kir 4 1  (Alomone Labs)


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    Alomone Labs kir 4 1
    Kir 4 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kir 4 1 - by Bioz Stars, 2022-05
    86/100 stars

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    Alomone Labs anti trpc6
    The intracellular localization of <t>TRPC6</t> contributes to reticular Ca 2+ -leakage in KL -overexpressing DDLPS cells, but is not necessary to increase cell death. ( A – F ) Variations of relative cytosolic Ca 2+ concentration were monitored by fluorescence videomicroscopy in Fluo2-loaded cells. Each experiment was repeated three times and the average of more than 20 single-cell traces was analyzed. The effect of OAG (50 µM) addition was evaluated in ( A ) IB115-empty vector or ( B ) IB115-KL cells bathed in HBSS medium ± 2 mM Ca 2+ , and in ( C ) IB115-KL cells pretreated with TG (10 nM) at 200 s in a Ca 2+ -free HBSS medium. ( D ) IB115 empty-vector and IB115-KL cells were bathed in a Ca 2+ -free HBSS medium and stimulated at 200 s by the specific activator of TRPC6 Hyp9 (1 μM). ( E ) OAG (50 µM) was added at 200 s to Ca 2+ -free HBSS medium on IB115-KL cells control or pretreated with inhibitors of TRPC6 (U73343 and larixyl acetate both at 1 μM, 1 h). ( F ) Ca 2+ -leakage was estimated by the application of 10 nM TG at 200 s on IB115-empty vector or IB115-KL cells, which were pretreated or not with larixyl acetate (1 μM, 1 h), in a Ca 2+ -free HBSS medium. ( G ) The abundance of TRPC6 was analyzed in IB115-empty vector and IB115-KL cells by western blotting after 48 h of incubation with no drugs, 10 nM TG, or 100 nM gemcitabine. Actin was used as a loading control. To compare TRPC6 abundance between conditions, results were all normalized to control conditions (IB115-empty vector, no treatment). Results shown are representative of three independent experiments. ( H , I ) Cell death was measured with a TMRM-staining analyzed by flow cytometry after 72 h incubation of IB115 cell lines, which were pretreated during 1 h with indicated concentrations of larixyl acetate and then treated with ( H ) 20 nM and 10 nM TG for the IB115-empty vector and IB115-KL cells, respectively (in order to have nearly similar cell death rates), or ( I ) 100 nM gemcitabine. Histograms sum up ( H ) four and ( I ) two independent experiments. Data shown correspond to medians (IQR).
    Anti Trpc6, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti trpc6/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti trpc6 - by Bioz Stars, 2022-05
    86/100 stars
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    93
    Alomone Labs trpc6
    Comparison of basal protein expression levels of store-operated Ca 2+ channels (SOCC) (STIM1/2, Orai1/2) and ROCC <t>(TRPC6)</t> and hypoxia-induced changes of protein expression of SOCC (STIM1/2, Orai1/2) and ROCC (TRPC6) in CASMC and PASMC. A and B : representative images ( A ) and summarized data ( B , means ± SE) showing Western blot analysis of STIM1, STIM2, Orai1, Orai2, and TRPC6 in CASMC and PASMC ( n = 5 separate experiments). β-Actin was used as a control. * P
    Trpc6, supplied by Alomone Labs, 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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    Image Search Results


