cross-linked CRP or with bovine α-thrombin at the indicated final concentrations, and the time course of RhoG activation was assessed by immunoblotting for RhoG in the pulldown samples. B , the GST-ELMO activation assay was then used to evaluate the effect of various pharmacologic inhibitors on RhoG activation in CRP-stimulated human platelets. Inhibitors were incubated with washed platelet suspensions for 10 min prior to stimulation. Compared with the appropriate vehicle control dimethyl sulfoxide ( DMSO ) or Tyrode's HEPES buffer ( Tyr ), RhoG activation was reduced by Su6656 (20 μ m ) and piceatannol ( Piceat ; 60 μ m ) but not by U73122 (10 μ m ), wortmannin ( Wort ; 100 n m ), BAPTA-AM ( B-AM ; 10 μ m ), BIM1 (10 μ m ), Abciximab ( Abcix ; 10 μg/ml), or AR-C66096 ( AR-C ; 1 μ m ). This suggests that RhoG activation in human platelets is dependent on Src family kinases and Syk activity and that RhoG activation is independent of PLC, PI3K, or PKC activity and does not require intracellular calcium or signaling downstream of α IIb β 3 or P2Y 12 receptors. All images are representative of three separate experiments. The white lines in the lower immunoblot panels denote gaps between different sections of the same membrane. C , the effects of these inhibitors on RhoG activation were quantified by densitometry using ImageJ. Bars represent the mean, and error bars represent S.E. from at least three membranes/group. The dotted gray line represents the mean level of the dimethyl sulfoxide controls.

    Journal: The Journal of Biological Chemistry

    Article Title: RhoG Protein Regulates Platelet Granule Secretion and Thrombus Formation in Mice *

    doi: 10.1074/jbc.M113.504100

    Figure Lengend Snippet: A , immunoblotting for RhoG on platelet whole cell lysates demonstrated RhoG expression in human platelets. GST-ELMO fusion protein pulldown assays were then used to assess activation of RhoG in human platelets. The fusion protein interacted specifically with the activated (GTP-bound) form of RhoG. Platelet whole cell lysates ( WCL ) were stimulated with cross-linked CRP or with bovine α-thrombin at the indicated final concentrations, and the time course of RhoG activation was assessed by immunoblotting for RhoG in the pulldown samples. B , the GST-ELMO activation assay was then used to evaluate the effect of various pharmacologic inhibitors on RhoG activation in CRP-stimulated human platelets. Inhibitors were incubated with washed platelet suspensions for 10 min prior to stimulation. Compared with the appropriate vehicle control dimethyl sulfoxide ( DMSO ) or Tyrode's HEPES buffer ( Tyr ), RhoG activation was reduced by Su6656 (20 μ m ) and piceatannol ( Piceat ; 60 μ m ) but not by U73122 (10 μ m ), wortmannin ( Wort ; 100 n m ), BAPTA-AM ( B-AM ; 10 μ m ), BIM1 (10 μ m ), Abciximab ( Abcix ; 10 μg/ml), or AR-C66096 ( AR-C ; 1 μ m ). This suggests that RhoG activation in human platelets is dependent on Src family kinases and Syk activity and that RhoG activation is independent of PLC, PI3K, or PKC activity and does not require intracellular calcium or signaling downstream of α IIb β 3 or P2Y 12 receptors. All images are representative of three separate experiments. The white lines in the lower immunoblot panels denote gaps between different sections of the same membrane. C , the effects of these inhibitors on RhoG activation were quantified by densitometry using ImageJ. Bars represent the mean, and error bars represent S.E. from at least three membranes/group. The dotted gray line represents the mean level of the dimethyl sulfoxide controls.

    Article Snippet: AR-C66096, BAPTA-AM, BIM1, MRS-2279, piceatannol, Su6656, U73122, and wortmannin were from Tocris (via R & D Systems, Oxford, United Kingdom).

    Techniques: Expressing, Activation Assay, Incubation, Activity Assay, Planar Chromatography

    673A Triggers Necroptosis in Ovarian CSCs (A) (i) FACS analysis of annexin-V and PI stain as indicators of apoptosis in FACS-sorted CD133 + (A2780) cells after treatment with 12.5 μM 673A, 1 μg/ml cisplatin, or 12.5 μM shikonin. (ii) Cell counts of FACS-sorted CD133 + A2780 cells after pretreatment with pan-caspase inhibitor (CI; 20 μM) and caspase 3 inhibitor (C3I; 20 μM) plus 72-h treatment with the indicated concentrations of 673A. (iii) FACS analysis of CD133 + expression after treatments with CI (20 μM) and 673A (12.5 μM). (B) Immunofluorescence (IF) DAPI nuclear stain (i) and TEM images (5,800×) (ii) of FACS-sorted CD133 + A2780 cells 24 h after treatment with 673A (12.5 μM) (red arrows, mitochondria; blue, rupture of plasma membrane). (C) IF of HMBG1 localization 24 h after 673A treatment; DAPI was used for nuclear stain. (D) Intracellular calcium level, using Fluo-4 FACS assay after 673A treatments (i). Cell counts (ii) and CD133 FACS analysis (iii) of A2780 cells after pretreatments with 20 μM BAPTA and treatments with 673A. (E) FACS of annexin-V-PE/DAPI in PEO4 cells after ALDH1A family member downregulation using siRNA (48 h). Error bars represent SDs n = 3 independent experiments with at least triplicate assays. Data are presented as mean ± SD with *p

