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  • 99
    ATCC raw264 7 cells
    Effects of SseI on host cell survival. (A and B) <t>RAW264.7</t> macrophages were infected with wild type (wt) or Δ sseI - Salmonella (MOI = 30; 30 min). 1.5 h p.i., cells were starved for 3.5 h in Earle’s balanced salt solution. The PI3K inhibitor LY29 (30 μM) was added 1.5 h p.i. where indicated. (A) The amount of LDH released into the medium was measured fluorometrically 5 h p.i.. Δ sseI- infected macrophages showed higher amounts of released LDH compared to wt- Salmonella infected cells. Data show means ±SEM from 3 independent experiments. Maximum LDH-release was obtained in the presence of 1% Triton X-100. Statistical significance was assessed using ANOVA (Bonferroni post-test). (B) Viability of RAW264.7 macrophages was fluorometrically measured 5 h p.i. by the ability of cells to reduce Resorufin. Experiments were performed in triplicates. Values are means ±SEM from at least 3 independent experiments. Statistical significance was assessed using ANOVA (Bonferroni post-test).
    Raw264 7 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 3228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    raw264 7 cells - by Bioz Stars, 2020-11
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    99
    Thermo Fisher viia 7 real time pcr system
    Effects of SseI on host cell survival. (A and B) <t>RAW264.7</t> macrophages were infected with wild type (wt) or Δ sseI - Salmonella (MOI = 30; 30 min). 1.5 h p.i., cells were starved for 3.5 h in Earle’s balanced salt solution. The PI3K inhibitor LY29 (30 μM) was added 1.5 h p.i. where indicated. (A) The amount of LDH released into the medium was measured fluorometrically 5 h p.i.. Δ sseI- infected macrophages showed higher amounts of released LDH compared to wt- Salmonella infected cells. Data show means ±SEM from 3 independent experiments. Maximum LDH-release was obtained in the presence of 1% Triton X-100. Statistical significance was assessed using ANOVA (Bonferroni post-test). (B) Viability of RAW264.7 macrophages was fluorometrically measured 5 h p.i. by the ability of cells to reduce Resorufin. Experiments were performed in triplicates. Values are means ±SEM from at least 3 independent experiments. Statistical significance was assessed using ANOVA (Bonferroni post-test).
    Viia 7 Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    viia 7 real time pcr system - by Bioz Stars, 2020-11
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    99
    Graph Pad Software Inc prism 7
    Effects of SseI on host cell survival. (A and B) <t>RAW264.7</t> macrophages were infected with wild type (wt) or Δ sseI - Salmonella (MOI = 30; 30 min). 1.5 h p.i., cells were starved for 3.5 h in Earle’s balanced salt solution. The PI3K inhibitor LY29 (30 μM) was added 1.5 h p.i. where indicated. (A) The amount of LDH released into the medium was measured fluorometrically 5 h p.i.. Δ sseI- infected macrophages showed higher amounts of released LDH compared to wt- Salmonella infected cells. Data show means ±SEM from 3 independent experiments. Maximum LDH-release was obtained in the presence of 1% Triton X-100. Statistical significance was assessed using ANOVA (Bonferroni post-test). (B) Viability of RAW264.7 macrophages was fluorometrically measured 5 h p.i. by the ability of cells to reduce Resorufin. Experiments were performed in triplicates. Values are means ±SEM from at least 3 independent experiments. Statistical significance was assessed using ANOVA (Bonferroni post-test).
    Prism 7, supplied by Graph Pad Software Inc, used in various techniques. Bioz Stars score: 99/100, based on 2309 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson 7 aad
    Knockdown of STIM1 with siRNA does not reduce apoptosis triggered by BIRD-2 in SU-DHL-4 cells. a Analysis of the cytosolic Ca 2+ response in mock-transfected SU-DHL-4 cells, or cells transfected with scrambled siRNA or with siRNA against STIM1, using Fura-2 AM. Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of three independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in b , and the time constant τ (s), which is shown in c . d Representative scatter plots from flow cytometry analysis detecting apoptosis in transfected SU-DHL-4 cells stained with Annexin V-FITC and <t>7-AAD.</t> Cells were treated for 2 h with 10 µM BIRD-2, after which apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. e Quantitative analysis of four independent experiments detecting apoptosis in mock-transfected SU-DHL-4 cells or cells transfected with scrambled siRNA or with STIM1 siRNA. Cells were treated for 2 h with 10 µM BIRD-2. In the scatter plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a one-way ANOVA. NS not significant.
    7 Aad, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 12084 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega caspase glo 3 7 assay
    Knockdown of STIM1 with siRNA does not reduce apoptosis triggered by BIRD-2 in SU-DHL-4 cells. a Analysis of the cytosolic Ca 2+ response in mock-transfected SU-DHL-4 cells, or cells transfected with scrambled siRNA or with siRNA against STIM1, using Fura-2 AM. Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of three independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in b , and the time constant τ (s), which is shown in c . d Representative scatter plots from flow cytometry analysis detecting apoptosis in transfected SU-DHL-4 cells stained with Annexin V-FITC and <t>7-AAD.</t> Cells were treated for 2 h with 10 µM BIRD-2, after which apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. e Quantitative analysis of four independent experiments detecting apoptosis in mock-transfected SU-DHL-4 cells or cells transfected with scrambled siRNA or with STIM1 siRNA. Cells were treated for 2 h with 10 µM BIRD-2. In the scatter plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a one-way ANOVA. NS not significant.
    Caspase Glo 3 7 Assay, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 5378 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dcfh da
    Knockdown of STIM1 with siRNA does not reduce apoptosis triggered by BIRD-2 in SU-DHL-4 cells. a Analysis of the cytosolic Ca 2+ response in mock-transfected SU-DHL-4 cells, or cells transfected with scrambled siRNA or with siRNA against STIM1, using Fura-2 AM. Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of three independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in b , and the time constant τ (s), which is shown in c . d Representative scatter plots from flow cytometry analysis detecting apoptosis in transfected SU-DHL-4 cells stained with Annexin V-FITC and <t>7-AAD.</t> Cells were treated for 2 h with 10 µM BIRD-2, after which apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. e Quantitative analysis of four independent experiments detecting apoptosis in mock-transfected SU-DHL-4 cells or cells transfected with scrambled siRNA or with STIM1 siRNA. Cells were treated for 2 h with 10 µM BIRD-2. In the scatter plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a one-way ANOVA. NS not significant.
    Dcfh Da, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1972 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GraphPad Prism Inc graphpad prism 7 0 software
    Western blotting analysis of LC3-I and LC3-II levels in the CHO cells before and after the EBSS treatment. ( A ) LC3-I and LC3-II protein levels were analyzed by performing Western blotting analysis. The cells were treated with EBSS for 0, 4, and 10 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. ( B ) Quantitative analysis of the results of Western blotting analysis are shown in ( A ) for LC3-II levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using <t>GraphPad</t> Prism 7.0 software and ImageJ 1.43. ( C ) Quantitative analysis of the results of Western blot analysis shown in (A) for LC3-II levels normalized using LC3-I levels. All values are represented as mean ± SD (* p
    Graphpad Prism 7 0 Software, supplied by GraphPad Prism Inc, used in various techniques. Bioz Stars score: 91/100, based on 2332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sodium bicarbonate
    Western blotting analysis of LC3-I and LC3-II levels in the CHO cells before and after the EBSS treatment. ( A ) LC3-I and LC3-II protein levels were analyzed by performing Western blotting analysis. The cells were treated with EBSS for 0, 4, and 10 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. ( B ) Quantitative analysis of the results of Western blotting analysis are shown in ( A ) for LC3-II levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using <t>GraphPad</t> Prism 7.0 software and ImageJ 1.43. ( C ) Quantitative analysis of the results of Western blot analysis shown in (A) for LC3-II levels normalized using LC3-I levels. All values are represented as mean ± SD (* p
    Sodium Bicarbonate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6432 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Carl Zeiss axio observer z1 microscope
    Western blotting analysis of LC3-I and LC3-II levels in the CHO cells before and after the EBSS treatment. ( A ) LC3-I and LC3-II protein levels were analyzed by performing Western blotting analysis. The cells were treated with EBSS for 0, 4, and 10 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. ( B ) Quantitative analysis of the results of Western blotting analysis are shown in ( A ) for LC3-II levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using <t>GraphPad</t> Prism 7.0 software and ImageJ 1.43. ( C ) Quantitative analysis of the results of Western blot analysis shown in (A) for LC3-II levels normalized using LC3-I levels. All values are represented as mean ± SD (* p
    Axio Observer Z1 Microscope, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 99/100, based on 6803 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bodipy 493 503
    Western blotting analysis of LC3-I and LC3-II levels in the CHO cells before and after the EBSS treatment. ( A ) LC3-I and LC3-II protein levels were analyzed by performing Western blotting analysis. The cells were treated with EBSS for 0, 4, and 10 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. ( B ) Quantitative analysis of the results of Western blotting analysis are shown in ( A ) for LC3-II levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using <t>GraphPad</t> Prism 7.0 software and ImageJ 1.43. ( C ) Quantitative analysis of the results of Western blot analysis shown in (A) for LC3-II levels normalized using LC3-I levels. All values are represented as mean ± SD (* p
    Bodipy 493 503, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    adobe systems adobe photoshop 7 0
    Transmission electron microscopy on tissue sections of cv. Grand Naine specifically stained for bacteria showing bacterial cells (indicated by an arrow) just inside the cell wall (cw) adjoining the plasma membrane captured at ×1400 (A), ×1200 (B), ×13 000 (C) or ×4800 (D) with a Tecnai™ G2 Spirit BioTWIN TEM, or at ×14 000 (E) using a JEOL 100S TEM with a further ×2 magnification in Adobe Photoshop 7.0. Intercellular space (ic) is obvious in (A), (B) and (C), and the plasma membrane (pm) envelope originating from the cell wall is indicated by the thin black arrow marked in ‘E’.
    Adobe Photoshop 7 0, supplied by adobe systems, used in various techniques. Bioz Stars score: 93/100, based on 4487 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    adobe photoshop 7 0 - by Bioz Stars, 2020-11
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    99
    Molecular Devices LLC softmax pro software
    Transmission electron microscopy on tissue sections of cv. Grand Naine specifically stained for bacteria showing bacterial cells (indicated by an arrow) just inside the cell wall (cw) adjoining the plasma membrane captured at ×1400 (A), ×1200 (B), ×13 000 (C) or ×4800 (D) with a Tecnai™ G2 Spirit BioTWIN TEM, or at ×14 000 (E) using a JEOL 100S TEM with a further ×2 magnification in Adobe Photoshop 7.0. Intercellular space (ic) is obvious in (A), (B) and (C), and the plasma membrane (pm) envelope originating from the cell wall is indicated by the thin black arrow marked in ‘E’.
    Softmax Pro Software, supplied by Molecular Devices LLC, used in various techniques. Bioz Stars score: 99/100, based on 4606 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pbs
    Transmission electron microscopy on tissue sections of cv. Grand Naine specifically stained for bacteria showing bacterial cells (indicated by an arrow) just inside the cell wall (cw) adjoining the plasma membrane captured at ×1400 (A), ×1200 (B), ×13 000 (C) or ×4800 (D) with a Tecnai™ G2 Spirit BioTWIN TEM, or at ×14 000 (E) using a JEOL 100S TEM with a further ×2 magnification in Adobe Photoshop 7.0. Intercellular space (ic) is obvious in (A), (B) and (C), and the plasma membrane (pm) envelope originating from the cell wall is indicated by the thin black arrow marked in ‘E’.
    Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 45702 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GraphPad Prism Inc graphpad prism version 7 0
