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Santa Cruz Biotechnology ethylene glycol tetraacetic acid egta
Ethylene Glycol Tetraacetic Acid Egta, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Low and moderate doses of ionizing radiation up to 2 Gy modulate transmigration and chemotaxis of activated macrophages, provoke an anti-inflammatory cytokine milieu, but do not impact upon viability and phagocytic function
Article Snippet: .. After 3 h of incubation at 37°C, cells that had transmigrated to the lower chamber or the lower side of the pore filter were harvested and lysed in 20 mM HEPES-K pH 7·4, 84 mM KCl, 10 mM MgCl2 , 0·2 mM ethylenediamine tetraacetic acid (EDTA), 0·2 mM ethylene glycol tetraacetic acid (EGTA) and 0·5% non-ionic, non-denaturating detergent IGEPAL CA-630 (Santa Cruz Biotechnologies, Heidelberg, Germany). .. Calcein fluorescence was measured at 485 nm excitation and 510 nm emission using a Synergy Mx plate reader (Biotek).

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Article Title: Tomato (Lycopersicon esculentum) Supplementation Induces Changes in Cardiac miRNA Expression, Reduces Oxidative Stress and Left Ventricular Mass, and Improves Diastolic Function
Article Snippet: Nrf-2, Type I and III Collagen Analysis Samples of LV were added to Tris-Triton buffer (10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 10% glycerol, 0.1% Sodium dodecyl sulfate (SDS), 0.5% deoxycholate, 1 nM EDTA, 1 mM EGTA and a mixture of protease inhibitors (1 mM sodium orthovanadate, 1 mM sodium fluoride and 1% leupeptin, aprotinin and pepstatin)) to detect type I (rabbit polyclonal IgG, sc8784R, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and III (mouse monoclonal IgG1, ab6310, Abcam, Inc., Cambridge, UK) collagen.