    Journal: Cell reports

    Article Title: A Pan-ALDH1A Inhibitor Induces Necroptosis in Ovarian Cancer Stem-like Cells

    doi: 10.1016/j.celrep.2019.02.032

    Figure Lengend Snippet: 673A Triggers Necroptosis in Ovarian CSCs (A) (i) FACS analysis of annexin-V and PI stain as indicators of apoptosis in FACS-sorted CD133 + (A2780) cells after treatment with 12.5 μM 673A, 1 μg/ml cisplatin, or 12.5 μM shikonin. (ii) Cell counts of FACS-sorted CD133 + A2780 cells after pretreatment with pan-caspase inhibitor (CI; 20 μM) and caspase 3 inhibitor (C3I; 20 μM) plus 72-h treatment with the indicated concentrations of 673A. (iii) FACS analysis of CD133 + expression after treatments with CI (20 μM) and 673A (12.5 μM). (B) Immunofluorescence (IF) DAPI nuclear stain (i) and TEM images (5,800×) (ii) of FACS-sorted CD133 + A2780 cells 24 h after treatment with 673A (12.5 μM) (red arrows, mitochondria; blue, rupture of plasma membrane). (C) IF of HMBG1 localization 24 h after 673A treatment; DAPI was used for nuclear stain. (D) Intracellular calcium level, using Fluo-4 FACS assay after 673A treatments (i). Cell counts (ii) and CD133 FACS analysis (iii) of A2780 cells after pretreatments with 20 μM BAPTA and treatments with 673A. (E) FACS of annexin-V-PE/DAPI in PEO4 cells after ALDH1A family member downregulation using siRNA (48 h). Error bars represent SDs n = 3 independent experiments with at least triplicate assays. Data are presented as mean ± SD with *p

    Article Snippet: The compounds and reagents used were Z-VAD-FMK (InvivoGen), BAPTA (Tocris), necrostatin-1 (Tocris), necrosulfonamide (Tocris), cisplatin (SICOR Pharmaceuticals), acetaldehyde, propionaldehyde, NAD+, and buffers for ALDH activity (Sigma Aldrich).

    Techniques: FACS, Staining, Expressing, Immunofluorescence, Transmission Electron Microscopy

    Ca 2+ -depletion causes defects in the localisation of CENP-F at the outer kinetochore. Fluorescence images of metaphase cells after control, BAPTA-AM and BAPTA (with ionomycin) treatments for the localisation of Aurora B ( a ), Mis12 ( b ), Hec1 ( c ) and CENP-F ( d ). Bar, 5 µm.

    Journal: Scientific Reports

    Article Title: Calcium depletion destabilises kinetochore fibres by the removal of CENP-F from the kinetochore

    doi: 10.1038/s41598-017-07777-6

    Figure Lengend Snippet: Ca 2+ -depletion causes defects in the localisation of CENP-F at the outer kinetochore. Fluorescence images of metaphase cells after control, BAPTA-AM and BAPTA (with ionomycin) treatments for the localisation of Aurora B ( a ), Mis12 ( b ), Hec1 ( c ) and CENP-F ( d ). Bar, 5 µm.

    Article Snippet: In brief, before the treatment with BAPTA-AM or BAPTA and ionomycin, cells were incubated with Fura-2 am for 45 min at room temperature and further 20 min at 37 °C to allow completely hydrolysis of AM ester.

    Techniques: Fluorescence

    Ca 2+ -depletion reduces the stability of spindle fibres. ( a ) HeLa WT cells arrested at metaphase using MG132 were treated with 25 µM BAPTA-AM or 10 mM BAPTA and 5 µM ionomycin to reduce intracellular Ca 2+ then stained with anti-Ac-tubulin antibody. ( b ) A graph presenting the relative Ac-tubulin intensity at kinetochore of each treatment. ( c ) Fluorescence images of metaphase cells in control, BAPTA-AM and BAPTA (with ionomycin) treatments after exposure to low temperature. ( d ) A graph presenting the relative cold-stable microtubule intensity at kinetochore of each treatment. ( e ) A bar graph showing the intensity ratio of Fura-2 excited at 340 and 380 nm measured in mitotic cells, which represent intracellular calcium levels. Error bars indicate standard deviations derived from three independent experiments. Bar, 5 µm.