    LSD1 acts cooperatively with Slug to inactivate ESR1 promoters by demethylating H3K4me2. ( a , b ) Western blot ( a ) and RT–PCR ( b ) analyze effects of LSD1 and Slug on ERα expression via Slug overexpression and LSD1 knockdown in MCF-7 cells. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of effects of LSD1 and Slug on ERα expression via Slug and LSD1 knockdown in MDA-MB-231 cells. ( e ) MDA-MB-231 cells expressing pHAGE-CMV-Flag-HA-LSD1 and pcDNA3.1-Slug-Flag or pcDNA3.1-ΔN-Slug-Flag was analyzed by IP. Cell lysates were immunoprecipitated with anti-HA antibody. Immunocomplexes were then immunoblotted using antibodies against Flag and HA. ( f ) A dual-luciferase reporter assay is used to investigate ESR1 promoter 2 activity in MDA-MB-231 cells by overexpressing wild-type or mutant Slug. ( g ) ChIP-qPCR analysis of LSD1 recruitment on ESR1 promoter in MDA-MB-231shSlug cells and their control cells. ( h ) ChIP-qPCR analysis of H3K4me2 recruitment on ESR1 promoter in MDA-MB-231shLSD1 cells and their control cells. All the ChIP-qPCR results represent % of input chromatin. ( i ) A dual-luciferase reporter assay is used to investigate ESR1 promoter activity in MDA-MB-231 cells after LSD1 knockdown. Statistical analysis is performed using <t>GraphPad</t> Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P
    Graphpad Prism Version 7 0, supplied by GraphPad Prism Inc, used in various techniques. Bioz Stars score: 92/100, based on 1274 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/graphpad prism version 7 0/product/GraphPad Prism Inc
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    graphpad prism version 7 0 - by Bioz Stars, 2020-11
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    99
    Thermo Fisher 2 7 dichlorodihydrofluorescein diacetate
    LSD1 acts cooperatively with Slug to inactivate ESR1 promoters by demethylating H3K4me2. ( a , b ) Western blot ( a ) and RT–PCR ( b ) analyze effects of LSD1 and Slug on ERα expression via Slug overexpression and LSD1 knockdown in MCF-7 cells. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of effects of LSD1 and Slug on ERα expression via Slug and LSD1 knockdown in MDA-MB-231 cells. ( e ) MDA-MB-231 cells expressing pHAGE-CMV-Flag-HA-LSD1 and pcDNA3.1-Slug-Flag or pcDNA3.1-ΔN-Slug-Flag was analyzed by IP. Cell lysates were immunoprecipitated with anti-HA antibody. Immunocomplexes were then immunoblotted using antibodies against Flag and HA. ( f ) A dual-luciferase reporter assay is used to investigate ESR1 promoter 2 activity in MDA-MB-231 cells by overexpressing wild-type or mutant Slug. ( g ) ChIP-qPCR analysis of LSD1 recruitment on ESR1 promoter in MDA-MB-231shSlug cells and their control cells. ( h ) ChIP-qPCR analysis of H3K4me2 recruitment on ESR1 promoter in MDA-MB-231shLSD1 cells and their control cells. All the ChIP-qPCR results represent % of input chromatin. ( i ) A dual-luciferase reporter assay is used to investigate ESR1 promoter activity in MDA-MB-231 cells after LSD1 knockdown. Statistical analysis is performed using <t>GraphPad</t> Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P
    2 7 Dichlorodihydrofluorescein Diacetate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/2 7 dichlorodihydrofluorescein diacetate/product/Thermo Fisher
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    99
    Thermo Fisher pbs phosphate buffered saline 10x ph 7 4
    LSD1 acts cooperatively with Slug to inactivate ESR1 promoters by demethylating H3K4me2. ( a , b ) Western blot ( a ) and RT–PCR ( b ) analyze effects of LSD1 and Slug on ERα expression via Slug overexpression and LSD1 knockdown in MCF-7 cells. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of effects of LSD1 and Slug on ERα expression via Slug and LSD1 knockdown in MDA-MB-231 cells. ( e ) MDA-MB-231 cells expressing pHAGE-CMV-Flag-HA-LSD1 and pcDNA3.1-Slug-Flag or pcDNA3.1-ΔN-Slug-Flag was analyzed by IP. Cell lysates were immunoprecipitated with anti-HA antibody. Immunocomplexes were then immunoblotted using antibodies against Flag and HA. ( f ) A dual-luciferase reporter assay is used to investigate ESR1 promoter 2 activity in MDA-MB-231 cells by overexpressing wild-type or mutant Slug. ( g ) ChIP-qPCR analysis of LSD1 recruitment on ESR1 promoter in MDA-MB-231shSlug cells and their control cells. ( h ) ChIP-qPCR analysis of H3K4me2 recruitment on ESR1 promoter in MDA-MB-231shLSD1 cells and their control cells. All the ChIP-qPCR results represent % of input chromatin. ( i ) A dual-luciferase reporter assay is used to investigate ESR1 promoter activity in MDA-MB-231 cells after LSD1 knockdown. Statistical analysis is performed using <t>GraphPad</t> Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P
    Pbs Phosphate Buffered Saline 10x Ph 7 4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pbs ph 7 4
    LSD1 acts cooperatively with Slug to inactivate ESR1 promoters by demethylating H3K4me2. ( a , b ) Western blot ( a ) and RT–PCR ( b ) analyze effects of LSD1 and Slug on ERα expression via Slug overexpression and LSD1 knockdown in MCF-7 cells. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of effects of LSD1 and Slug on ERα expression via Slug and LSD1 knockdown in MDA-MB-231 cells. ( e ) MDA-MB-231 cells expressing pHAGE-CMV-Flag-HA-LSD1 and pcDNA3.1-Slug-Flag or pcDNA3.1-ΔN-Slug-Flag was analyzed by IP. Cell lysates were immunoprecipitated with anti-HA antibody. Immunocomplexes were then immunoblotted using antibodies against Flag and HA. ( f ) A dual-luciferase reporter assay is used to investigate ESR1 promoter 2 activity in MDA-MB-231 cells by overexpressing wild-type or mutant Slug. ( g ) ChIP-qPCR analysis of LSD1 recruitment on ESR1 promoter in MDA-MB-231shSlug cells and their control cells. ( h ) ChIP-qPCR analysis of H3K4me2 recruitment on ESR1 promoter in MDA-MB-231shLSD1 cells and their control cells. All the ChIP-qPCR results represent % of input chromatin. ( i ) A dual-luciferase reporter assay is used to investigate ESR1 promoter activity in MDA-MB-231 cells after LSD1 knockdown. Statistical analysis is performed using <t>GraphPad</t> Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P
    Pbs Ph 7 4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 880 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson 7 amino actinomycin d
    Elevated Mcl-1 expression protects Fbw7-deficient T-ALL cell lines from ABT-737-induced apoptosis a , Cell viability assays showing that Fbw7-deficient T-ALL cell lines were more sensitive to sorafenib, but resistant to ABT-737 treatment. T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib or ABT-737 for 48 hours before performing the cell viability assays. Data was shown as mean ± SD for three independent experiments. b , Immunoblot analysis of the indicated human T-ALL cell lines with or without ABT-737 (0.8 µM) treatment. c , Specific depletion of endogenous Mcl-1 expression restored ABT-737 sensitivity in the indicated Fbw7-deficient T-ALL cell lines. Various T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of ABT-737 for 48 hours before performing the cell viability assays, or with or without ABT-737 (0.8 µM) treatment for 24 hours before collecting whole cell lysates for immunoblot analysis with the indicated antibodies. For cell viability assays, data was shown as mean ± SD for three independent experiments. d , <t>7-Amino-Actinomycin</t> D (7-AAD)/Annexin V double-staining FACS analysis to detect the percentage of ABT-737-induced apoptosis in the indicated Fbw7-deficient T-ALL cell lines where the endogenous Mcl-1 was depleted by lentiviral shRNA treatment (lentiviral shGFP was used as a negative control). Various T-ALL cells were cultured in 10% FBS-containing medium with or without ABT-737 (0.8 µM) treatment for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells. e , 7-AAD/Annexin V double-staining FACS analysis to demonstrate that sorafenib treatment restores ABT-737 sensitivity to Fbw7-deficient HPB-ALL cells. HPB-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib and/or ABT-737 for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells.
    7 Amino Actinomycin D, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 3930 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher quantstudio 7 flex real time pcr system
    Elevated Mcl-1 expression protects Fbw7-deficient T-ALL cell lines from ABT-737-induced apoptosis a , Cell viability assays showing that Fbw7-deficient T-ALL cell lines were more sensitive to sorafenib, but resistant to ABT-737 treatment. T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib or ABT-737 for 48 hours before performing the cell viability assays. Data was shown as mean ± SD for three independent experiments. b , Immunoblot analysis of the indicated human T-ALL cell lines with or without ABT-737 (0.8 µM) treatment. c , Specific depletion of endogenous Mcl-1 expression restored ABT-737 sensitivity in the indicated Fbw7-deficient T-ALL cell lines. Various T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of ABT-737 for 48 hours before performing the cell viability assays, or with or without ABT-737 (0.8 µM) treatment for 24 hours before collecting whole cell lysates for immunoblot analysis with the indicated antibodies. For cell viability assays, data was shown as mean ± SD for three independent experiments. d , <t>7-Amino-Actinomycin</t> D (7-AAD)/Annexin V double-staining FACS analysis to detect the percentage of ABT-737-induced apoptosis in the indicated Fbw7-deficient T-ALL cell lines where the endogenous Mcl-1 was depleted by lentiviral shRNA treatment (lentiviral shGFP was used as a negative control). Various T-ALL cells were cultured in 10% FBS-containing medium with or without ABT-737 (0.8 µM) treatment for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells. e , 7-AAD/Annexin V double-staining FACS analysis to demonstrate that sorafenib treatment restores ABT-737 sensitivity to Fbw7-deficient HPB-ALL cells. HPB-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib and/or ABT-737 for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells.
    Quantstudio 7 Flex Real Time Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2283 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Elevated Mcl-1 expression protects Fbw7-deficient T-ALL cell lines from ABT-737-induced apoptosis a , Cell viability assays showing that Fbw7-deficient T-ALL cell lines were more sensitive to sorafenib, but resistant to ABT-737 treatment. T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib or ABT-737 for 48 hours before performing the cell viability assays. Data was shown as mean ± SD for three independent experiments. b , Immunoblot analysis of the indicated human T-ALL cell lines with or without ABT-737 (0.8 µM) treatment. c , Specific depletion of endogenous Mcl-1 expression restored ABT-737 sensitivity in the indicated Fbw7-deficient T-ALL cell lines. Various T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of ABT-737 for 48 hours before performing the cell viability assays, or with or without ABT-737 (0.8 µM) treatment for 24 hours before collecting whole cell lysates for immunoblot analysis with the indicated antibodies. For cell viability assays, data was shown as mean ± SD for three independent experiments. d , <t>7-Amino-Actinomycin</t> D (7-AAD)/Annexin V double-staining FACS analysis to detect the percentage of ABT-737-induced apoptosis in the indicated Fbw7-deficient T-ALL cell lines where the endogenous Mcl-1 was depleted by lentiviral shRNA treatment (lentiviral shGFP was used as a negative control). Various T-ALL cells were cultured in 10% FBS-containing medium with or without ABT-737 (0.8 µM) treatment for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells. e , 7-AAD/Annexin V double-staining FACS analysis to demonstrate that sorafenib treatment restores ABT-737 sensitivity to Fbw7-deficient HPB-ALL cells. HPB-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib and/or ABT-737 for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells.
    Graphpad Prism Version 7 00, supplied by GraphPad Prism Inc, used in various techniques. Bioz Stars score: 90/100, based on 977 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of SseI on host cell survival. (A and B) RAW264.7 macrophages were infected with wild type (wt) or Δ sseI - Salmonella (MOI = 30; 30 min). 1.5 h p.i., cells were starved for 3.5 h in Earle’s balanced salt solution. The PI3K inhibitor LY29 (30 μM) was added 1.5 h p.i. where indicated. (A) The amount of LDH released into the medium was measured fluorometrically 5 h p.i.. Δ sseI- infected macrophages showed higher amounts of released LDH compared to wt- Salmonella infected cells. Data show means ±SEM from 3 independent experiments. Maximum LDH-release was obtained in the presence of 1% Triton X-100. Statistical significance was assessed using ANOVA (Bonferroni post-test). (B) Viability of RAW264.7 macrophages was fluorometrically measured 5 h p.i. by the ability of cells to reduce Resorufin. Experiments were performed in triplicates. Values are means ±SEM from at least 3 independent experiments. Statistical significance was assessed using ANOVA (Bonferroni post-test).