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    Santa Cruz Biotechnology hrpe 1 cell lysates
    MB d -accumulation is high in stem cells and subpopulations of cancer cells and does not correlate with cell doubling time. ( a ) Percent of cells that accumulate MB d s ( > 1) in a range of different cell types, as indicated. Below, doubling-times of representative cell lines aligned with MB d -accumulation data. Data are presented as mean ± s.d.; Cell lines are examined in triplicate (MCF-10A, DLD-1, MCF-10AT, MCF-7, H1, and H9), or quadruplicate (e.v. B6 MEFs, HeLa, SAOS-2, and MCF-10CA1a), except <t>hRPE-1</t> ( n =6), U2OS ( n =7) and NCC-IT ( n =8). Horizontal line, cell lines with different MB d -accumulation potential (14-fold) but similar doubling time. ( b ) Cells pulse-chased with EdU show a decrease in EdU intensity (x-axis) over time (y-axis), reflecting dilution of dye after cell divisions. ( c, d ) After a 96-hr chase period, EdU levels were compared between cells with MB d numbers of > 1, 1, and 0 (y-axis) in HeLa (c) and SAOS-2 cells (d). In both cases, no significant differences were noted (c, p=0.2101; d, p=0.5609, one-way ANOVA, with at least 800 cells analyzed for each experiment, n =3), indicating similar cycling rates among different subpopulations of cells. (b-d) Each graph is a representative experiment. Cells analyzed shown by green points, median depicted by vertical red lines, and horizontal red lines with ticks illustrate the interquartile range.
    Hrpe 1 Cell Lysates, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology presynaptic egta
    Numerical simulation of the VGCC-SV coupling distance reveals subpools of synaptic vesicles. a <t>EGTA</t> effect on EPSC amplitudes (EGTA/control) was plotted against the number of swellings (circles) and fitted (black) with the equation ± 95% confidence interval (dashed; see Equation (3) in Results). n Sw is the number of swellings, RRP St and RRP Sw are the RRP (readily releasable pool) sizes in entire stalks and per swelling, Inh St and Inh Sw are the inhibitory effects of EGTA in stalks and swellings, respectively. The gray zone (right axis) exhibits the occupancy of tightly coupled vesicles. b The least mean squares (LMS) of the fit exhibit the best EGTA effect in swellings ( X -ordinate) and stalks ( Y -ordinate). c The magnitude of inhibition by EGTA is a function of distance between vesicular Ca 2+ sensor and voltage-gated Ca 2+ channel (VGCC) cluster perimeter. Horizontal dashed lines indicate the EGTA inhibition in stalks (0.88) and in swellings (0.44). Vertical shaded regions indicate the range of distances between the sensor location and nearest open VGCC matching EGTA inhibition for different number of VGCCs in cluster. d Estimated coupling distance of loose and tight SVs were plotted against the number of VGCCs in cluster. e We describe a modular model of the heterogeneity in synaptic function. Variable proportions of distinct morphological modules (i.e. swelling and stalk) define functional heterogeneity. The stalk module (black) contains synaptic vesicles (SVs) with tighter coupling (14 nm; tight nanodomain) to larger VGCC clusters present in lower density. The swelling module (red) contains SVs with looser coupling (30–50 nm; loose nanodomain) to smaller clusters present in higher density. <t>Septin</t> 5 (Sept5) differentiates the coupling distances. Simple calyces mostly contain stalk modules while complex calyces are composed of a mixture of stalk and swelling modules. Arrows represent the contribution of the modules
    Presynaptic Egta, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology egta am
    Starvation induces lysosomal Ca 2+ release via MCOLN1. (a) Starvation does not induce bulk cytosolic Ca 2+ elevation in HeLa cells transfected with the Ca 2+ -sensitive probe aequorin. Bulk cytosolic [Ca 2+ ] was monitored during perfusion with complete L-15 medium, HBSS and HBSS supplemented with 100µM histamine as indicated. n=8 coverslips from two independent transfections (b) Stable HeLa TFEB-GFP cells were left untreated or pretreated for 30 minutes with the Ca 2+ chelators <t>BAPTA-AM</t> or <t>EGTA-AM</t> (5µM each). After washing, cells were left untreated or starved for 1 hr. After treatment, cells were fixed and a HC imaging analysis was performed. The plot shows the percentage of TFEB nuclear translocation in BAPTA-treated cells compared with untreated and EGTA treated (mean ± s.d., n=3 independent experiments). Scale bar 10µm. (c) Average [Ca 2+ ] cyt evoked by maximal histamine stimulation in WT HeLa cells. Agonist stimulation was carried out in complete L-15 medium or after a three minutes starvation with HBSS (2.45 ± 0.19 µM, HeLa WT in L-15 medium; 2.406 ± 0.23 µM, HeLa WT in HBSS; n=8 coverslips from two independent transfections). (d) Representative traces of the cytosolic GCaMP6s and the perilysosomal ML1-GCaMP3 calcium probes. HeLa cells were transfected with the indicated probe and ratiometric imaging (474 and 410 nm excitation) was performed. Cells were continuously perfused with the indicated solutions. The plot in the middle represents the average perilysosomal calcium peak values induced by perfusion of the indicated buffer, as recorded by the GCaMP3-ML1 probe (R: 1.149 ± 0.051, L-15; 1.508 ± 0.060, HBSS; 7.500 ± 0.456, histamine; n= 18 cells from two independent transfections). The plot on the right represents the average cytosolic calcium peak values induced by perfusion of the indicated buffer, as recorded by the GCaMP6s probe (R: 0.404 ± 0.026, L-15; 0.389 ± 0.022, HBSS; 7.473 ± 0.428, histamine; n = 12 cells from two independent transfections). (e) Ca 2+ release (measured with deltaF/F 0 fluorescence intensity) was detected right after the L15 medium (containing 2 mM [Ca 2+ ], aminoacids and 10% FBS) was switched to Tyrode’s solution (2mM [Ca 2+ ]) in HEK293 cells stably expressing GCaMP3-ML1 36 . The agonist of MCOLN1 ML-SA1 (10µM) was applied to induce MCOLN1-mediated Ca 2+ release. n=3 independent experiments. (f) Similarly the lysosomotropic drug GPN blunted starvation-mediated calcium release detected by GCaMP3-ML1. n=3 independent experiments, *, p
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    89
    Santa Cruz Biotechnology ethylene glycol tetra acetic acid egta
    Effect of caspase or MMP inhibition on rat hepatocyte spheroid formation. Isolated primary rat hepatocytes were cultured under rocked suspension conditions using control (anti-mouse IgG), 2.5 mM <t>EGTA,</t> or <t>E-cadherin</t> blocking antibody (Ecad Ab) ±
    Ethylene Glycol Tetra Acetic Acid Egta, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MB d -accumulation is high in stem cells and subpopulations of cancer cells and does not correlate with cell doubling time. ( a ) Percent of cells that accumulate MB d s ( > 1) in a range of different cell types, as indicated. Below, doubling-times of representative cell lines aligned with MB d -accumulation data. Data are presented as mean ± s.d.; Cell lines are examined in triplicate (MCF-10A, DLD-1, MCF-10AT, MCF-7, H1, and H9), or quadruplicate (e.v. B6 MEFs, HeLa, SAOS-2, and MCF-10CA1a), except hRPE-1 ( n =6), U2OS ( n =7) and NCC-IT ( n =8). Horizontal line, cell lines with different MB d -accumulation potential (14-fold) but similar doubling time. ( b ) Cells pulse-chased with EdU show a decrease in EdU intensity (x-axis) over time (y-axis), reflecting dilution of dye after cell divisions. ( c, d ) After a 96-hr chase period, EdU levels were compared between cells with MB d numbers of > 1, 1, and 0 (y-axis) in HeLa (c) and SAOS-2 cells (d). In both cases, no significant differences were noted (c, p=0.2101; d, p=0.5609, one-way ANOVA, with at least 800 cells analyzed for each experiment, n =3), indicating similar cycling rates among different subpopulations of cells. (b-d) Each graph is a representative experiment. Cells analyzed shown by green points, median depicted by vertical red lines, and horizontal red lines with ticks illustrate the interquartile range.