    Journal: Scientific Reports

    Article Title: Calcium depletion destabilises kinetochore fibres by the removal of CENP-F from the kinetochore

    doi: 10.1038/s41598-017-07777-6

    Figure Lengend Snippet: Ca 2+ -depletion reduces the stability of spindle fibres. ( a ) HeLa WT cells arrested at metaphase using MG132 were treated with 25 µM BAPTA-AM or 10 mM BAPTA and 5 µM ionomycin to reduce intracellular Ca 2+ then stained with anti-Ac-tubulin antibody. ( b ) A graph presenting the relative Ac-tubulin intensity at kinetochore of each treatment. ( c ) Fluorescence images of metaphase cells in control, BAPTA-AM and BAPTA (with ionomycin) treatments after exposure to low temperature. ( d ) A graph presenting the relative cold-stable microtubule intensity at kinetochore of each treatment. ( e ) A bar graph showing the intensity ratio of Fura-2 excited at 340 and 380 nm measured in mitotic cells, which represent intracellular calcium levels. Error bars indicate standard deviations derived from three independent experiments. Bar, 5 µm.

    Article Snippet: In brief, before the treatment with BAPTA-AM or BAPTA and ionomycin, cells were incubated with Fura-2 am for 45 min at room temperature and further 20 min at 37 °C to allow completely hydrolysis of AM ester.

    Techniques: Staining, Fluorescence, Derivative Assay

    ACYP2 promotes STAT3 phosphorylation in glioma cells through regulating intracellular Ca 2+ homeostasis and calpain activity. Cells transfected with the indicated shRNAs were treated with 5 μM BAPTA-AM for 6 h ( a ), 10 μM calpeptin for 10 h ( b ), 10 μM Na3VO4 for 1 h ( c ), or the vehicle. Western blot analysis was then performed to test their effect on STAT3 phosphorylation. GAPDH was used as a loading control. d , Cells transfected with the indicated shRNAs were treated with the vehicle or 10 μM Na3VO4 for 1 h, and the MTT assay was then carried out to evaluate their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). Cells stably knocking down ACYP2 and control cells were transfected with siRNA targeting PTP1B or not. e , Western blot analysis was then performed to evaluate their effect on STAT3 phosphorylation. f , MTT assay was performed to assess their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). **, P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: ACYP2 contributes to malignant progression of glioma through promoting Ca2+ efflux and subsequently activating c-Myc and STAT3 signals

    doi: 10.1186/s13046-020-01607-w

    Figure Lengend Snippet: ACYP2 promotes STAT3 phosphorylation in glioma cells through regulating intracellular Ca 2+ homeostasis and calpain activity. Cells transfected with the indicated shRNAs were treated with 5 μM BAPTA-AM for 6 h ( a ), 10 μM calpeptin for 10 h ( b ), 10 μM Na3VO4 for 1 h ( c ), or the vehicle. Western blot analysis was then performed to test their effect on STAT3 phosphorylation. GAPDH was used as a loading control. d , Cells transfected with the indicated shRNAs were treated with the vehicle or 10 μM Na3VO4 for 1 h, and the MTT assay was then carried out to evaluate their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). Cells stably knocking down ACYP2 and control cells were transfected with siRNA targeting PTP1B or not. e , Western blot analysis was then performed to evaluate their effect on STAT3 phosphorylation. f , MTT assay was performed to assess their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). **, P

    Article Snippet: This could be effectively reversed upon BAPTA-AM or calpeptin treatment.

    Techniques: Activity Assay, Transfection, Western Blot, MTT Assay, Stable Transfection

    Transcriptional activation of c-Myc by ACYP2 in glioma cells through regulating intracellular Ca 2+ homeostasis and calpain activity. a , Western blot analysis was carried out to determine the effect of ectopic expression of ACYP2 in SHG44 and A172 cells on the levels of transcriptionally active c-Myc and its downstream target NCL. Tubulin and GAPDH were used as loading controls. Cells transfected with the indicated shRNAs were treated with the vehicle or 10 μM calpeptin for 10 h. b , Western blot analysis was performed to evaluate their effect on protein expression of c-Myc and its target NCL. c , qRT-PCR assay was used to evaluate their effect on mRNA levels of ACYP2 and NCL . GAPDH was used as a loading control in western blot analysis. 18S rRNA was used as a reference gene in qRT-PCR assay. d , Cells transfected with the indicated shRNAs were treated with the vehicle or 5 μM BAPTA-AM for 6 h. Western blot analysis was then performed to investigate their effect on the expression of c-Myc and its downstream target NCL. GAPDH was used as a loading control. e , The indicated cells were treated with the vehicle or 100 μM 10,058-F4 for 48 h, and the MTT assay was then used to evaluate their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). **, P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: ACYP2 contributes to malignant progression of glioma through promoting Ca2+ efflux and subsequently activating c-Myc and STAT3 signals