    Journal: PLoS Pathogens

    Article Title: Salmonella Typhimurium effector SseI inhibits chemotaxis and increases host cell survival by deamidation of heterotrimeric Gi proteins

    doi: 10.1371/journal.ppat.1007248

    Figure Lengend Snippet: Effects of SseI on host cell survival. (A and B) RAW264.7 macrophages were infected with wild type (wt) or Δ sseI - Salmonella (MOI = 30; 30 min). 1.5 h p.i., cells were starved for 3.5 h in Earle’s balanced salt solution. The PI3K inhibitor LY29 (30 μM) was added 1.5 h p.i. where indicated. (A) The amount of LDH released into the medium was measured fluorometrically 5 h p.i.. Δ sseI- infected macrophages showed higher amounts of released LDH compared to wt- Salmonella infected cells. Data show means ±SEM from 3 independent experiments. Maximum LDH-release was obtained in the presence of 1% Triton X-100. Statistical significance was assessed using ANOVA (Bonferroni post-test). (B) Viability of RAW264.7 macrophages was fluorometrically measured 5 h p.i. by the ability of cells to reduce Resorufin. Experiments were performed in triplicates. Values are means ±SEM from at least 3 independent experiments. Statistical significance was assessed using ANOVA (Bonferroni post-test).

    Article Snippet: RAW264.7 cells (male, ATCC TIB-71) were cultured in DMEM supplemented with 5% FCS and 1% P/S.

    Techniques: Infection

    Cellular effects of SseI during infection. (A) Fluorescence microscopy of fixed cells. RAW264.7 cells were infected with wild type (wt) S . Typhimurium or a Δ sseI -strain at a MOI of 1 for 5 h. Deamidation of Gα was detected utilizing the GαQE antibody and an Alexa 568-conjugated secondary antibody. Salmonella were identified by Salmonella O antiserum and an Alexa 488-conjugated secondary antibody. Deamidated Gα is depicted in red and Salmonella in green. Z planes showing Salmonella were maximum projected into one image. Scale bars = 5 μm. Quantification of images (right panel). Gα protein deamidation of Salmonella -infected or uninfected cells was calculated by determining the average intensity of Alexa 568 fluorescence of the whole cell area in the Z plane with internalized Salmonella . Significance was determined by two-tailed Student’s t -test. Data are means ±SEM. (n = 10 cells). (B-D) Serum-starved RAW264.7 cells were infected with a MOI of 30 for 30 min. (B) Time-resolved immunoblot analysis. At indicated times p.i., cells were lysed and processed for immunoblotting. Blotting membranes were treated with indicated antibodies (GαQE, p-Akt and Akt). Representative blots of 3 independent experiments are shown. Equal loading was verified by detection of Akt, presence of Salmonella was verified by Salmonella -O antigen staining. Right panel shows the quantification of p-Akt (Ser473) labeling over time after infection with wt- and Δ sseI - Salmonella strains from 3 independent experiments. Chemiluminescence intensity, given as area units [AU], was determined for each band. Statistical significance was assessed by Mann Whitney test. (C) Only macrophages infected with wild type (wt) Salmonella show strong phosphorylation of Akt. Cells were infected with pEGFP expressing wt- or ΔsseI-Salmonella Typhimurium for 30 min. 5 h p.i. cells were fixed and processed for immunofluorescence microscopy. Cells were stained for phospho-Akt Ser473 (red) and nuclei were stained with DAPI (blue). Scale bars = 20 μm. (D) Addition of the PI3K inhibitor LY29 leads to concentration dependent inhibition of Akt phosphorylation in wild type (wt) Salmonella- infected macrophages. 1.5 h p.i. cells were treated with the indicated concentrations of LY29 for 3.5 h. Thereafter, cells were lysed. Lysates were processed for immunoblotting with indicated antibodies (Akt, p-Akt(Ser473)). (E) Quantification of p-Akt (Ser473) immunoblots of infected cells treated with increased concentrations of LY29. Experiments were performed as depicted in (D). Intensity of labeled bands was normalized to untreated (con) cells. Values are means ±SEM from at least 3 independent experiments.