    Journal: Nature cell biology

    Article Title: Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

    doi: 10.1038/ncb2332

    Figure Lengend Snippet: MB d -accumulation is high in stem cells and subpopulations of cancer cells and does not correlate with cell doubling time. ( a ) Percent of cells that accumulate MB d s ( > 1) in a range of different cell types, as indicated. Below, doubling-times of representative cell lines aligned with MB d -accumulation data. Data are presented as mean ± s.d.; Cell lines are examined in triplicate (MCF-10A, DLD-1, MCF-10AT, MCF-7, H1, and H9), or quadruplicate (e.v. B6 MEFs, HeLa, SAOS-2, and MCF-10CA1a), except hRPE-1 ( n =6), U2OS ( n =7) and NCC-IT ( n =8). Horizontal line, cell lines with different MB d -accumulation potential (14-fold) but similar doubling time. ( b ) Cells pulse-chased with EdU show a decrease in EdU intensity (x-axis) over time (y-axis), reflecting dilution of dye after cell divisions. ( c, d ) After a 96-hr chase period, EdU levels were compared between cells with MB d numbers of > 1, 1, and 0 (y-axis) in HeLa (c) and SAOS-2 cells (d). In both cases, no significant differences were noted (c, p=0.2101; d, p=0.5609, one-way ANOVA, with at least 800 cells analyzed for each experiment, n =3), indicating similar cycling rates among different subpopulations of cells. (b-d) Each graph is a representative experiment. Cells analyzed shown by green points, median depicted by vertical red lines, and horizontal red lines with ticks illustrate the interquartile range.

    Article Snippet: hRPE-1 cell lysates (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 4°C) were pre-cleared for 1 hour with protein G-plus conjugated agarose beads (Santa Cruz) at 4°C, incubated with 2 μg normal IgG, anti-Cep55 or anti-NBR1 antibodies for 3 hours at 4°C, and incubated overnight at 4°C with 25 μl protein G-plus beads.