    doi: 10.1186/s13046-020-01607-w

    Figure Lengend Snippet: Transcriptional activation of c-Myc by ACYP2 in glioma cells through regulating intracellular Ca 2+ homeostasis and calpain activity. a , Western blot analysis was carried out to determine the effect of ectopic expression of ACYP2 in SHG44 and A172 cells on the levels of transcriptionally active c-Myc and its downstream target NCL. Tubulin and GAPDH were used as loading controls. Cells transfected with the indicated shRNAs were treated with the vehicle or 10 μM calpeptin for 10 h. b , Western blot analysis was performed to evaluate their effect on protein expression of c-Myc and its target NCL. c , qRT-PCR assay was used to evaluate their effect on mRNA levels of ACYP2 and NCL . GAPDH was used as a loading control in western blot analysis. 18S rRNA was used as a reference gene in qRT-PCR assay. d , Cells transfected with the indicated shRNAs were treated with the vehicle or 5 μM BAPTA-AM for 6 h. Western blot analysis was then performed to investigate their effect on the expression of c-Myc and its downstream target NCL. GAPDH was used as a loading control. e , The indicated cells were treated with the vehicle or 100 μM 10,058-F4 for 48 h, and the MTT assay was then used to evaluate their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). **, P

    Article Snippet: This could be effectively reversed upon BAPTA-AM or calpeptin treatment.

    Techniques: Activation Assay, Activity Assay, Western Blot, Expressing, Transfection, Quantitative RT-PCR, MTT Assay

    ACYP2 promotes glioma cell proliferation through altering intracellular Ca 2+ homeostasis and calpain activity. a , Representative images showing free intracellular Ca 2+ in the indicated cells under the confocal microscope (left panel). Green color represents staining of free intracellular Ca 2+ . Histogram represents mean ± SD of the fluorescence intensity from five microscopic fields in each group, as shown in right panel. Scale bars, 200 μm. b , Free intracellular Ca 2+ measured by flow cytometer was shown in the middle and right panels. The data were presented as mean ± SD ( n = 3). c , Cells were transfected with the indicated siRNAs and treated with the vehicle or 5 μM BAPTA-AM for 6 h, the MTT assay was then carried out to evaluate their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). d , Calpain activity was measured in the indicated cells using a calpain activity assay kit. Active Calpain I and calpain inhibitor Z-LLY-FMK were used as positive and negative control, respectively. Data are presented as mean ± SD ( n = 3). e , Cells were transfected with the indicated siRNAs and treated with the vehicle or 10 μM calpeptin for 12 h, and the MTT assay was then carried out to evaluate their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). *, P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: ACYP2 contributes to malignant progression of glioma through promoting Ca2+ efflux and subsequently activating c-Myc and STAT3 signals

    doi: 10.1186/s13046-020-01607-w

    Figure Lengend Snippet: ACYP2 promotes glioma cell proliferation through altering intracellular Ca 2+ homeostasis and calpain activity. a , Representative images showing free intracellular Ca 2+ in the indicated cells under the confocal microscope (left panel). Green color represents staining of free intracellular Ca 2+ . Histogram represents mean ± SD of the fluorescence intensity from five microscopic fields in each group, as shown in right panel. Scale bars, 200 μm. b , Free intracellular Ca 2+ measured by flow cytometer was shown in the middle and right panels. The data were presented as mean ± SD ( n = 3). c , Cells were transfected with the indicated siRNAs and treated with the vehicle or 5 μM BAPTA-AM for 6 h, the MTT assay was then carried out to evaluate their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). d , Calpain activity was measured in the indicated cells using a calpain activity assay kit. Active Calpain I and calpain inhibitor Z-LLY-FMK were used as positive and negative control, respectively. Data are presented as mean ± SD ( n = 3). e , Cells were transfected with the indicated siRNAs and treated with the vehicle or 10 μM calpeptin for 12 h, and the MTT assay was then carried out to evaluate their effect on cell proliferation. The data were presented as mean ± SD ( n = 3). *, P

    Article Snippet: This could be effectively reversed upon BAPTA-AM or calpeptin treatment.