    Journal: PLoS Pathogens

    Article Title: Salmonella Typhimurium effector SseI inhibits chemotaxis and increases host cell survival by deamidation of heterotrimeric Gi proteins

    doi: 10.1371/journal.ppat.1007248

    Figure Lengend Snippet: Cellular effects of SseI during infection. (A) Fluorescence microscopy of fixed cells. RAW264.7 cells were infected with wild type (wt) S . Typhimurium or a Δ sseI -strain at a MOI of 1 for 5 h. Deamidation of Gα was detected utilizing the GαQE antibody and an Alexa 568-conjugated secondary antibody. Salmonella were identified by Salmonella O antiserum and an Alexa 488-conjugated secondary antibody. Deamidated Gα is depicted in red and Salmonella in green. Z planes showing Salmonella were maximum projected into one image. Scale bars = 5 μm. Quantification of images (right panel). Gα protein deamidation of Salmonella -infected or uninfected cells was calculated by determining the average intensity of Alexa 568 fluorescence of the whole cell area in the Z plane with internalized Salmonella . Significance was determined by two-tailed Student’s t -test. Data are means ±SEM. (n = 10 cells). (B-D) Serum-starved RAW264.7 cells were infected with a MOI of 30 for 30 min. (B) Time-resolved immunoblot analysis. At indicated times p.i., cells were lysed and processed for immunoblotting. Blotting membranes were treated with indicated antibodies (GαQE, p-Akt and Akt). Representative blots of 3 independent experiments are shown. Equal loading was verified by detection of Akt, presence of Salmonella was verified by Salmonella -O antigen staining. Right panel shows the quantification of p-Akt (Ser473) labeling over time after infection with wt- and Δ sseI - Salmonella strains from 3 independent experiments. Chemiluminescence intensity, given as area units [AU], was determined for each band. Statistical significance was assessed by Mann Whitney test. (C) Only macrophages infected with wild type (wt) Salmonella show strong phosphorylation of Akt. Cells were infected with pEGFP expressing wt- or ΔsseI-Salmonella Typhimurium for 30 min. 5 h p.i. cells were fixed and processed for immunofluorescence microscopy. Cells were stained for phospho-Akt Ser473 (red) and nuclei were stained with DAPI (blue). Scale bars = 20 μm. (D) Addition of the PI3K inhibitor LY29 leads to concentration dependent inhibition of Akt phosphorylation in wild type (wt) Salmonella- infected macrophages. 1.5 h p.i. cells were treated with the indicated concentrations of LY29 for 3.5 h. Thereafter, cells were lysed. Lysates were processed for immunoblotting with indicated antibodies (Akt, p-Akt(Ser473)). (E) Quantification of p-Akt (Ser473) immunoblots of infected cells treated with increased concentrations of LY29. Experiments were performed as depicted in (D). Intensity of labeled bands was normalized to untreated (con) cells. Values are means ±SEM from at least 3 independent experiments.

    Article Snippet: RAW264.7 cells (male, ATCC TIB-71) were cultured in DMEM supplemented with 5% FCS and 1% P/S.

    Techniques: Infection, Fluorescence, Microscopy, Two Tailed Test, Staining, Labeling, MANN-WHITNEY, Expressing, Immunofluorescence, Concentration Assay, Inhibition, Western Blot

    L-plastin induces [Ca 2+ ] i  oscillations and sustained NFATc1 translocation in osteoclast precursors. (A-B) Fura2-loaded RANKL-primed RAW 264.7 cells were imaged at baseline, 50 μg/ml recombinant L-plastin (rLPL) or same amount of media, vehicle (Veh) was added to control for mechanical perturbations, and changes in [Ca 2+ ] i  were imaged for additional 60-120 seconds. (A)  Top : representative [Ca 2+ ] i  recordings ( top , 10- second bar indicates time scale) in seven individual cells per experimental condition.  Bottom : For each cell, the average variation in [Ca 2+ ] i , measured as a standard deviation of [Ca 2+ ] i , levels was determined and presented in ascending order. (B) Average [Ca 2+ ] i  ( top ) and variation (standard deviations of [Ca 2+ ] i  level,  bottom ). Data are means ± SEM,  N  = 33-63 cells/condition, from four independent experiments, ** P

    Journal: Translational Oncology

    Article Title: Exosomal Release of L-Plastin by Breast Cancer Cells Facilitates Metastatic Bone Osteolysis

    doi: 10.1016/j.tranon.2018.11.014

    Figure Lengend Snippet: L-plastin induces [Ca 2+ ] i oscillations and sustained NFATc1 translocation in osteoclast precursors. (A-B) Fura2-loaded RANKL-primed RAW 264.7 cells were imaged at baseline, 50 μg/ml recombinant L-plastin (rLPL) or same amount of media, vehicle (Veh) was added to control for mechanical perturbations, and changes in [Ca 2+ ] i were imaged for additional 60-120 seconds. (A) Top : representative [Ca 2+ ] i recordings ( top , 10- second bar indicates time scale) in seven individual cells per experimental condition. Bottom : For each cell, the average variation in [Ca 2+ ] i , measured as a standard deviation of [Ca 2+ ] i , levels was determined and presented in ascending order. (B) Average [Ca 2+ ] i ( top ) and variation (standard deviations of [Ca 2+ ] i level, bottom ). Data are means ± SEM, N  = 33-63 cells/condition, from four independent experiments, ** P

    Article Snippet: RAW 264.7 cells (TIB-71, American Type Culture Collection) were cultured in DMEM supplemented with L-glutamine, 1 mM pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% FBS.

    Techniques: Translocation Assay, Recombinant, Standard Deviation

    L-plastin is an osteoclastogenic factor secreted from MDA-MB-231 breast cancer cells. (A) Immunoblotting for I, L, and T-plastin (IPL, LPL, and TPL) in MDA-MB-231 cell lysates (CL) and conditioned medium (CM). Equal loading was visualized by Ponceau staining (lower gels). (B-D) L-plastin was transiently depleted in MDA-MB-231 cells (MD-231) using siRNA for L-plastin specific oligos (siLPL) or negative control (siNC). (B) L-plastin mRNA  (top)  and protein  (bottom)  expression in parental (−) and siLPL cells. (C, D) RAW 264.7 cells were primed with RANKL (50 ng/ml) for 2 days and then cultured for additional 2 days without treatment (negative control, NC), with RANKL (50 ng/ml, PC), or with 10% CM from siNC or siLPL MDA-MB-231. Average numbers (C) and representative images (D) of osteoclasts (OC) formed in indicated conditions. Scale bar of 100 μm applies to both images. Data are means ± SD,  N  = 2. (E, F) Protein expressions of L-plastin in whole cell lysates (E) and CM (F) in human erythroleukemia cells K562, human embryonic kidney cells 293 (HEK), normal skin fibroblasts (NSF), prostate carcinoma cells (PC3), human breast carcinoma cells MCF7 and MDA-MB-231 (231), and mouse breast carcinoma cells (4T1) were determined using immunoblotting. Protein loading was assessed using α-tubulin for cell lysates (E) and Ponceau stain for CM (F).

    Journal: Translational Oncology

    Article Title: Exosomal Release of L-Plastin by Breast Cancer Cells Facilitates Metastatic Bone Osteolysis

    doi: 10.1016/j.tranon.2018.11.014

    Figure Lengend Snippet: L-plastin is an osteoclastogenic factor secreted from MDA-MB-231 breast cancer cells. (A) Immunoblotting for I, L, and T-plastin (IPL, LPL, and TPL) in MDA-MB-231 cell lysates (CL) and conditioned medium (CM). Equal loading was visualized by Ponceau staining (lower gels). (B-D) L-plastin was transiently depleted in MDA-MB-231 cells (MD-231) using siRNA for L-plastin specific oligos (siLPL) or negative control (siNC). (B) L-plastin mRNA (top) and protein (bottom) expression in parental (−) and siLPL cells. (C, D) RAW 264.7 cells were primed with RANKL (50 ng/ml) for 2 days and then cultured for additional 2 days without treatment (negative control, NC), with RANKL (50 ng/ml, PC), or with 10% CM from siNC or siLPL MDA-MB-231. Average numbers (C) and representative images (D) of osteoclasts (OC) formed in indicated conditions. Scale bar of 100 μm applies to both images. Data are means ± SD, N  = 2. (E, F) Protein expressions of L-plastin in whole cell lysates (E) and CM (F) in human erythroleukemia cells K562, human embryonic kidney cells 293 (HEK), normal skin fibroblasts (NSF), prostate carcinoma cells (PC3), human breast carcinoma cells MCF7 and MDA-MB-231 (231), and mouse breast carcinoma cells (4T1) were determined using immunoblotting. Protein loading was assessed using α-tubulin for cell lysates (E) and Ponceau stain for CM (F).

    Article Snippet: RAW 264.7 cells (TIB-71, American Type Culture Collection) were cultured in DMEM supplemented with L-glutamine, 1 mM pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% FBS.

    Techniques: Multiple Displacement Amplification, Staining, Negative Control, Expressing, Cell Culture

    Recombinant L-plastin induces osteoclast differentiation from RANKL-primed precursors. (A-C) RAW 264.7 cells were primed with RANKL (50 ng/ml) for 2 days and then cultured for an additional 2 days without RANKL treatment (negative control, NC), with RANKL (50 ng/ml, positive control, PC), with human recombinant L-plastin (rLPL) at different concentrations (5, 10, or 25 μg/ml) (A), or with human recombinant I-plastin-1 (rIPL), L-plastin (rLPL), T-plastin (rTPL), or control protein (rCP, an unrelated protein produced by the same process) (5-10 μg/ml), (B) and average osteoclast numbers were assessed. Data are means ± SEM.,  N  = 3-11 independent experiments, *** P

    Journal: Translational Oncology

    Article Title: Exosomal Release of L-Plastin by Breast Cancer Cells Facilitates Metastatic Bone Osteolysis

    doi: 10.1016/j.tranon.2018.11.014

    Figure Lengend Snippet: Recombinant L-plastin induces osteoclast differentiation from RANKL-primed precursors. (A-C) RAW 264.7 cells were primed with RANKL (50 ng/ml) for 2 days and then cultured for an additional 2 days without RANKL treatment (negative control, NC), with RANKL (50 ng/ml, positive control, PC), with human recombinant L-plastin (rLPL) at different concentrations (5, 10, or 25 μg/ml) (A), or with human recombinant I-plastin-1 (rIPL), L-plastin (rLPL), T-plastin (rTPL), or control protein (rCP, an unrelated protein produced by the same process) (5-10 μg/ml), (B) and average osteoclast numbers were assessed. Data are means ± SEM., N  = 3-11 independent experiments, *** P

    Article Snippet: RAW 264.7 cells (TIB-71, American Type Culture Collection) were cultured in DMEM supplemented with L-glutamine, 1 mM pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% FBS.