    Techniques:

    Autophagy controls intracellular MB d levels. ( a ) Single-plane confocal images of MB d s within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MB d markers: Cep55, MKLP1, or mgcRACGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and mgcRACGAP (red) are co-localized (magenta) in the autophagosome (green), suggesting that MB d s are sorted into autophagosomes. Bars, 2 μm. ( b ) Decreasing autophagy levels by deletion of Atg5 gene (left, MEFs) or depletion of Atg7 by siRNA (right, HeLa) significantly increases the percent of MB d + cells (p=0.0019 and p=0.021, respectively, n =3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of Atg7 (asterisk). ( c ) Rapamycin (Rapa) and lithium chloride (LiCl) co-treatment induces autophagy and decreases the percent of MB d + cells (left, HeLa; p=0.0056, n =3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by over-expression of Flag-tagged BECN1 reduces the percent of MB d + cells (right, MCF-7; p=0.0008, n =4) ( d ) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d/PepA. U, uninhibited. I, inhibited. Below, the average of the percent change in LC3-II levels after lysosomal inhibition from 3 experiments. α-tubulin, loading control. ( e ) Quantification of autophagic flux from 3 experiments in different cell lines. Normal dividing cells (MB d -poor) typically have high autophagic flux, whereas stem and cancer cells (MB d -rich) have low autophagic flux. The data are presented as mean ± s.d. (b-e).

    Journal: Nature cell biology

    Article Title: Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity

    doi: 10.1038/ncb2332

    Figure Lengend Snippet: Autophagy controls intracellular MB d levels. ( a ) Single-plane confocal images of MB d s within LC3-positive autophagosomes in MEFs expressing GFP-LC3 (left) and in hRPE-1 cells stained for endogenous LC3 (right). MB d markers: Cep55, MKLP1, or mgcRACGAP. Autophagosomes: GFP-LC3 or LC3. Note that MKLP1 (blue) and mgcRACGAP (red) are co-localized (magenta) in the autophagosome (green), suggesting that MB d s are sorted into autophagosomes. Bars, 2 μm. ( b ) Decreasing autophagy levels by deletion of Atg5 gene (left, MEFs) or depletion of Atg7 by siRNA (right, HeLa) significantly increases the percent of MB d + cells (p=0.0019 and p=0.021, respectively, n =3). Immunoblots confirm loss of the Atg5-Atg12 conjugation in mutant cells and depletion of Atg7 (asterisk). ( c ) Rapamycin (Rapa) and lithium chloride (LiCl) co-treatment induces autophagy and decreases the percent of MB d + cells (left, HeLa; p=0.0056, n =3). Immunoblots showing increased LC3-II levels confirm autophagy induction. Induction of autophagy by over-expression of Flag-tagged BECN1 reduces the percent of MB d + cells (right, MCF-7; p=0.0008, n =4) ( d ) Representative immunoblots showing high autophagy levels in normal cells and low levels in stem cells and cancer cells. Autophagic flux (autophagic activity) was measured by changes in the levels of LC3-II, in the presence or absence of lysosomal inhibitors E64d/PepA. U, uninhibited. I, inhibited. Below, the average of the percent change in LC3-II levels after lysosomal inhibition from 3 experiments. α-tubulin, loading control. ( e ) Quantification of autophagic flux from 3 experiments in different cell lines. Normal dividing cells (MB d -poor) typically have high autophagic flux, whereas stem and cancer cells (MB d -rich) have low autophagic flux. The data are presented as mean ± s.d. (b-e).

    Article Snippet: hRPE-1 cell lysates (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 4°C) were pre-cleared for 1 hour with protein G-plus conjugated agarose beads (Santa Cruz) at 4°C, incubated with 2 μg normal IgG, anti-Cep55 or anti-NBR1 antibodies for 3 hours at 4°C, and incubated overnight at 4°C with 25 μl protein G-plus beads.

    Techniques: Expressing, Staining, Western Blot, Conjugation Assay, Mutagenesis, Over Expression, Activity Assay, Inhibition