    The intracellular localization of TRPC6 contributes to reticular Ca 2+ -leakage in KL -overexpressing DDLPS cells, but is not necessary to increase cell death. ( A – F ) Variations of relative cytosolic Ca 2+ concentration were monitored by fluorescence videomicroscopy in Fluo2-loaded cells. Each experiment was repeated three times and the average of more than 20 single-cell traces was analyzed. The effect of OAG (50 µM) addition was evaluated in ( A ) IB115-empty vector or ( B ) IB115-KL cells bathed in HBSS medium ± 2 mM Ca 2+ , and in ( C ) IB115-KL cells pretreated with TG (10 nM) at 200 s in a Ca 2+ -free HBSS medium. ( D ) IB115 empty-vector and IB115-KL cells were bathed in a Ca 2+ -free HBSS medium and stimulated at 200 s by the specific activator of TRPC6 Hyp9 (1 μM). ( E ) OAG (50 µM) was added at 200 s to Ca 2+ -free HBSS medium on IB115-KL cells control or pretreated with inhibitors of TRPC6 (U73343 and larixyl acetate both at 1 μM, 1 h). ( F ) Ca 2+ -leakage was estimated by the application of 10 nM TG at 200 s on IB115-empty vector or IB115-KL cells, which were pretreated or not with larixyl acetate (1 μM, 1 h), in a Ca 2+ -free HBSS medium. ( G ) The abundance of TRPC6 was analyzed in IB115-empty vector and IB115-KL cells by western blotting after 48 h of incubation with no drugs, 10 nM TG, or 100 nM gemcitabine. Actin was used as a loading control. To compare TRPC6 abundance between conditions, results were all normalized to control conditions (IB115-empty vector, no treatment). Results shown are representative of three independent experiments. ( H , I ) Cell death was measured with a TMRM-staining analyzed by flow cytometry after 72 h incubation of IB115 cell lines, which were pretreated during 1 h with indicated concentrations of larixyl acetate and then treated with ( H ) 20 nM and 10 nM TG for the IB115-empty vector and IB115-KL cells, respectively (in order to have nearly similar cell death rates), or ( I ) 100 nM gemcitabine. Histograms sum up ( H ) four and ( I ) two independent experiments. Data shown correspond to medians (IQR).

    Journal: Cancers

    Article Title: The Role of the Anti-Aging Protein Klotho in IGF-1 Signaling and Reticular Calcium Leak: Impact on the Chemosensitivity of Dedifferentiated Liposarcomas

    doi: 10.3390/cancers10110439

    Figure Lengend Snippet: The intracellular localization of TRPC6 contributes to reticular Ca 2+ -leakage in KL -overexpressing DDLPS cells, but is not necessary to increase cell death. ( A – F ) Variations of relative cytosolic Ca 2+ concentration were monitored by fluorescence videomicroscopy in Fluo2-loaded cells. Each experiment was repeated three times and the average of more than 20 single-cell traces was analyzed. The effect of OAG (50 µM) addition was evaluated in ( A ) IB115-empty vector or ( B ) IB115-KL cells bathed in HBSS medium ± 2 mM Ca 2+ , and in ( C ) IB115-KL cells pretreated with TG (10 nM) at 200 s in a Ca 2+ -free HBSS medium. ( D ) IB115 empty-vector and IB115-KL cells were bathed in a Ca 2+ -free HBSS medium and stimulated at 200 s by the specific activator of TRPC6 Hyp9 (1 μM). ( E ) OAG (50 µM) was added at 200 s to Ca 2+ -free HBSS medium on IB115-KL cells control or pretreated with inhibitors of TRPC6 (U73343 and larixyl acetate both at 1 μM, 1 h). ( F ) Ca 2+ -leakage was estimated by the application of 10 nM TG at 200 s on IB115-empty vector or IB115-KL cells, which were pretreated or not with larixyl acetate (1 μM, 1 h), in a Ca 2+ -free HBSS medium. ( G ) The abundance of TRPC6 was analyzed in IB115-empty vector and IB115-KL cells by western blotting after 48 h of incubation with no drugs, 10 nM TG, or 100 nM gemcitabine. Actin was used as a loading control. To compare TRPC6 abundance between conditions, results were all normalized to control conditions (IB115-empty vector, no treatment). Results shown are representative of three independent experiments. ( H , I ) Cell death was measured with a TMRM-staining analyzed by flow cytometry after 72 h incubation of IB115 cell lines, which were pretreated during 1 h with indicated concentrations of larixyl acetate and then treated with ( H ) 20 nM and 10 nM TG for the IB115-empty vector and IB115-KL cells, respectively (in order to have nearly similar cell death rates), or ( I ) 100 nM gemcitabine. Histograms sum up ( H ) four and ( I ) two independent experiments. Data shown correspond to medians (IQR).

    Article Snippet: Anti-TRPC6 (#ACC-120) was provided by Alomone Labs (Jerusalem, Israel).