    Techniques: Recombinant, Cell Culture, Negative Control, Positive Control, Produced

    Knockdown of STIM1 with siRNA does not reduce apoptosis triggered by BIRD-2 in SU-DHL-4 cells. a Analysis of the cytosolic Ca 2+ response in mock-transfected SU-DHL-4 cells, or cells transfected with scrambled siRNA or with siRNA against STIM1, using Fura-2 AM. Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of three independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in b , and the time constant τ (s), which is shown in c . d Representative scatter plots from flow cytometry analysis detecting apoptosis in transfected SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were treated for 2 h with 10 µM BIRD-2, after which apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. e Quantitative analysis of four independent experiments detecting apoptosis in mock-transfected SU-DHL-4 cells or cells transfected with scrambled siRNA or with STIM1 siRNA. Cells were treated for 2 h with 10 µM BIRD-2. In the scatter plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a one-way ANOVA. NS not significant.

    Journal: Cell Death Discovery

    Article Title: Extracellular and ER-stored Ca2+ contribute to BIRD-2-induced cell death in diffuse large B-cell lymphoma cells

    doi: 10.1038/s41420-018-0118-6

    Figure Lengend Snippet: Knockdown of STIM1 with siRNA does not reduce apoptosis triggered by BIRD-2 in SU-DHL-4 cells. a Analysis of the cytosolic Ca 2+ response in mock-transfected SU-DHL-4 cells, or cells transfected with scrambled siRNA or with siRNA against STIM1, using Fura-2 AM. Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of three independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in b , and the time constant τ (s), which is shown in c . d Representative scatter plots from flow cytometry analysis detecting apoptosis in transfected SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were treated for 2 h with 10 µM BIRD-2, after which apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. e Quantitative analysis of four independent experiments detecting apoptosis in mock-transfected SU-DHL-4 cells or cells transfected with scrambled siRNA or with STIM1 siRNA. Cells were treated for 2 h with 10 µM BIRD-2. In the scatter plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a one-way ANOVA. NS not significant.

    Article Snippet: Subsequently, cells were pelleted by centrifugation and incubated with Annexin V-FITC (Life Technologies, Carlsbad, CA, USA, V13245) and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA, 555815).

    Techniques: Transfection, Flow Cytometry, Cytometry, Staining

    BIRD-2-triggered cell death is reduced by chelating extracellular Ca 2+ with EGTA in SU-DHL-4 cells. a Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 3 mM EGTA 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. b Quantitative analysis detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without a pre-treatment of 30 min with 3 mM EGTA. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. Data are represented as the mean ± SEM of five independent experiments. Statistically significant differences were determined with a one-way ANOVA (* p

    Journal: Cell Death Discovery

    Article Title: Extracellular and ER-stored Ca2+ contribute to BIRD-2-induced cell death in diffuse large B-cell lymphoma cells

    doi: 10.1038/s41420-018-0118-6

    Figure Lengend Snippet: BIRD-2-triggered cell death is reduced by chelating extracellular Ca 2+ with EGTA in SU-DHL-4 cells. a Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 3 mM EGTA 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. b Quantitative analysis detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without a pre-treatment of 30 min with 3 mM EGTA. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. Data are represented as the mean ± SEM of five independent experiments. Statistically significant differences were determined with a one-way ANOVA (* p

    Article Snippet: Subsequently, cells were pelleted by centrifugation and incubated with Annexin V-FITC (Life Technologies, Carlsbad, CA, USA, V13245) and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA, 555815).

    Techniques: Flow Cytometry, Cytometry, Staining

    BIRD-2-triggered apoptosis is reduced by depleting the ER Ca 2+ store in SU-DHL-4 cells. a Analysis of the cytosolic Ca 2+ response in SU-DHL-4 cells using Fura-2 AM. After the addition of 3 mM EGTA, 1 or 10 µM TG or 10 µM CPA were added to deplete the ER Ca 2+ store. The curves represent the mean ± SEM of three independent experiments. The TG/CPA-releasable Ca 2+ is quantified by measuring the AUC ( F 340 / F 380 × s), which is shown in b . c Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 1 µM TG 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. d Quantitative analysis of five independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without a pre-treatment of 30 min with 1 µM TG. e Quantitative analysis of five independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without a pre-treatment of 30 min with 10 µM CPA. f A representative western blot of four independent experiments showing the expression levels of total eIF2α and p-eIF2α in SU-DHL-4 cells treated for 4 h with 5µg/ml tunicamycin. The expression level of vinculin was used as a loading control. g Quantification of the p-eIF2α/total eIF2α-protein levels in SU-DHL-4 relative to vehicle-treated cells, which was set at 1. h Quantitative analysis of three independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without a pre-treatment of 4 h with 5µg/ml tunicamycin. In d , e , and h the ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. In the dot plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a one-way ANOVA (* p

    Journal: Cell Death Discovery

    Article Title: Extracellular and ER-stored Ca2+ contribute to BIRD-2-induced cell death in diffuse large B-cell lymphoma cells

    doi: 10.1038/s41420-018-0118-6

    Figure Lengend Snippet: BIRD-2-triggered apoptosis is reduced by depleting the ER Ca 2+ store in SU-DHL-4 cells. a Analysis of the cytosolic Ca 2+ response in SU-DHL-4 cells using Fura-2 AM. After the addition of 3 mM EGTA, 1 or 10 µM TG or 10 µM CPA were added to deplete the ER Ca 2+ store. The curves represent the mean ± SEM of three independent experiments. The TG/CPA-releasable Ca 2+ is quantified by measuring the AUC ( F 340 / F 380 × s), which is shown in b . c Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 1 µM TG 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. d Quantitative analysis of five independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without a pre-treatment of 30 min with 1 µM TG. e Quantitative analysis of five independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without a pre-treatment of 30 min with 10 µM CPA. f A representative western blot of four independent experiments showing the expression levels of total eIF2α and p-eIF2α in SU-DHL-4 cells treated for 4 h with 5µg/ml tunicamycin. The expression level of vinculin was used as a loading control. g Quantification of the p-eIF2α/total eIF2α-protein levels in SU-DHL-4 relative to vehicle-treated cells, which was set at 1. h Quantitative analysis of three independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without a pre-treatment of 4 h with 5µg/ml tunicamycin. In d , e , and h the ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. In the dot plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a one-way ANOVA (* p

    Article Snippet: Subsequently, cells were pelleted by centrifugation and incubated with Annexin V-FITC (Life Technologies, Carlsbad, CA, USA, V13245) and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA, 555815).

    Techniques: Flow Cytometry, Cytometry, Staining, Western Blot, Expressing

    DPB162-AE reduces the cytotoxic effects of BIRD-2 in SU-DHL-4 cells. a Analysis of Ca 2+ influx after store depletion in SU-DHL-4 cells using the ratiometric Ca 2+ indicator Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or with 10 µM DPB162-AE (blue line). To deplete the ER Ca 2+ store, 3 mM EGTA and 1 µM thapsigargin (TG) were added as indicated. After store depletion, Ca 2+ influx was triggered by adding 10 mM CaCl 2 . The curves represent the mean ± SEM of four independent experiments. Quantification of the Ca 2+ influx is provided as the peak amplitude (∆ F 340 / F 380 ) in b , whereas the TG-releasable Ca 2+ is quantified as the AUC ( F 340 / F 380 × s) in c . d Analysis of the cytosolic Ca 2+ response in SU-DHL-4 cells using Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or 10 µM DPB162-AE (blue line). Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of five independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in e , and the time constant τ (s), which is shown in f . g Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 10 µM DPB162-AE 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. h Quantitative analysis of five independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without pre-treatment with 10 µM DPB162-AE. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. In the dot plots, data are represented as the mean ± SEM of at least four independent experiments. Statistically significant differences were determined with a paired two-tailed Student’s t test or a one-way ANOVA, as appropriate (* p

    Journal: Cell Death Discovery

    Article Title: Extracellular and ER-stored Ca2+ contribute to BIRD-2-induced cell death in diffuse large B-cell lymphoma cells

    doi: 10.1038/s41420-018-0118-6

    Figure Lengend Snippet: DPB162-AE reduces the cytotoxic effects of BIRD-2 in SU-DHL-4 cells. a Analysis of Ca 2+ influx after store depletion in SU-DHL-4 cells using the ratiometric Ca 2+ indicator Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or with 10 µM DPB162-AE (blue line). To deplete the ER Ca 2+ store, 3 mM EGTA and 1 µM thapsigargin (TG) were added as indicated. After store depletion, Ca 2+ influx was triggered by adding 10 mM CaCl 2 . The curves represent the mean ± SEM of four independent experiments. Quantification of the Ca 2+ influx is provided as the peak amplitude (∆ F 340 / F 380 ) in b , whereas the TG-releasable Ca 2+ is quantified as the AUC ( F 340 / F 380 × s) in c . d Analysis of the cytosolic Ca 2+ response in SU-DHL-4 cells using Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or 10 µM DPB162-AE (blue line). Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of five independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in e , and the time constant τ (s), which is shown in f . g Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 10 µM DPB162-AE 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. h Quantitative analysis of five independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without pre-treatment with 10 µM DPB162-AE. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. In the dot plots, data are represented as the mean ± SEM of at least four independent experiments. Statistically significant differences were determined with a paired two-tailed Student’s t test or a one-way ANOVA, as appropriate (* p

    Article Snippet: Subsequently, cells were pelleted by centrifugation and incubated with Annexin V-FITC (Life Technologies, Carlsbad, CA, USA, V13245) and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA, 555815).