    Numerical simulation of the VGCC-SV coupling distance reveals subpools of synaptic vesicles. a EGTA effect on EPSC amplitudes (EGTA/control) was plotted against the number of swellings (circles) and fitted (black) with the equation ± 95% confidence interval (dashed; see Equation (3) in Results). n Sw is the number of swellings, RRP St and RRP Sw are the RRP (readily releasable pool) sizes in entire stalks and per swelling, Inh St and Inh Sw are the inhibitory effects of EGTA in stalks and swellings, respectively. The gray zone (right axis) exhibits the occupancy of tightly coupled vesicles. b The least mean squares (LMS) of the fit exhibit the best EGTA effect in swellings ( X -ordinate) and stalks ( Y -ordinate). c The magnitude of inhibition by EGTA is a function of distance between vesicular Ca 2+ sensor and voltage-gated Ca 2+ channel (VGCC) cluster perimeter. Horizontal dashed lines indicate the EGTA inhibition in stalks (0.88) and in swellings (0.44). Vertical shaded regions indicate the range of distances between the sensor location and nearest open VGCC matching EGTA inhibition for different number of VGCCs in cluster. d Estimated coupling distance of loose and tight SVs were plotted against the number of VGCCs in cluster. e We describe a modular model of the heterogeneity in synaptic function. Variable proportions of distinct morphological modules (i.e. swelling and stalk) define functional heterogeneity. The stalk module (black) contains synaptic vesicles (SVs) with tighter coupling (14 nm; tight nanodomain) to larger VGCC clusters present in lower density. The swelling module (red) contains SVs with looser coupling (30–50 nm; loose nanodomain) to smaller clusters present in higher density. Septin 5 (Sept5) differentiates the coupling distances. Simple calyces mostly contain stalk modules while complex calyces are composed of a mixture of stalk and swelling modules. Arrows represent the contribution of the modules

    Journal: Nature Communications

    Article Title: Underpinning heterogeneity in synaptic transmission by presynaptic ensembles of distinct morphological modules

    doi: 10.1038/s41467-019-08452-2

    Figure Lengend Snippet: Numerical simulation of the VGCC-SV coupling distance reveals subpools of synaptic vesicles. a EGTA effect on EPSC amplitudes (EGTA/control) was plotted against the number of swellings (circles) and fitted (black) with the equation ± 95% confidence interval (dashed; see Equation (3) in Results). n Sw is the number of swellings, RRP St and RRP Sw are the RRP (readily releasable pool) sizes in entire stalks and per swelling, Inh St and Inh Sw are the inhibitory effects of EGTA in stalks and swellings, respectively. The gray zone (right axis) exhibits the occupancy of tightly coupled vesicles. b The least mean squares (LMS) of the fit exhibit the best EGTA effect in swellings ( X -ordinate) and stalks ( Y -ordinate). c The magnitude of inhibition by EGTA is a function of distance between vesicular Ca 2+ sensor and voltage-gated Ca 2+ channel (VGCC) cluster perimeter. Horizontal dashed lines indicate the EGTA inhibition in stalks (0.88) and in swellings (0.44). Vertical shaded regions indicate the range of distances between the sensor location and nearest open VGCC matching EGTA inhibition for different number of VGCCs in cluster. d Estimated coupling distance of loose and tight SVs were plotted against the number of VGCCs in cluster. e We describe a modular model of the heterogeneity in synaptic function. Variable proportions of distinct morphological modules (i.e. swelling and stalk) define functional heterogeneity. The stalk module (black) contains synaptic vesicles (SVs) with tighter coupling (14 nm; tight nanodomain) to larger VGCC clusters present in lower density. The swelling module (red) contains SVs with looser coupling (30–50 nm; loose nanodomain) to smaller clusters present in higher density. Septin 5 (Sept5) differentiates the coupling distances. Simple calyces mostly contain stalk modules while complex calyces are composed of a mixture of stalk and swelling modules. Arrows represent the contribution of the modules

    Article Snippet: To test the effect of presynaptic EGTA or SP20 antibody (1:1000, Mouse-anti-Septin 5, 200 μg/ml; Santa Cruz Biotech, Cat# sc-65512, Lot# K1407, RRID:AB_1129364) injection on synaptic transmission, we performed whole-cell voltage-clamp recordings of the excitatory postsynaptic currents (EPSC) from the principle cells.