    Techniques: Activity Assay, Microscopy, Staining, Fluorescence, Flow Cytometry, Transfection, MTT Assay, Negative Control

    Effect of seawater exposure on the average fluorescence intensity in A549 cells. (A) Seawater exposure evoked a rapid fluorescence intensity increase that represented [Ca 2+ ]c elevation. Left panel, A549 cells without stimulation; right panel, A549 cells exposed to seawater. (B) Quantification of fluorescence intensity following seawater treatment. (C) Pretreatment with BAPTA-AM may reverse the elevation of [Ca 2+ ]c. (D) Chelation of calcium with EGTA reduced the rise of [Ca 2+ ]c triggered by seawater. (E) Average fluorescence intensities for all groups. * P

    Journal: Molecular Medicine Reports

    Article Title: Activation of TRPV1-dependent calcium oscillation exacerbates seawater inhalation-induced acute lung injury

    doi: 10.3892/mmr.2016.4804

    Figure Lengend Snippet: Effect of seawater exposure on the average fluorescence intensity in A549 cells. (A) Seawater exposure evoked a rapid fluorescence intensity increase that represented [Ca 2+ ]c elevation. Left panel, A549 cells without stimulation; right panel, A549 cells exposed to seawater. (B) Quantification of fluorescence intensity following seawater treatment. (C) Pretreatment with BAPTA-AM may reverse the elevation of [Ca 2+ ]c. (D) Chelation of calcium with EGTA reduced the rise of [Ca 2+ ]c triggered by seawater. (E) Average fluorescence intensities for all groups. * P

    Article Snippet: These alterations were alleviated when cells were treated with BAPTA-AM or EGTA ( ).

    Techniques: Fluorescence

    Effect of chelation of calcium on the secretion of TNF-α and IL-1β in A549 cells. (A) TNF-α and (B) IL-1β concentrations following control, seawater, EGTA and BAPTA-AM treatments. * P

    Journal: Molecular Medicine Reports

    Article Title: Activation of TRPV1-dependent calcium oscillation exacerbates seawater inhalation-induced acute lung injury

    doi: 10.3892/mmr.2016.4804

    Figure Lengend Snippet: Effect of chelation of calcium on the secretion of TNF-α and IL-1β in A549 cells. (A) TNF-α and (B) IL-1β concentrations following control, seawater, EGTA and BAPTA-AM treatments. * P

    Article Snippet: These alterations were alleviated when cells were treated with BAPTA-AM or EGTA ( ).

    Techniques:

    Effect of BAPTA-AM on RuV infection. (A) Endocytic infection. RuV or SFV was prebound to Vero cells on ice for 1.5 h in binding medium, shifted to 37°C for 20 min in medium containing 2 mM CaCl 2 and the indicated concentration of BAPTA-AM, and

    Journal: Journal of Virology

    Article Title: Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

    doi: 10.1128/JVI.00634-16

    Figure Lengend Snippet: Effect of BAPTA-AM on RuV infection. (A) Endocytic infection. RuV or SFV was prebound to Vero cells on ice for 1.5 h in binding medium, shifted to 37°C for 20 min in medium containing 2 mM CaCl 2 and the indicated concentration of BAPTA-AM, and

    Article Snippet: RuV infection was strongly inhibited by the presence of BAPTA-AM during virus uptake, with an ∼85% decrease being observed with 50 μM BAPTA-AM.

    Techniques: Infection, Binding Assay, Concentration Assay

    Gene expression regulated by celastrol-mediated Ca 2+ mobilization analysis in RASF. RT-qPCR independent validation from RASF cells untreated (control), or treated with 1 μM Celastrol (Cel), and 10 μM BAPTA/AM (BM) for 24 h. Gene expressions were normalized to GAPDH, relative to control, and analyzed using the 2 −ΔΔCT method. The data is represented as the mean ± SD. ** P ≤ 0.01; *** P ≤ 0.001 compared with control. # P ≤ 0.05; ## P ≤ 0.01; ### P ≤ 0.001 compared with Celastrol.

    Journal: Frontiers in Pharmacology

    Article Title: The Calcium-Induced Regulation in the Molecular and Transcriptional Circuitry of Human Inflammatory Response and Autoimmunity

    doi: 10.3389/fphar.2017.00962

    Figure Lengend Snippet: Gene expression regulated by celastrol-mediated Ca 2+ mobilization analysis in RASF. RT-qPCR independent validation from RASF cells untreated (control), or treated with 1 μM Celastrol (Cel), and 10 μM BAPTA/AM (BM) for 24 h. Gene expressions were normalized to GAPDH, relative to control, and analyzed using the 2 −ΔΔCT method. The data is represented as the mean ± SD. ** P ≤ 0.01; *** P ≤ 0.001 compared with control. # P ≤ 0.05; ## P ≤ 0.01; ### P ≤ 0.001 compared with Celastrol.

    Article Snippet: RNA extraction & cDNA synthesis RNA was extracted using RNeasy Mini Kit (Qiagen, USA) from RASFs untreated (control), treated with celastrol [1 μM], or celastrol in the presence of BAPTA/AM [10 μM] (Santa Cruz, USA) for 24 h. RNA concentration was determined using the NanoDrop 2000c Spectrophotometer (Thermo Scientific) and 1 μg of RNA was used to synthesize cDNA with RT2 First Strand Kit (Qiagen, USA).