    Techniques: Concentration Assay, Fluorescence, Plasmid Preparation, Western Blot, Incubation, Staining, Flow Cytometry, Cytometry

    Comparison of basal protein expression levels of store-operated Ca 2+ channels (SOCC) (STIM1/2, Orai1/2) and ROCC (TRPC6) and hypoxia-induced changes of protein expression of SOCC (STIM1/2, Orai1/2) and ROCC (TRPC6) in CASMC and PASMC. A and B : representative images ( A ) and summarized data ( B , means ± SE) showing Western blot analysis of STIM1, STIM2, Orai1, Orai2, and TRPC6 in CASMC and PASMC ( n = 5 separate experiments). β-Actin was used as a control. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Hypoxia selectively upregulates cation channels and increases cytosolic [Ca2+] in pulmonary, but not coronary, arterial smooth muscle cells

    doi: 10.1152/ajpcell.00272.2017

    Figure Lengend Snippet: Comparison of basal protein expression levels of store-operated Ca 2+ channels (SOCC) (STIM1/2, Orai1/2) and ROCC (TRPC6) and hypoxia-induced changes of protein expression of SOCC (STIM1/2, Orai1/2) and ROCC (TRPC6) in CASMC and PASMC. A and B : representative images ( A ) and summarized data ( B , means ± SE) showing Western blot analysis of STIM1, STIM2, Orai1, Orai2, and TRPC6 in CASMC and PASMC ( n = 5 separate experiments). β-Actin was used as a control. * P

    Article Snippet: After blocking with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween 20 for 1 h at room temperature, the membranes were incubated at 4°C overnight with primary antibody against STIM1 (diluted 1:1,000; catalog no. 4119, Prosci, Poway, CA) , STIM2 (diluted 1:1,000; catalog no. S8572, Sigma, St. Louis, MO) , Orai1 (diluted 1:1,000; catalog no. ACC-060, Alomone, Jerusalem, Israel) ( , ), Orai2 (diluted 1:1,000; catalog no. ACC-061, Alomone) , and TRPC6 (diluted 1:1,000; catalog no. ACC-120, Alomone) ( ).

    Techniques: Expressing, Western Blot

    Melatonin downregulates TRPC6 expression in MDA-MB-231 cells. MCF10A ( A , E and I ) and MDA-MB-231 cells ( B – D , F – G and J – L ) were treated for 48, 72, and 168 h with melatonin (10–1000 nM) or the vehicle (control) and lysed. Whole-cell lysates were subjected to 10% SDS-PAGE and western blotting with the anti-TRPC6 ( A , B , E , F , I , and J ), anti-PMCA ( C , G , and K ), anti-Orai1 ( D , H and L ), or anti-β-actin antibodies, as described in Experimental procedures . Blots are representative of five to ten separate experiments. Scatter plots represent TRPC6, Orai1, or PMCA expression. Analysis of statistical significance was performed using one-way ANOVA (0.001

    Journal: The Journal of Biological Chemistry

    Article Title: Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells

    doi: 10.1074/jbc.RA120.015769

    Figure Lengend Snippet: Melatonin downregulates TRPC6 expression in MDA-MB-231 cells. MCF10A ( A , E and I ) and MDA-MB-231 cells ( B – D , F – G and J – L ) were treated for 48, 72, and 168 h with melatonin (10–1000 nM) or the vehicle (control) and lysed. Whole-cell lysates were subjected to 10% SDS-PAGE and western blotting with the anti-TRPC6 ( A , B , E , F , I , and J ), anti-PMCA ( C , G , and K ), anti-Orai1 ( D , H and L ), or anti-β-actin antibodies, as described in Experimental procedures . Blots are representative of five to ten separate experiments. Scatter plots represent TRPC6, Orai1, or PMCA expression. Analysis of statistical significance was performed using one-way ANOVA (0.001

    Article Snippet: Rabbit polyclonal anti-TRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone.