    Techniques: Inhibition, Functional Assay

    Synaptic strength is more sensitivity to EGTA and downregulated by septin 5 in complex calyces. a Presynaptic compound action potential currents (pre-I AP ) and EPSCs (I EPSC ; axonal stimulation) were tested in control (black, simple calyx; red, complex calyx) and after EGTA injection (blue, 10 mM, 3–5 min loading). SD 1 (interval between peak 1 of pre-I AP and EPSC onset) and SD 2 (interval between peak 2 of pre-I AP and EPSC onset; dashed lines) are synaptic delays. b Bar graphs show EGTA effect on EPSC amplitudes (simple, control, 10.05 ± 0.64 nA, EGTA, 8.06 ± 0.55 nA, n = 8/7, t = 5.762, p = 0.0007, df = 7, paired t -test [p.t.t.]; complex, control, 14.44 ± 1.76 nA, EGTA, 8.77 ± 0.93 nA, n = 8/8, t = 5.558, p = 0.0009, df = 7, p.t.t.). The difference in EGTA effect on simple and complex calyces was quantified as EGTA/control (simple, 0.802 ± 0.032, n = 8/7, complex, 0.616 ± 0.042, n = 8/8, t = 3.552, p = 0.0032, df = 14, unpaired t -test [u.t.t.]). c EPSC amplitudes for all EGTA injected synapses were plotted against swelling number (black triangles, control; blue dots, EGTA) and fitted linearly (mean ± 95% confidence interval). d , Pre-I AP and I EPSC were tested in control (black, simple calyx; red, complex calyx) and after septin 5 antibody injection (blue, SP20, 1:1000, 3–5 min loading). e Bar graphs show SP20 effect on EPSC amplitudes 10–15 min after presynaptic pipette removal (simple, control, 12.53 ± 0.77 nA, SP20, 12.46 ± 0.70 nA, n = 6/4, t = 0.1137, p = 0.914, df = 5, p.t.t.; complex, control, 13.83 ± 0.76 nA, SP20, 18.29 ± 1.17 nA, n = 10/8, t = 6.808, p

    Journal: Nature Communications

    Article Title: Underpinning heterogeneity in synaptic transmission by presynaptic ensembles of distinct morphological modules

    doi: 10.1038/s41467-019-08452-2

    Figure Lengend Snippet: Synaptic strength is more sensitivity to EGTA and downregulated by septin 5 in complex calyces. a Presynaptic compound action potential currents (pre-I AP ) and EPSCs (I EPSC ; axonal stimulation) were tested in control (black, simple calyx; red, complex calyx) and after EGTA injection (blue, 10 mM, 3–5 min loading). SD 1 (interval between peak 1 of pre-I AP and EPSC onset) and SD 2 (interval between peak 2 of pre-I AP and EPSC onset; dashed lines) are synaptic delays. b Bar graphs show EGTA effect on EPSC amplitudes (simple, control, 10.05 ± 0.64 nA, EGTA, 8.06 ± 0.55 nA, n = 8/7, t = 5.762, p = 0.0007, df = 7, paired t -test [p.t.t.]; complex, control, 14.44 ± 1.76 nA, EGTA, 8.77 ± 0.93 nA, n = 8/8, t = 5.558, p = 0.0009, df = 7, p.t.t.). The difference in EGTA effect on simple and complex calyces was quantified as EGTA/control (simple, 0.802 ± 0.032, n = 8/7, complex, 0.616 ± 0.042, n = 8/8, t = 3.552, p = 0.0032, df = 14, unpaired t -test [u.t.t.]). c EPSC amplitudes for all EGTA injected synapses were plotted against swelling number (black triangles, control; blue dots, EGTA) and fitted linearly (mean ± 95% confidence interval). d , Pre-I AP and I EPSC were tested in control (black, simple calyx; red, complex calyx) and after septin 5 antibody injection (blue, SP20, 1:1000, 3–5 min loading). e Bar graphs show SP20 effect on EPSC amplitudes 10–15 min after presynaptic pipette removal (simple, control, 12.53 ± 0.77 nA, SP20, 12.46 ± 0.70 nA, n = 6/4, t = 0.1137, p = 0.914, df = 5, p.t.t.; complex, control, 13.83 ± 0.76 nA, SP20, 18.29 ± 1.17 nA, n = 10/8, t = 6.808, p

    Article Snippet: To test the effect of presynaptic EGTA or SP20 antibody (1:1000, Mouse-anti-Septin 5, 200 μg/ml; Santa Cruz Biotech, Cat# sc-65512, Lot# K1407, RRID:AB_1129364) injection on synaptic transmission, we performed whole-cell voltage-clamp recordings of the excitatory postsynaptic currents (EPSC) from the principle cells.