    Techniques: Expressing, Quantitative RT-PCR

    (A) Celastrol induced calcium dynamic change in RASFs. Cells treated with 1 μM celastrol were loaded with FLIPR Calcium 6 dye. Real time Ca 2+ kinetic was monitored with FLIPR Tetra instrument. Data from the chart represent mean values ± SD. of three independent experiments. (B) Single cell imaging visualized celastrol-mobilized cytosolic calcium level in RASFs. Cells treated with 1 μM celastrol were loaded with FLIPR Calcium 6 dye. Calcium signal was monitored by Applied Precision DeltaVision Elite in real-time mode (see Supplementary Video ). Chart represents the mean intensity of fluorescence signal at 525 nm. (C) Scatter plot for inflammatory and immunity genes fold regulation values from celastrol stimulated RASFs relative to unstimulated RASFs (Control): genes not regulated (black), up-regulated genes (red), and down-regulated genes (green) with threshold lines of 1.5 and −1.5. (D) Scatter plot for the genes identified as up-regulated or down-regulated with celastrol treatment (in C). Dots represent the genes fold regulation values from RASFs treated with celastrol and BAPTA/AM relative to untreated control: genes not regulated (black), up-regulated genes (red), and down-regulated genes (green) with threshold lines of 1.5 and −1.5 (Ct

    Journal: Frontiers in Pharmacology

    Article Title: The Calcium-Induced Regulation in the Molecular and Transcriptional Circuitry of Human Inflammatory Response and Autoimmunity

    doi: 10.3389/fphar.2017.00962

    Figure Lengend Snippet: (A) Celastrol induced calcium dynamic change in RASFs. Cells treated with 1 μM celastrol were loaded with FLIPR Calcium 6 dye. Real time Ca 2+ kinetic was monitored with FLIPR Tetra instrument. Data from the chart represent mean values ± SD. of three independent experiments. (B) Single cell imaging visualized celastrol-mobilized cytosolic calcium level in RASFs. Cells treated with 1 μM celastrol were loaded with FLIPR Calcium 6 dye. Calcium signal was monitored by Applied Precision DeltaVision Elite in real-time mode (see Supplementary Video ). Chart represents the mean intensity of fluorescence signal at 525 nm. (C) Scatter plot for inflammatory and immunity genes fold regulation values from celastrol stimulated RASFs relative to unstimulated RASFs (Control): genes not regulated (black), up-regulated genes (red), and down-regulated genes (green) with threshold lines of 1.5 and −1.5. (D) Scatter plot for the genes identified as up-regulated or down-regulated with celastrol treatment (in C). Dots represent the genes fold regulation values from RASFs treated with celastrol and BAPTA/AM relative to untreated control: genes not regulated (black), up-regulated genes (red), and down-regulated genes (green) with threshold lines of 1.5 and −1.5 (Ct

    Article Snippet: RNA extraction & cDNA synthesis RNA was extracted using RNeasy Mini Kit (Qiagen, USA) from RASFs untreated (control), treated with celastrol [1 μM], or celastrol in the presence of BAPTA/AM [10 μM] (Santa Cruz, USA) for 24 h. RNA concentration was determined using the NanoDrop 2000c Spectrophotometer (Thermo Scientific) and 1 μg of RNA was used to synthesize cDNA with RT2 First Strand Kit (Qiagen, USA).

    Techniques: Imaging, Fluorescence

    Ca 2+ /CaMKII-dependent LTA-mediated activation of JNK and c-Jun in RBA-1 cells . (A) Cells were pretreated with BAPTA (upper part) or TG (lower part) for 1 h and then incubated with LTA for the indicated time intervals. Cell lysates were assayed by western blot using anti-phospho-CaMKII antibody. (B) Cells were pretreated CaMI or KN-62 for 1 h and then incubated with LTA for the indicated time intervals. Cell lysates were assayed by western blot using an anti-phospho-JNK or phospho-c-Jun antibody. The membranes were stripped and re-probed with anti-GAPDH antibody as a control. Data are expressed as mean of three independent experiments (n = 3). * P

    Journal: Journal of Neuroinflammation

    Article Title: Calmodulin kinase II-dependent transactivation of PDGF receptors mediates astrocytic MMP-9 expression and cell motility induced by lipoteichoic acid

    doi: 10.1186/1742-2094-7-84

    Figure Lengend Snippet: Ca 2+ /CaMKII-dependent LTA-mediated activation of JNK and c-Jun in RBA-1 cells . (A) Cells were pretreated with BAPTA (upper part) or TG (lower part) for 1 h and then incubated with LTA for the indicated time intervals. Cell lysates were assayed by western blot using anti-phospho-CaMKII antibody. (B) Cells were pretreated CaMI or KN-62 for 1 h and then incubated with LTA for the indicated time intervals. Cell lysates were assayed by western blot using an anti-phospho-JNK or phospho-c-Jun antibody. The membranes were stripped and re-probed with anti-GAPDH antibody as a control. Data are expressed as mean of three independent experiments (n = 3). * P