    Techniques: Expressing, Multiple Displacement Amplification, SDS Page, Western Blot

    Overexpression of TRPC6 attenuates melatonin-evoked inhibition of store-operated Ca 2+ entry in MDA-MB-231 cells. A , left panel , MDA-MB-231 cells were transfected with TRPC6dn-YFP or TRPC6-YFP expression plasmids. Forty-eight hours later, TRPC6 expression was determined by fluorescence microscopy. A , right panel , MDA-MB-231 cells were transfected with TRPC6dn-YFP or TRPC6-YFP expression plasmids or empty vector (control). Forty-eight hours later cells were loaded with fura-2. Fura-2-loaded cells were perfused with a Ca 2+ -free medium (100 μM EGTA added) and then stimulated with TG (1 μM) followed by reintroduction of external Ca 2+ (final concentration 1 mM) to initiate Ca 2+ entry. Scatter plots represent the quantification of TG-evoked Ca 2+ release and entry determined as described in Experimental procedures . Data are presented as mean ± SD. Dots represent a single experiment including 20–30 cells. Analysis of statistical significance was performed using one-way ANOVA (F = 1.82; p = 0.19 for Ca 2+ release and F = 546.5 and p

    Journal: The Journal of Biological Chemistry

    Article Title: Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells

    doi: 10.1074/jbc.RA120.015769

    Figure Lengend Snippet: Overexpression of TRPC6 attenuates melatonin-evoked inhibition of store-operated Ca 2+ entry in MDA-MB-231 cells. A , left panel , MDA-MB-231 cells were transfected with TRPC6dn-YFP or TRPC6-YFP expression plasmids. Forty-eight hours later, TRPC6 expression was determined by fluorescence microscopy. A , right panel , MDA-MB-231 cells were transfected with TRPC6dn-YFP or TRPC6-YFP expression plasmids or empty vector (control). Forty-eight hours later cells were loaded with fura-2. Fura-2-loaded cells were perfused with a Ca 2+ -free medium (100 μM EGTA added) and then stimulated with TG (1 μM) followed by reintroduction of external Ca 2+ (final concentration 1 mM) to initiate Ca 2+ entry. Scatter plots represent the quantification of TG-evoked Ca 2+ release and entry determined as described in Experimental procedures . Data are presented as mean ± SD. Dots represent a single experiment including 20–30 cells. Analysis of statistical significance was performed using one-way ANOVA (F = 1.82; p = 0.19 for Ca 2+ release and F = 546.5 and p

    Article Snippet: Rabbit polyclonal anti-TRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone.

    Techniques: Over Expression, Inhibition, Multiple Displacement Amplification, Transfection, Expressing, Fluorescence, Microscopy, Plasmid Preparation, Concentration Assay

    Effect of melatonin on calcium mobilization, viability, and migration in MDA-MB-468 cells. A , MDA-MB-468 cells were treated for 72 h with melatonin (100–1000 nM) or the vehicle (control) and lysed. Whole-cell lysates were subjected to 10% SDS-PAGE and western blotting with anti-TRPC6, anti-PMCA, anti-Orai1, or anti-β-actin antibody, as described in Experimental procedures . Blots are representative of five to six separate experiments. Scatter plots represent TRPC6, Orai1 or PMCA expression. Analysis of statistical significance was performed using one-way ANOVA (F = 10.72, 0.16, and 0.85 and p = 0.001, 0.84, and 0.44 for TRPC6, PMCA, and Orai1, respectively) with post-hoc Dunnett's test for TRPC6 (∗ p

    Journal: The Journal of Biological Chemistry

    Article Title: Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells

    doi: 10.1074/jbc.RA120.015769

    Figure Lengend Snippet: Effect of melatonin on calcium mobilization, viability, and migration in MDA-MB-468 cells. A , MDA-MB-468 cells were treated for 72 h with melatonin (100–1000 nM) or the vehicle (control) and lysed. Whole-cell lysates were subjected to 10% SDS-PAGE and western blotting with anti-TRPC6, anti-PMCA, anti-Orai1, or anti-β-actin antibody, as described in Experimental procedures . Blots are representative of five to six separate experiments. Scatter plots represent TRPC6, Orai1 or PMCA expression. Analysis of statistical significance was performed using one-way ANOVA (F = 10.72, 0.16, and 0.85 and p = 0.001, 0.84, and 0.44 for TRPC6, PMCA, and Orai1, respectively) with post-hoc Dunnett's test for TRPC6 (∗ p

    Article Snippet: Rabbit polyclonal anti-TRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone.