    Techniques: Injection, Transferring

    Starvation induces lysosomal Ca 2+ release via MCOLN1. (a) Starvation does not induce bulk cytosolic Ca 2+ elevation in HeLa cells transfected with the Ca 2+ -sensitive probe aequorin. Bulk cytosolic [Ca 2+ ] was monitored during perfusion with complete L-15 medium, HBSS and HBSS supplemented with 100µM histamine as indicated. n=8 coverslips from two independent transfections (b) Stable HeLa TFEB-GFP cells were left untreated or pretreated for 30 minutes with the Ca 2+ chelators BAPTA-AM or EGTA-AM (5µM each). After washing, cells were left untreated or starved for 1 hr. After treatment, cells were fixed and a HC imaging analysis was performed. The plot shows the percentage of TFEB nuclear translocation in BAPTA-treated cells compared with untreated and EGTA treated (mean ± s.d., n=3 independent experiments). Scale bar 10µm. (c) Average [Ca 2+ ] cyt evoked by maximal histamine stimulation in WT HeLa cells. Agonist stimulation was carried out in complete L-15 medium or after a three minutes starvation with HBSS (2.45 ± 0.19 µM, HeLa WT in L-15 medium; 2.406 ± 0.23 µM, HeLa WT in HBSS; n=8 coverslips from two independent transfections). (d) Representative traces of the cytosolic GCaMP6s and the perilysosomal ML1-GCaMP3 calcium probes. HeLa cells were transfected with the indicated probe and ratiometric imaging (474 and 410 nm excitation) was performed. Cells were continuously perfused with the indicated solutions. The plot in the middle represents the average perilysosomal calcium peak values induced by perfusion of the indicated buffer, as recorded by the GCaMP3-ML1 probe (R: 1.149 ± 0.051, L-15; 1.508 ± 0.060, HBSS; 7.500 ± 0.456, histamine; n= 18 cells from two independent transfections). The plot on the right represents the average cytosolic calcium peak values induced by perfusion of the indicated buffer, as recorded by the GCaMP6s probe (R: 0.404 ± 0.026, L-15; 0.389 ± 0.022, HBSS; 7.473 ± 0.428, histamine; n = 12 cells from two independent transfections). (e) Ca 2+ release (measured with deltaF/F 0 fluorescence intensity) was detected right after the L15 medium (containing 2 mM [Ca 2+ ], aminoacids and 10% FBS) was switched to Tyrode’s solution (2mM [Ca 2+ ]) in HEK293 cells stably expressing GCaMP3-ML1 36 . The agonist of MCOLN1 ML-SA1 (10µM) was applied to induce MCOLN1-mediated Ca 2+ release. n=3 independent experiments. (f) Similarly the lysosomotropic drug GPN blunted starvation-mediated calcium release detected by GCaMP3-ML1. n=3 independent experiments, *, p

    Journal: Nature cell biology

    Article Title: Lysosomal calcium signaling regulates autophagy via calcineurin and TFEB