    Article Snippet: BAPTA/AM, thapsigargin (TG), calmidazolium chloride (CaMI), KN-62, AG1296, LY294002, SP600125, and tanshinone IIA (TSIIA) were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Activation Assay, Incubation, Western Blot

    c-Jun/AP-1 binding element is essential for LTA-induced MMP-9 expression through a Ca 2+ /CaMKII/PDGFR/PI3K/JNK pathway in RBA-1 cells . (A) Time dependence of LTA-stimulated c-Jun/AP-1 binding activity. RBA-1 cells were incubated with 50 mg/ml LTA for the indicated time intervals, or cells were pretreated with TG, KN-62, AG1296, SP600125, or TSIIA for 1 h and then incubated with 50 mg/ml LTA for 30 min. c-Jun/AP-1 binding activity was analyzed by chromatin-IP (ChIP)-PCR assay. (B) Cells were transiently cotransfected with pGL-MMP9-Luc and pGal encoding for b-galactosidase for 24 h. The pGL-MMP-9-Luc-transfected cells were pretreated with TG, KN-62, AG1296, SP600125, or TSIIA for 1 h and then incubated with LTA for 16 h. (C) Activation of wild-type (WT) and AP-1-mutated (mt-AP1) MMP-9 promoter constructs by LTA. Cells were cotransfected with respective promoter constructs for 24 h and then incubated with 50 μg/ml LTA for 16 h. The values for beetle luciferase were normalized to that of b-galactosidase activity. (D) Cells were pretreated with CaMI, KN-62, BAPTA, or TG for 1 h and then incubated with LTA for 16 h. Total RNA were extracted and analyzed by RT-PCR. Data are expressed as mean ± SEM (B, C) or mean (A, D) of three independent experiments (n = 3). * P

    Journal: Journal of Neuroinflammation

    Article Title: Calmodulin kinase II-dependent transactivation of PDGF receptors mediates astrocytic MMP-9 expression and cell motility induced by lipoteichoic acid

    doi: 10.1186/1742-2094-7-84

    Figure Lengend Snippet: c-Jun/AP-1 binding element is essential for LTA-induced MMP-9 expression through a Ca 2+ /CaMKII/PDGFR/PI3K/JNK pathway in RBA-1 cells . (A) Time dependence of LTA-stimulated c-Jun/AP-1 binding activity. RBA-1 cells were incubated with 50 mg/ml LTA for the indicated time intervals, or cells were pretreated with TG, KN-62, AG1296, SP600125, or TSIIA for 1 h and then incubated with 50 mg/ml LTA for 30 min. c-Jun/AP-1 binding activity was analyzed by chromatin-IP (ChIP)-PCR assay. (B) Cells were transiently cotransfected with pGL-MMP9-Luc and pGal encoding for b-galactosidase for 24 h. The pGL-MMP-9-Luc-transfected cells were pretreated with TG, KN-62, AG1296, SP600125, or TSIIA for 1 h and then incubated with LTA for 16 h. (C) Activation of wild-type (WT) and AP-1-mutated (mt-AP1) MMP-9 promoter constructs by LTA. Cells were cotransfected with respective promoter constructs for 24 h and then incubated with 50 μg/ml LTA for 16 h. The values for beetle luciferase were normalized to that of b-galactosidase activity. (D) Cells were pretreated with CaMI, KN-62, BAPTA, or TG for 1 h and then incubated with LTA for 16 h. Total RNA were extracted and analyzed by RT-PCR. Data are expressed as mean ± SEM (B, C) or mean (A, D) of three independent experiments (n = 3). * P

    Article Snippet: BAPTA/AM, thapsigargin (TG), calmidazolium chloride (CaMI), KN-62, AG1296, LY294002, SP600125, and tanshinone IIA (TSIIA) were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Binding Assay, Expressing, Activity Assay, Incubation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Transfection, Activation Assay, Construct, Luciferase, Reverse Transcription Polymerase Chain Reaction

    LTA induces MMP-9 expression via Ca 2+ /CaMKII-dependent transactivation of PDGFR in RBA-1 cells . (A) Cells were pretreated with AG1296 (10 μM) or LY294002 (30 μM) for 1 h and then incubated with 50 mg/ml LTA for the indicated time intervals. (B, C) Cells were pretreated with TLR2 neutralizing antibody (TLR2 nAb), BAPTA, TG, CaMI, or KN-62 for 1 h and then incubated with 50 mg/ml LTA for the indicated time intervals. Cell lysates were subjected to SDS-PAGE and blotted using an anti-phospho-JNK or anti-phospho-c-Jun (A) and anti-phospho-PDGFR (B, C) or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM (B, C) or mean (A) of three independent experiments (n = 3). * P