    Techniques: Migration, Multiple Displacement Amplification, SDS Page, Western Blot, Expressing

    Effect of pharmacological inhibition or silencing of TRPC6 on Ca 2+ entry in the presence of melatonin in MDA-MB-231 cells. A , Fura-2-loaded MDA-MB-231 cells were pretreated for 10 min with SAR7334 (1 μM). Cells were then stimulated in the presence of 1 mM extracellular Ca 2+ with OAG (100 μM). Scatter plots represent the quantification of OAG-induced Ca 2+ entry determined as described in Experimental procedures . Data are presented as mean ± SD. Dots represent a single experiment including 20–30 cells. Analysis of statistical significance was performed using Student's t -test (∗ p

    Journal: The Journal of Biological Chemistry

    Article Title: Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells

    doi: 10.1074/jbc.RA120.015769

    Figure Lengend Snippet: Effect of pharmacological inhibition or silencing of TRPC6 on Ca 2+ entry in the presence of melatonin in MDA-MB-231 cells. A , Fura-2-loaded MDA-MB-231 cells were pretreated for 10 min with SAR7334 (1 μM). Cells were then stimulated in the presence of 1 mM extracellular Ca 2+ with OAG (100 μM). Scatter plots represent the quantification of OAG-induced Ca 2+ entry determined as described in Experimental procedures . Data are presented as mean ± SD. Dots represent a single experiment including 20–30 cells. Analysis of statistical significance was performed using Student's t -test (∗ p

    Article Snippet: Rabbit polyclonal anti-TRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone.

    Techniques: Inhibition, Multiple Displacement Amplification

    Effect of melatonin on TRPC6 synthesis and degradation in MDA-MB-231 cells. A and B , MDA-MB-231 cells were treated for 48 or 72 h with melatonin (100 nM) or the vehicle (control). The expression of TRPC6 ( A ) or miR26a-5p ( B ) was determined as described in Experimental procedures . Scatter plots represent relative expression as compared with control (n = 4–7). Analysis of statistical significance was performed using one-way ANOVA (F and p values were 0.41 and 0.74, respectively, for [ A ] and 0.17 and 0.91, respectively, for [ B ]). C , MDA-MB-231 cells were treated for 72 h with melatonin (100 nM) or the vehicle (control) and lysed. Sixteen hours before the end of the treatment, cells were treated with 10 μM MG132 or the vehicle. Whole-cell lysates were immunoprecipitated with anti-TRPC6 antibody. Immunoprecipitates were subjected to 10% SDS-PAGE and subsequent western blotting with specific anti-TRPC6 (left panel), antiubiquitin (right panel), or anti-β-actin antibody, as indicated. The panels show results from one experiment representative of three others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Bar graphs represent the expression of TRPC6 (left panel) and the quantification of TRPC6-associated ubiquitin. Results are presented as arbitrary optical density units and expressed as percentage of control. Analysis of statistical significance was performed using one-way ANOVA (F = 14.71 ( p = 0.0003) and 71.28 ( p

    Journal: The Journal of Biological Chemistry

    Article Title: Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells

    doi: 10.1074/jbc.RA120.015769

    Figure Lengend Snippet: Effect of melatonin on TRPC6 synthesis and degradation in MDA-MB-231 cells. A and B , MDA-MB-231 cells were treated for 48 or 72 h with melatonin (100 nM) or the vehicle (control). The expression of TRPC6 ( A ) or miR26a-5p ( B ) was determined as described in Experimental procedures . Scatter plots represent relative expression as compared with control (n = 4–7). Analysis of statistical significance was performed using one-way ANOVA (F and p values were 0.41 and 0.74, respectively, for [ A ] and 0.17 and 0.91, respectively, for [ B ]). C , MDA-MB-231 cells were treated for 72 h with melatonin (100 nM) or the vehicle (control) and lysed. Sixteen hours before the end of the treatment, cells were treated with 10 μM MG132 or the vehicle. Whole-cell lysates were immunoprecipitated with anti-TRPC6 antibody. Immunoprecipitates were subjected to 10% SDS-PAGE and subsequent western blotting with specific anti-TRPC6 (left panel), antiubiquitin (right panel), or anti-β-actin antibody, as indicated. The panels show results from one experiment representative of three others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Bar graphs represent the expression of TRPC6 (left panel) and the quantification of TRPC6-associated ubiquitin. Results are presented as arbitrary optical density units and expressed as percentage of control. Analysis of statistical significance was performed using one-way ANOVA (F = 14.71 ( p = 0.0003) and 71.28 ( p