    doi: 10.1038/ncb3114

    Figure Lengend Snippet: Starvation induces lysosomal Ca 2+ release via MCOLN1. (a) Starvation does not induce bulk cytosolic Ca 2+ elevation in HeLa cells transfected with the Ca 2+ -sensitive probe aequorin. Bulk cytosolic [Ca 2+ ] was monitored during perfusion with complete L-15 medium, HBSS and HBSS supplemented with 100µM histamine as indicated. n=8 coverslips from two independent transfections (b) Stable HeLa TFEB-GFP cells were left untreated or pretreated for 30 minutes with the Ca 2+ chelators BAPTA-AM or EGTA-AM (5µM each). After washing, cells were left untreated or starved for 1 hr. After treatment, cells were fixed and a HC imaging analysis was performed. The plot shows the percentage of TFEB nuclear translocation in BAPTA-treated cells compared with untreated and EGTA treated (mean ± s.d., n=3 independent experiments). Scale bar 10µm. (c) Average [Ca 2+ ] cyt evoked by maximal histamine stimulation in WT HeLa cells. Agonist stimulation was carried out in complete L-15 medium or after a three minutes starvation with HBSS (2.45 ± 0.19 µM, HeLa WT in L-15 medium; 2.406 ± 0.23 µM, HeLa WT in HBSS; n=8 coverslips from two independent transfections). (d) Representative traces of the cytosolic GCaMP6s and the perilysosomal ML1-GCaMP3 calcium probes. HeLa cells were transfected with the indicated probe and ratiometric imaging (474 and 410 nm excitation) was performed. Cells were continuously perfused with the indicated solutions. The plot in the middle represents the average perilysosomal calcium peak values induced by perfusion of the indicated buffer, as recorded by the GCaMP3-ML1 probe (R: 1.149 ± 0.051, L-15; 1.508 ± 0.060, HBSS; 7.500 ± 0.456, histamine; n= 18 cells from two independent transfections). The plot on the right represents the average cytosolic calcium peak values induced by perfusion of the indicated buffer, as recorded by the GCaMP6s probe (R: 0.404 ± 0.026, L-15; 0.389 ± 0.022, HBSS; 7.473 ± 0.428, histamine; n = 12 cells from two independent transfections). (e) Ca 2+ release (measured with deltaF/F 0 fluorescence intensity) was detected right after the L15 medium (containing 2 mM [Ca 2+ ], aminoacids and 10% FBS) was switched to Tyrode’s solution (2mM [Ca 2+ ]) in HEK293 cells stably expressing GCaMP3-ML1 36 . The agonist of MCOLN1 ML-SA1 (10µM) was applied to induce MCOLN1-mediated Ca 2+ release. n=3 independent experiments. (f) Similarly the lysosomotropic drug GPN blunted starvation-mediated calcium release detected by GCaMP3-ML1. n=3 independent experiments, *, p

    Article Snippet: Drugs and cellular treatments The following drugs were used: Torin 1 (1µM, otherwise indicated) from Tocris Bioscience; FK506 (5 µM, otherwise indicated), cyclosporine A (10 µM, otherwise indicated), Chloroquine (100 µM) thapsigargin (300 nM), ionomycin (1µM), and EGTA from Sigma; BAPTA-AM (5-25 µM) from Invitrogen; EGTA-AM (5 µM) from Santa Cruz Biotechnology; Bafilomycin A1 (300 nM); SF51 (200 µM) from VITAS-M Laboratory, LTD. Starvation medium was HBSS, 10 mM Hepes.

    Techniques: Transfection, Imaging, Translocation Assay, Fluorescence, Stable Transfection, Expressing

    Effect of caspase or MMP inhibition on rat hepatocyte spheroid formation. Isolated primary rat hepatocytes were cultured under rocked suspension conditions using control (anti-mouse IgG), 2.5 mM EGTA, or E-cadherin blocking antibody (Ecad Ab) ±

    Journal: Cell transplantation

    Article Title: E-Cadherin Protects Primary Hepatocyte Spheroids From Cell Death by a Caspase-Independent Mechanism

    doi: 10.3727/096368909X474258

    Figure Lengend Snippet: Effect of caspase or MMP inhibition on rat hepatocyte spheroid formation. Isolated primary rat hepatocytes were cultured under rocked suspension conditions using control (anti-mouse IgG), 2.5 mM EGTA, or E-cadherin blocking antibody (Ecad Ab) ±

    Article Snippet: Freshly isolated hepatocytes were cultured for 24 h under control conditions, 200 mg/ml mouse IgG1 (2C11) (Santa Cruz), 10 μM Q-Val-Asp-OPh (QVD-OPH) (MP Biochemicals), 10 mM l -carnitine hydrochloride (Sigma), or E-cadherin inhibitory conditions using either 2.5 mM ethylene glycol tetra-acetic acid (EGTA) or 10 μg/ml E-cadherin inhibitory antibody (Santa Cruz).

    Techniques: Inhibition, Isolation, Cell Culture, Blocking Assay