    Techniques: Flow Cytometry, Cytometry, Staining, Two Tailed Test

    SOCE inhibition with YM-58483 does not reduce cell death triggered by BIRD-2 in SU-DHL-4 cells. a Analysis of Ca 2+ influx after store depletion in SU-DHL-4 cells using the ratiometric Ca 2+ indicator Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or with 3 µM YM-58483 (blue line). To deplete the ER Ca 2+ store, 3 mM EGTA and 1 µM TG were added as indicated. After store depletion, Ca 2+ influx was triggered by adding 10 mM CaCl 2 . The curves represent the mean ± SEM of three independent experiments. Quantification of the Ca 2+ influx is provided as the peak amplitude (∆ F 340 / F 380 ) in b , whereas the TG-releasable Ca 2+ is quantified as the AUC ( F 340 / F 380 × s) in c . d Dose–response curve of YM-58483 on SERCA2b ATPase activity (%). The Ca 2+ -dependent ATPase activity was measured at maximal (free [Ca 2+ ] 3.16 µM) and submaximal (free [Ca 2+ ] 0.316 µM) [Ca 2+ ] for different treatments, including vehicle (control) and different [YM-58483]. Data were normalized to the values obtained in the control condition at maximal [Ca 2+ ], which was set at 100%. Data are represented at the mean ± SEM of three independent experiments. e Single-cell analysis of the ER Ca 2+ levels in HeLa cells transfected with G-CEPIA1 er plasmid. Cells were treated with vehicle (gray curve), 1 µM TG (black curve), or 3 µM YM-58483 (blue curve) 60 s after the addition of 3 mM EGTA. Data are represented as the mean ± SEM of three independent experiments ( n > 100 cells/condition). f Analysis of the cytosolic Ca 2+ response in SU-DHL-4 cells using Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or 3 µM YM-58483 (blue line). Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of four independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in g , and the time constant τ (s), which is shown in h . i Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 3 µM YM-58483 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. j Quantitative analysis of three independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without pre-treatment with 3 µM YM-58483. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. In the dot plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a paired two-tailed Student’s t test or a one-way ANOVA, as appropriate (** p

    Journal: Cell Death Discovery

    Article Title: Extracellular and ER-stored Ca2+ contribute to BIRD-2-induced cell death in diffuse large B-cell lymphoma cells

    doi: 10.1038/s41420-018-0118-6

    Figure Lengend Snippet: SOCE inhibition with YM-58483 does not reduce cell death triggered by BIRD-2 in SU-DHL-4 cells. a Analysis of Ca 2+ influx after store depletion in SU-DHL-4 cells using the ratiometric Ca 2+ indicator Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or with 3 µM YM-58483 (blue line). To deplete the ER Ca 2+ store, 3 mM EGTA and 1 µM TG were added as indicated. After store depletion, Ca 2+ influx was triggered by adding 10 mM CaCl 2 . The curves represent the mean ± SEM of three independent experiments. Quantification of the Ca 2+ influx is provided as the peak amplitude (∆ F 340 / F 380 ) in b , whereas the TG-releasable Ca 2+ is quantified as the AUC ( F 340 / F 380 × s) in c . d Dose–response curve of YM-58483 on SERCA2b ATPase activity (%). The Ca 2+ -dependent ATPase activity was measured at maximal (free [Ca 2+ ] 3.16 µM) and submaximal (free [Ca 2+ ] 0.316 µM) [Ca 2+ ] for different treatments, including vehicle (control) and different [YM-58483]. Data were normalized to the values obtained in the control condition at maximal [Ca 2+ ], which was set at 100%. Data are represented at the mean ± SEM of three independent experiments. e Single-cell analysis of the ER Ca 2+ levels in HeLa cells transfected with G-CEPIA1 er plasmid. Cells were treated with vehicle (gray curve), 1 µM TG (black curve), or 3 µM YM-58483 (blue curve) 60 s after the addition of 3 mM EGTA. Data are represented as the mean ± SEM of three independent experiments ( n > 100 cells/condition). f Analysis of the cytosolic Ca 2+ response in SU-DHL-4 cells using Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or 3 µM YM-58483 (blue line). Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of four independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in g , and the time constant τ (s), which is shown in h . i Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 3 µM YM-58483 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. j Quantitative analysis of three independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without pre-treatment with 3 µM YM-58483. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. In the dot plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a paired two-tailed Student’s t test or a one-way ANOVA, as appropriate (** p

    Article Snippet: Subsequently, cells were pelleted by centrifugation and incubated with Annexin V-FITC (Life Technologies, Carlsbad, CA, USA, V13245) and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA, 555815).

    Techniques: Inhibition, Activity Assay, Single-cell Analysis, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Staining, Two Tailed Test

    SOCE inhibition with GSK-7975A does not reduce apoptosis induced by BIRD-2 in SU-DHL-4 cells. a Analysis of Ca 2+ influx after store depletion in SU-DHL-4 cells using the ratiometric Ca 2+ indicator Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or with 3 µM GSK-7975A (blue line). To deplete the ER Ca 2+ store, 3 mM EGTA and 1 µM TG were added as indicated. After store depletion, Ca 2+ influx was triggered by adding 10 mM CaCl 2 . The curves represent the mean ± SEM of three independent experiments. Quantification of the Ca 2+ influx is provided as the peak amplitude (∆ F 340 / F 380 ) in b , whereas the TG-releasable Ca 2+ is quantified as the AUC ( F 340 / F 380 × s) in c . d Dose–response curve of GSK-7975A on SERCA2b ATPase activity (%). The Ca 2+ -dependent ATPase activity was measured at maximal (free [Ca 2+ ] 3.16 µM) and submaximal (free [Ca 2+ ] 0.316 µM) [Ca 2+ ] for different treatments, including vehicle (control) and different [GSK-7975A]. Data were normalized to the values obtained in the control condition at maximal [Ca 2+ ], which was set at 100%. Data are represented at the mean ± SEM of three independent experiments. e Single-cell analysis of the ER Ca 2+ levels in HeLa cells transfected with G-CEPIA1 er plasmid. Cells were treated with vehicle (gray curve), 1 µM TG (black curve), or 3 µM GSK-7975A (blue curve) 60 s after the addition of 3 mM EGTA. Data are represented as the mean ± SEM of three independent experiments ( n > 100 cells/condition). f Analysis of the cytosolic Ca 2+ response in SU-DHL-4 cells using Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or 3 µM GSK-7975A (blue line). Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of four independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in g , and the time constant τ (s), which is shown in h . i Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 3 µM GSK-7975A 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. j Quantitative analysis of four independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without pre-treatment with 3 µM GSK-7975A. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. In the dot plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a paired two-tailed Student’s t test or a one-way ANOVA, as appropriate (** p

    Journal: Cell Death Discovery

    Article Title: Extracellular and ER-stored Ca2+ contribute to BIRD-2-induced cell death in diffuse large B-cell lymphoma cells

    doi: 10.1038/s41420-018-0118-6

    Figure Lengend Snippet: SOCE inhibition with GSK-7975A does not reduce apoptosis induced by BIRD-2 in SU-DHL-4 cells. a Analysis of Ca 2+ influx after store depletion in SU-DHL-4 cells using the ratiometric Ca 2+ indicator Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or with 3 µM GSK-7975A (blue line). To deplete the ER Ca 2+ store, 3 mM EGTA and 1 µM TG were added as indicated. After store depletion, Ca 2+ influx was triggered by adding 10 mM CaCl 2 . The curves represent the mean ± SEM of three independent experiments. Quantification of the Ca 2+ influx is provided as the peak amplitude (∆ F 340 / F 380 ) in b , whereas the TG-releasable Ca 2+ is quantified as the AUC ( F 340 / F 380 × s) in c . d Dose–response curve of GSK-7975A on SERCA2b ATPase activity (%). The Ca 2+ -dependent ATPase activity was measured at maximal (free [Ca 2+ ] 3.16 µM) and submaximal (free [Ca 2+ ] 0.316 µM) [Ca 2+ ] for different treatments, including vehicle (control) and different [GSK-7975A]. Data were normalized to the values obtained in the control condition at maximal [Ca 2+ ], which was set at 100%. Data are represented at the mean ± SEM of three independent experiments. e Single-cell analysis of the ER Ca 2+ levels in HeLa cells transfected with G-CEPIA1 er plasmid. Cells were treated with vehicle (gray curve), 1 µM TG (black curve), or 3 µM GSK-7975A (blue curve) 60 s after the addition of 3 mM EGTA. Data are represented as the mean ± SEM of three independent experiments ( n > 100 cells/condition). f Analysis of the cytosolic Ca 2+ response in SU-DHL-4 cells using Fura-2 AM. Cells were pre-treated for 30 min with vehicle (black line) or 3 µM GSK-7975A (blue line). Addition of 10 µM BIRD-2 is indicated by the dotted line. The curves represent the mean ± SEM of four independent experiments. The BIRD-2-provoked cytosolic Ca 2+ rise is quantified by measuring the peak amplitude (∆ F 340 / F 380 ), shown in g , and the time constant τ (s), which is shown in h . i Representative scatter plots from flow cytometry analysis detecting apoptosis in SU-DHL-4 cells stained with Annexin V-FITC and 7-AAD. Cells were pre-treated with or without 3 µM GSK-7975A 30 min prior to application of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was detected by measuring the Annexin V-FITC-positive fraction. j Quantitative analysis of four independent experiments detecting apoptosis in SU-DHL-4 cells treated for 2 h with 10 µM BIRD-2, with or without pre-treatment with 3 µM GSK-7975A. The ∆ apoptotic fraction is plotted, which corresponds to the apoptotic fraction corrected for the percentage of apoptosis detected in untreated cells. In the dot plots, data are represented as the mean ± SEM of at least three independent experiments. Statistically significant differences were determined with a paired two-tailed Student’s t test or a one-way ANOVA, as appropriate (** p

    Article Snippet: Subsequently, cells were pelleted by centrifugation and incubated with Annexin V-FITC (Life Technologies, Carlsbad, CA, USA, V13245) and 7-AAD (Becton Dickinson, Franklin Lakes, NJ, USA, 555815).