    Article Snippet: Rabbit polyclonal anti-TRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone.

    Techniques: Multiple Displacement Amplification, Expressing, Immunoprecipitation, SDS Page, Western Blot

    Expression of exogenous TRPC6 reverses the antiproliferative effect of melatonin in MDA-MB-231 cells. MDA-MB-231 cells were treated for 48, 72, and 168 h with melatonin (10–1000 nM) or the vehicle (control). Forty-eight hours before the end of the treatment period, cells were transfected with TRPC6-YFP ( B , E , and H ) or TRPC6dn-YFP ( C , F , and I ) expression plasmid or empty vector ( A , D and G ). Cell proliferation was assessed at time = 0, 24, 48, and 72 h using the BrdU cell proliferation assay kit, as described in Experimental procedures . Scatter plots represent cell proliferation 0, 24, 48, and 72 h after the onset of the experiment, presented as BrdU uptake rate (n = 6). Analysis of statistical significance was performed using two-way ANOVA (for A , D , G : 0 h, F values were 0.14, 0.14, and 1.35 and p values were 0.93, 0.86, and 0.24 for concentration, time, and the interaction respectively; for A , D , G : 24 h, F values were 593.8, 1570, and 199.6 and p values were

    Journal: The Journal of Biological Chemistry

    Article Title: Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells

    doi: 10.1074/jbc.RA120.015769

    Figure Lengend Snippet: Expression of exogenous TRPC6 reverses the antiproliferative effect of melatonin in MDA-MB-231 cells. MDA-MB-231 cells were treated for 48, 72, and 168 h with melatonin (10–1000 nM) or the vehicle (control). Forty-eight hours before the end of the treatment period, cells were transfected with TRPC6-YFP ( B , E , and H ) or TRPC6dn-YFP ( C , F , and I ) expression plasmid or empty vector ( A , D and G ). Cell proliferation was assessed at time = 0, 24, 48, and 72 h using the BrdU cell proliferation assay kit, as described in Experimental procedures . Scatter plots represent cell proliferation 0, 24, 48, and 72 h after the onset of the experiment, presented as BrdU uptake rate (n = 6). Analysis of statistical significance was performed using two-way ANOVA (for A , D , G : 0 h, F values were 0.14, 0.14, and 1.35 and p values were 0.93, 0.86, and 0.24 for concentration, time, and the interaction respectively; for A , D , G : 24 h, F values were 593.8, 1570, and 199.6 and p values were

    Article Snippet: Rabbit polyclonal anti-TRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone.

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation, BrdU Cell Proliferation Assay, Concentration Assay