    Journal: Journal of Neuroinflammation

    Article Title: Calmodulin kinase II-dependent transactivation of PDGF receptors mediates astrocytic MMP-9 expression and cell motility induced by lipoteichoic acid

    doi: 10.1186/1742-2094-7-84

    Figure Lengend Snippet: LTA induces MMP-9 expression via Ca 2+ /CaMKII-dependent transactivation of PDGFR in RBA-1 cells . (A) Cells were pretreated with AG1296 (10 μM) or LY294002 (30 μM) for 1 h and then incubated with 50 mg/ml LTA for the indicated time intervals. (B, C) Cells were pretreated with TLR2 neutralizing antibody (TLR2 nAb), BAPTA, TG, CaMI, or KN-62 for 1 h and then incubated with 50 mg/ml LTA for the indicated time intervals. Cell lysates were subjected to SDS-PAGE and blotted using an anti-phospho-JNK or anti-phospho-c-Jun (A) and anti-phospho-PDGFR (B, C) or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM (B, C) or mean (A) of three independent experiments (n = 3). * P

    Article Snippet: BAPTA/AM, thapsigargin (TG), calmidazolium chloride (CaMI), KN-62, AG1296, LY294002, SP600125, and tanshinone IIA (TSIIA) were from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Incubation, SDS Page

    Peroxynitrite negatively regulates mitochondrial degradation. (a) and (b) SH-SY5Y cells overexpressing parkin were treated with BAPTA-AM (10 μM) (a) or FeTPPS (5 μM) (b) with CCCP (10 μM) for 3 hr, and lysates were analysed by SNO-RAC. The quantity of S-nitrosylated parkin, as measured by scanning densitometry, is expressed as a percentage of control, normalized with respect to total parkin. Data shown are mean ± SE (n = 3); *p

    Journal: Scientific Reports

    Article Title: S-nitrosylation regulates mitochondrial quality control via activation of parkin

    doi: 10.1038/srep02202

    Figure Lengend Snippet: Peroxynitrite negatively regulates mitochondrial degradation. (a) and (b) SH-SY5Y cells overexpressing parkin were treated with BAPTA-AM (10 μM) (a) or FeTPPS (5 μM) (b) with CCCP (10 μM) for 3 hr, and lysates were analysed by SNO-RAC. The quantity of S-nitrosylated parkin, as measured by scanning densitometry, is expressed as a percentage of control, normalized with respect to total parkin. Data shown are mean ± SE (n = 3); *p

    Article Snippet: BAPTA/AM and FeTPPS were obtained from Merck-Millipore (Darmstadt, Germany).

    Techniques:

    Effect of AD2900 on proliferation is dependent on cAMP reduction and calcium signal transduction The effect of Edelfosine (Et-18-OCH 3 ) (Et), BAPTA-AM (BAPTA), or 8-bromo-cAMP (8-b-CAMP) on the inhibition of PBMC proliferation was tested in a 5-day MLR experiment. The cells were incubated with 1 μM Edelfosine, 0.2 μM BAPTA-AM, or 5 μM 8-bromo-cAMP for 1 hour before the addition of sphingolipid analogues, 2 μM AD2900 (AD) (A) , FTY720 (FTY) (B) , or S1P (C) . Significances are compared to AD2900/FTY/S1P without preincubation, respectively. Graphs summarize the results of at least four independent experiments. Results of Student’s t -test: *(P

    Journal: Oncotarget

    Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

    doi: 10.18632/oncotarget.18626

    Figure Lengend Snippet: Effect of AD2900 on proliferation is dependent on cAMP reduction and calcium signal transduction The effect of Edelfosine (Et-18-OCH 3 ) (Et), BAPTA-AM (BAPTA), or 8-bromo-cAMP (8-b-CAMP) on the inhibition of PBMC proliferation was tested in a 5-day MLR experiment. The cells were incubated with 1 μM Edelfosine, 0.2 μM BAPTA-AM, or 5 μM 8-bromo-cAMP for 1 hour before the addition of sphingolipid analogues, 2 μM AD2900 (AD) (A) , FTY720 (FTY) (B) , or S1P (C) . Significances are compared to AD2900/FTY/S1P without preincubation, respectively. Graphs summarize the results of at least four independent experiments. Results of Student’s t -test: *(P

    Article Snippet: Et-18-OCH3, BAPTA-AM, and 8-bromo-cAMP were obtained from ENZO life sciences (NY, USA) and dissolved according to the manufacturer’s instructions.

    Techniques: Transduction, Inhibition, Incubation

    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