    Techniques: Inhibition, Activity Assay, Single-cell Analysis, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Staining, Two Tailed Test

    Western blotting analysis of LC3-I and LC3-II levels in the CHO cells before and after the EBSS treatment. ( A ) LC3-I and LC3-II protein levels were analyzed by performing Western blotting analysis. The cells were treated with EBSS for 0, 4, and 10 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. ( B ) Quantitative analysis of the results of Western blotting analysis are shown in ( A ) for LC3-II levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using GraphPad Prism 7.0 software and ImageJ 1.43. ( C ) Quantitative analysis of the results of Western blot analysis shown in (A) for LC3-II levels normalized using LC3-I levels. All values are represented as mean ± SD (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Silkworm Storage Protein 1 Inhibits Autophagy-Mediated Apoptosis

    doi: 10.3390/ijms20020318

    Figure Lengend Snippet: Western blotting analysis of LC3-I and LC3-II levels in the CHO cells before and after the EBSS treatment. ( A ) LC3-I and LC3-II protein levels were analyzed by performing Western blotting analysis. The cells were treated with EBSS for 0, 4, and 10 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. ( B ) Quantitative analysis of the results of Western blotting analysis are shown in ( A ) for LC3-II levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using GraphPad Prism 7.0 software and ImageJ 1.43. ( C ) Quantitative analysis of the results of Western blot analysis shown in (A) for LC3-II levels normalized using LC3-I levels. All values are represented as mean ± SD (* p

    Article Snippet: Statistical analysis of Western blotting analysis data obtained from three independent experiments was performed using GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA) and ImageJ 1.43.

    Techniques: Western Blot, Software

    The mRNA and protein levels of Beclin-1 after the EBSS treatment. ( A ) Quantitative PCR analysis of Beclin-1 mRNA levels in the CHO/CTRL and CHO/SP1 cells treated with EBSS for 0, 12, and 18 h. ( B ) Western blotting analysis of Beclin-1 protein levels in the CHO cells treated with EBSS for 0, 4, and 10 h. GAPDH was used as an internal control. ( C ) Quantitative analysis of the results of Western blotting analysis shown in ( B ) for Beclin-1 levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using GraphPad Prism 7.0 software and ImageJ 1.43. All values are represented as mean ± SD (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Silkworm Storage Protein 1 Inhibits Autophagy-Mediated Apoptosis

    doi: 10.3390/ijms20020318

    Figure Lengend Snippet: The mRNA and protein levels of Beclin-1 after the EBSS treatment. ( A ) Quantitative PCR analysis of Beclin-1 mRNA levels in the CHO/CTRL and CHO/SP1 cells treated with EBSS for 0, 12, and 18 h. ( B ) Western blotting analysis of Beclin-1 protein levels in the CHO cells treated with EBSS for 0, 4, and 10 h. GAPDH was used as an internal control. ( C ) Quantitative analysis of the results of Western blotting analysis shown in ( B ) for Beclin-1 levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using GraphPad Prism 7.0 software and ImageJ 1.43. All values are represented as mean ± SD (* p

    Article Snippet: Statistical analysis of Western blotting analysis data obtained from three independent experiments was performed using GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA) and ImageJ 1.43.

    Techniques: Real-time Polymerase Chain Reaction, Western Blot, Software

    The mRNA and protein levels of the key regulator ATG7 during EBSS-induced autophagy. ( A ) Quantitative PCR analysis of ATG7 mRNA levels in the CHO/CTRL and CHO/SP1 cells treated with EBSS for 0, 12, and 18 h. ( B ) Western blotting analysis of ATG7 protein levels in the CHO cells treated with EBSS for 0, 4, and 10 h. GAPDH was used as an internal control ( C ) Quantitative analysis of the results of Western blot analysis shown in ( B ) for ATG7 levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using GraphPad Prism 7.0 software and ImageJ 1.43. All values are represented as mean ± SD (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Silkworm Storage Protein 1 Inhibits Autophagy-Mediated Apoptosis

    doi: 10.3390/ijms20020318

    Figure Lengend Snippet: The mRNA and protein levels of the key regulator ATG7 during EBSS-induced autophagy. ( A ) Quantitative PCR analysis of ATG7 mRNA levels in the CHO/CTRL and CHO/SP1 cells treated with EBSS for 0, 12, and 18 h. ( B ) Western blotting analysis of ATG7 protein levels in the CHO cells treated with EBSS for 0, 4, and 10 h. GAPDH was used as an internal control ( C ) Quantitative analysis of the results of Western blot analysis shown in ( B ) for ATG7 levels normalized using the GAPDH levels. Statistical analysis of data obtained from three independent experiments was performed using GraphPad Prism 7.0 software and ImageJ 1.43. All values are represented as mean ± SD (* p

    Article Snippet: Statistical analysis of Western blotting analysis data obtained from three independent experiments was performed using GraphPad Prism 7.0 software (GraphPad, San Diego, CA, USA) and ImageJ 1.43.

    Techniques: Real-time Polymerase Chain Reaction, Western Blot, Software

    Hemagglutination inhibition (HAI) serum antibody and survival after H1N1 influenza virus challenge. HAI titers were determined for each group of mice ( n = 11) from blood collected at week 12 postvaccination. Mice were vaccinated with either V4, V5, or V6 HA VLP vaccines, and the collected sera were tested against a panel of 9 H1N1 influenza viruses. Values are the geometric mean titers plus standard errors of the means (SEM) (error bars). The dotted lines indicate the 1:40 to 1:80 HAI titer range. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Journal: Journal of Virology

    Article Title: N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies

    doi: 10.1128/JVI.01432-19

    Figure Lengend Snippet: Hemagglutination inhibition (HAI) serum antibody and survival after H1N1 influenza virus challenge. HAI titers were determined for each group of mice ( n = 11) from blood collected at week 12 postvaccination. Mice were vaccinated with either V4, V5, or V6 HA VLP vaccines, and the collected sera were tested against a panel of 9 H1N1 influenza viruses. Values are the geometric mean titers plus standard errors of the means (SEM) (error bars). The dotted lines indicate the 1:40 to 1:80 HAI titer range. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Article Snippet: All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P< 0.0001).

    Techniques: HI Assay, Mouse Assay, Software

    Survival after H1N1 influenza virus challenges. BALB/c mice (11 mice/group) were vaccinated on days 0, 28, and 56 with each vaccine plus the AF03 adjuvant and infected on week 13 with 1 × 10 6 PFU of the A/California/07/2009 H1N1 influenza virus. (A) Kaplan-Meier survival curves for vaccinated mice challenged with each influenza virus. The percent survival per vaccine group is listed in parenthesis. (B) Viral lung titers are listed as PFU per gram of lung tissue on the y axis. Vaccines used for vaccination are listed on the x axis. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Journal: Journal of Virology

    Article Title: N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies

    doi: 10.1128/JVI.01432-19

    Figure Lengend Snippet: Survival after H1N1 influenza virus challenges. BALB/c mice (11 mice/group) were vaccinated on days 0, 28, and 56 with each vaccine plus the AF03 adjuvant and infected on week 13 with 1 × 10 6 PFU of the A/California/07/2009 H1N1 influenza virus. (A) Kaplan-Meier survival curves for vaccinated mice challenged with each influenza virus. The percent survival per vaccine group is listed in parenthesis. (B) Viral lung titers are listed as PFU per gram of lung tissue on the y axis. Vaccines used for vaccination are listed on the x axis. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Article Snippet: All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P< 0.0001).

    Techniques: Mouse Assay, Infection, Software

    Hemagglutination inhibition (HAI) serum antibody titers induced by vaccination of mice with V1, V2, or V3 VLP vaccines. HAI titers were determined for each group of mice ( n = 11) from blood collected at week 12 postvaccination. Mice were vaccinated with either COBRA, VIPER, or wild-type HA VLP vaccines and the collected sera were tested against a panel of 9 H1N1 influenza viruses. Values are the geometric mean titers plus standard errors of the means (SEM) (error bars). The dotted lines indicate the 1:40 to 1:80 HAI titer range. (A) Bris/07 VLP; (B) CA/09 VLP; (C) X6 VLP; (D) P1 VLP; (E) V1; (F) V2 VLP; (G) V3 VLP; (H) mock. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Journal: Journal of Virology

    Article Title: N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies

    doi: 10.1128/JVI.01432-19

    Figure Lengend Snippet: Hemagglutination inhibition (HAI) serum antibody titers induced by vaccination of mice with V1, V2, or V3 VLP vaccines. HAI titers were determined for each group of mice ( n = 11) from blood collected at week 12 postvaccination. Mice were vaccinated with either COBRA, VIPER, or wild-type HA VLP vaccines and the collected sera were tested against a panel of 9 H1N1 influenza viruses. Values are the geometric mean titers plus standard errors of the means (SEM) (error bars). The dotted lines indicate the 1:40 to 1:80 HAI titer range. (A) Bris/07 VLP; (B) CA/09 VLP; (C) X6 VLP; (D) P1 VLP; (E) V1; (F) V2 VLP; (G) V3 VLP; (H) mock. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Article Snippet: All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P< 0.0001).

    Techniques: HI Assay, Mouse Assay, Combined Bisulfite Restriction Analysis Assay, Software

    Hemagglutination inhibition (HAI) serum antibody. HAI titers were determined for each group of preimmune ferrets ( n = 4) from blood collected at day 125 postvaccination. Preimmune ferrets were vaccinated as follows: (A) mock; (B) CA/07 rHA vaccine; (C) P1 rHA vaccine; (D) X6 rHA vaccine; (E) V3 rHA vaccine; (F) V6 rHA vaccine; (G) V12 rHA vaccine. The collected sera were tested against a panel of 9 H1N1 influenza viruses. Values are the geometric mean titers plus standard errors of the means (SEM) (error bars). The dotted lines indicate the 1:40 to 1:80 HAI titer range. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Journal: Journal of Virology

    Article Title: N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies

    doi: 10.1128/JVI.01432-19

    Figure Lengend Snippet: Hemagglutination inhibition (HAI) serum antibody. HAI titers were determined for each group of preimmune ferrets ( n = 4) from blood collected at day 125 postvaccination. Preimmune ferrets were vaccinated as follows: (A) mock; (B) CA/07 rHA vaccine; (C) P1 rHA vaccine; (D) X6 rHA vaccine; (E) V3 rHA vaccine; (F) V6 rHA vaccine; (G) V12 rHA vaccine. The collected sera were tested against a panel of 9 H1N1 influenza viruses. Values are the geometric mean titers plus standard errors of the means (SEM) (error bars). The dotted lines indicate the 1:40 to 1:80 HAI titer range. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Article Snippet: All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P< 0.0001).