    The intracellular localization of TRPC6 contributes to reticular Ca 2+ -leakage in KL -overexpressing DDLPS cells, but is not necessary to increase cell death. ( A – F ) Variations of relative cytosolic Ca 2+ concentration were monitored by fluorescence videomicroscopy in Fluo2-loaded cells. Each experiment was repeated three times and the average of more than 20 single-cell traces was analyzed. The effect of OAG (50 µM) addition was evaluated in ( A ) IB115-empty vector or ( B ) IB115-KL cells bathed in HBSS medium ± 2 mM Ca 2+ , and in ( C ) IB115-KL cells pretreated with TG (10 nM) at 200 s in a Ca 2+ -free HBSS medium. ( D ) IB115 empty-vector and IB115-KL cells were bathed in a Ca 2+ -free HBSS medium and stimulated at 200 s by the specific activator of TRPC6 Hyp9 (1 μM). ( E ) OAG (50 µM) was added at 200 s to Ca 2+ -free HBSS medium on IB115-KL cells control or pretreated with inhibitors of TRPC6 (U73343 and larixyl acetate both at 1 μM, 1 h). ( F ) Ca 2+ -leakage was estimated by the application of 10 nM TG at 200 s on IB115-empty vector or IB115-KL cells, which were pretreated or not with larixyl acetate (1 μM, 1 h), in a Ca 2+ -free HBSS medium. ( G ) The abundance of TRPC6 was analyzed in IB115-empty vector and IB115-KL cells by western blotting after 48 h of incubation with no drugs, 10 nM TG, or 100 nM gemcitabine. Actin was used as a loading control. To compare TRPC6 abundance between conditions, results were all normalized to control conditions (IB115-empty vector, no treatment). Results shown are representative of three independent experiments. ( H , I ) Cell death was measured with a TMRM-staining analyzed by flow cytometry after 72 h incubation of IB115 cell lines, which were pretreated during 1 h with indicated concentrations of larixyl acetate and then treated with ( H ) 20 nM and 10 nM TG for the IB115-empty vector and IB115-KL cells, respectively (in order to have nearly similar cell death rates), or ( I ) 100 nM gemcitabine. Histograms sum up ( H ) four and ( I ) two independent experiments. Data shown correspond to medians (IQR).

    Journal: Cancers

    Article Title: The Role of the Anti-Aging Protein Klotho in IGF-1 Signaling and Reticular Calcium Leak: Impact on the Chemosensitivity of Dedifferentiated Liposarcomas

    doi: 10.3390/cancers10110439

    Figure Lengend Snippet: The intracellular localization of TRPC6 contributes to reticular Ca 2+ -leakage in KL -overexpressing DDLPS cells, but is not necessary to increase cell death. ( A – F ) Variations of relative cytosolic Ca 2+ concentration were monitored by fluorescence videomicroscopy in Fluo2-loaded cells. Each experiment was repeated three times and the average of more than 20 single-cell traces was analyzed. The effect of OAG (50 µM) addition was evaluated in ( A ) IB115-empty vector or ( B ) IB115-KL cells bathed in HBSS medium ± 2 mM Ca 2+ , and in ( C ) IB115-KL cells pretreated with TG (10 nM) at 200 s in a Ca 2+ -free HBSS medium. ( D ) IB115 empty-vector and IB115-KL cells were bathed in a Ca 2+ -free HBSS medium and stimulated at 200 s by the specific activator of TRPC6 Hyp9 (1 μM). ( E ) OAG (50 µM) was added at 200 s to Ca 2+ -free HBSS medium on IB115-KL cells control or pretreated with inhibitors of TRPC6 (U73343 and larixyl acetate both at 1 μM, 1 h). ( F ) Ca 2+ -leakage was estimated by the application of 10 nM TG at 200 s on IB115-empty vector or IB115-KL cells, which were pretreated or not with larixyl acetate (1 μM, 1 h), in a Ca 2+ -free HBSS medium. ( G ) The abundance of TRPC6 was analyzed in IB115-empty vector and IB115-KL cells by western blotting after 48 h of incubation with no drugs, 10 nM TG, or 100 nM gemcitabine. Actin was used as a loading control. To compare TRPC6 abundance between conditions, results were all normalized to control conditions (IB115-empty vector, no treatment). Results shown are representative of three independent experiments. ( H , I ) Cell death was measured with a TMRM-staining analyzed by flow cytometry after 72 h incubation of IB115 cell lines, which were pretreated during 1 h with indicated concentrations of larixyl acetate and then treated with ( H ) 20 nM and 10 nM TG for the IB115-empty vector and IB115-KL cells, respectively (in order to have nearly similar cell death rates), or ( I ) 100 nM gemcitabine. Histograms sum up ( H ) four and ( I ) two independent experiments. Data shown correspond to medians (IQR).

    Article Snippet: Anti-TRPC6 (#ACC-120) was provided by Alomone Labs (Jerusalem, Israel).

    Techniques: Concentration Assay, Fluorescence, Plasmid Preparation, Western Blot, Incubation, Staining, Flow Cytometry