    Techniques: HI Assay, Software

    Hemagglutination inhibition (HAI) serum antibody. HAI titers were determined for each group of mice ( n = 11) from blood collected at week 12 postvaccination. Mice were vaccinated as follows: (A) mock; (B) Bris/07 VLP vaccine; (C) CA/09 VLP vaccine; (D) X6 VLP vaccine; (E) V9 VLP vaccine; (F) V10 VLP vaccine; (G) V11 VLP vaccine; (H) V12 VLP vaccine. The collected sera were tested against a panel of 9 H1N1 influenza viruses. The dotted lines indicate the 1:40 to 1:80 HAI titer range. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Journal: Journal of Virology

    Article Title: N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies

    doi: 10.1128/JVI.01432-19

    Figure Lengend Snippet: Hemagglutination inhibition (HAI) serum antibody. HAI titers were determined for each group of mice ( n = 11) from blood collected at week 12 postvaccination. Mice were vaccinated as follows: (A) mock; (B) Bris/07 VLP vaccine; (C) CA/09 VLP vaccine; (D) X6 VLP vaccine; (E) V9 VLP vaccine; (F) V10 VLP vaccine; (G) V11 VLP vaccine; (H) V12 VLP vaccine. The collected sera were tested against a panel of 9 H1N1 influenza viruses. The dotted lines indicate the 1:40 to 1:80 HAI titer range. All data are reported as absolute mean values ± SEM. HAI titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Article Snippet: All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P< 0.0001).

    Techniques: HI Assay, Mouse Assay, Software

    Challenge with A/California/07/2009 H1N1 influenza virus. Vaccinated preimmune ferrets (4 ferrets/group) were infected on day 132 with 1 × 10 6 PFU/ml of the A/California/07/2009 H1N1 influenza virus. (A) Percent original weight per vaccine group. (B) Viral nasal wash titers are listed as PFU on the y axis for each individual ferret collected at days 1, 3, and 5 postinfection. All data are reported as absolute mean values ± SEM. Weight losses among the different vaccinated groups were compared using nonparametric two-way ANOVA, and viral lung titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Journal: Journal of Virology

    Article Title: N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies

    doi: 10.1128/JVI.01432-19

    Figure Lengend Snippet: Challenge with A/California/07/2009 H1N1 influenza virus. Vaccinated preimmune ferrets (4 ferrets/group) were infected on day 132 with 1 × 10 6 PFU/ml of the A/California/07/2009 H1N1 influenza virus. (A) Percent original weight per vaccine group. (B) Viral nasal wash titers are listed as PFU on the y axis for each individual ferret collected at days 1, 3, and 5 postinfection. All data are reported as absolute mean values ± SEM. Weight losses among the different vaccinated groups were compared using nonparametric two-way ANOVA, and viral lung titers were compared using nonparametric one-way ANOVA. All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of

    Article Snippet: All statistical analysis was performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA), and a P value of < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P< 0.0001).

    Techniques: Infection, Software

    Transmission electron microscopy on tissue sections of cv. Grand Naine specifically stained for bacteria showing bacterial cells (indicated by an arrow) just inside the cell wall (cw) adjoining the plasma membrane captured at ×1400 (A), ×1200 (B), ×13 000 (C) or ×4800 (D) with a Tecnai™ G2 Spirit BioTWIN TEM, or at ×14 000 (E) using a JEOL 100S TEM with a further ×2 magnification in Adobe Photoshop 7.0. Intercellular space (ic) is obvious in (A), (B) and (C), and the plasma membrane (pm) envelope originating from the cell wall is indicated by the thin black arrow marked in ‘E’.

    Journal: AoB Plants

    Article Title: Microscopic elucidation of abundant endophytic bacteria colonizing the cell wall–plasma membrane peri-space in the shoot-tip tissue of banana

    doi: 10.1093/aobpla/plt011

    Figure Lengend Snippet: Transmission electron microscopy on tissue sections of cv. Grand Naine specifically stained for bacteria showing bacterial cells (indicated by an arrow) just inside the cell wall (cw) adjoining the plasma membrane captured at ×1400 (A), ×1200 (B), ×13 000 (C) or ×4800 (D) with a Tecnai™ G2 Spirit BioTWIN TEM, or at ×14 000 (E) using a JEOL 100S TEM with a further ×2 magnification in Adobe Photoshop 7.0. Intercellular space (ic) is obvious in (A), (B) and (C), and the plasma membrane (pm) envelope originating from the cell wall is indicated by the thin black arrow marked in ‘E’.

    Article Snippet: The images were captured with LAS software and further processed with Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, USA) software.

    Techniques: Transmission Assay, Electron Microscopy, Staining, Transmission Electron Microscopy

    Expression of EGFR and EGF in NG2 +  cells of the postnatal brain. Immunostaining of sagittal sections from P8 CNP-EGFP mouse brains. aSVZ, Anterior SVZ; CTX, cerebral cortex. NG2 +  cells express high levels of EGFR in the SVZ ( a ), RMS ( b ), and SCWM ( c ), respectively. Conversely, coronal sections show that, in cerebral cortex ( d ), NG2 +  cells express levels of EGFR undetectable by immunohistochemistry. The individual cells selected for multi-marker illustration are indicated as boxed areas. Additional arrows indicate triple-positive cells. Dotted lines indicate the borders of the RMS. Optical sections ( Z  = 0.5 μm;  X  = 30 μm) of confocal epifluorescence images were sequentially acquired using a 60× oil objective (NA, 1.40) with Bio-Rad LaserSharp version 3.2 software. Confocal Assistant 4.02 was used to merge images. Merged images were processed in Photoshop 7.0 with minimal manipulations of contrast. Scale bar, 50 μm.  e ,  f , Total protein extracts from FACS-purified NG2 +  cells were analyzed by Western blot with selective anti-EGFR ( e ) and anti-EGF ( f ) antibodies. SVZ NG2 +  cells express EGFR and EGF levels higher than cortical cells. SVZ tissue was used as a positive control. The blotted membrane was reprobed with anti-actin antibodies to determine equal protein loading (bottom panels).

    Journal: The Journal of Neuroscience

    Article Title: Overexpression of the Epidermal Growth Factor Receptor Confers Migratory Properties to Nonmigratory Postnatal Neural Progenitors

    doi: 10.1523/JNEUROSCI.2981-05.2005

    Figure Lengend Snippet: Expression of EGFR and EGF in NG2 + cells of the postnatal brain. Immunostaining of sagittal sections from P8 CNP-EGFP mouse brains. aSVZ, Anterior SVZ; CTX, cerebral cortex. NG2 + cells express high levels of EGFR in the SVZ ( a ), RMS ( b ), and SCWM ( c ), respectively. Conversely, coronal sections show that, in cerebral cortex ( d ), NG2 + cells express levels of EGFR undetectable by immunohistochemistry. The individual cells selected for multi-marker illustration are indicated as boxed areas. Additional arrows indicate triple-positive cells. Dotted lines indicate the borders of the RMS. Optical sections ( Z = 0.5 μm; X = 30 μm) of confocal epifluorescence images were sequentially acquired using a 60× oil objective (NA, 1.40) with Bio-Rad LaserSharp version 3.2 software. Confocal Assistant 4.02 was used to merge images. Merged images were processed in Photoshop 7.0 with minimal manipulations of contrast. Scale bar, 50 μm. e , f , Total protein extracts from FACS-purified NG2 + cells were analyzed by Western blot with selective anti-EGFR ( e ) and anti-EGF ( f ) antibodies. SVZ NG2 + cells express EGFR and EGF levels higher than cortical cells. SVZ tissue was used as a positive control. The blotted membrane was reprobed with anti-actin antibodies to determine equal protein loading (bottom panels).

    Article Snippet: Merged images were processed in Photoshop 7.0 (Adobe Systems, San Jose, CA) with minimal manipulations of contrast.

    Techniques: Expressing, Immunostaining, Immunohistochemistry, Marker, Software, FACS, Purification, Western Blot, Positive Control

    LSD1 acts cooperatively with Slug to inactivate ESR1 promoters by demethylating H3K4me2. ( a , b ) Western blot ( a ) and RT–PCR ( b ) analyze effects of LSD1 and Slug on ERα expression via Slug overexpression and LSD1 knockdown in MCF-7 cells. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of effects of LSD1 and Slug on ERα expression via Slug and LSD1 knockdown in MDA-MB-231 cells. ( e ) MDA-MB-231 cells expressing pHAGE-CMV-Flag-HA-LSD1 and pcDNA3.1-Slug-Flag or pcDNA3.1-ΔN-Slug-Flag was analyzed by IP. Cell lysates were immunoprecipitated with anti-HA antibody. Immunocomplexes were then immunoblotted using antibodies against Flag and HA. ( f ) A dual-luciferase reporter assay is used to investigate ESR1 promoter 2 activity in MDA-MB-231 cells by overexpressing wild-type or mutant Slug. ( g ) ChIP-qPCR analysis of LSD1 recruitment on ESR1 promoter in MDA-MB-231shSlug cells and their control cells. ( h ) ChIP-qPCR analysis of H3K4me2 recruitment on ESR1 promoter in MDA-MB-231shLSD1 cells and their control cells. All the ChIP-qPCR results represent % of input chromatin. ( i ) A dual-luciferase reporter assay is used to investigate ESR1 promoter activity in MDA-MB-231 cells after LSD1 knockdown. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Journal: Oncogenesis

    Article Title: The zinc-finger transcriptional factor Slug transcriptionally downregulates ERα by recruiting lysine-specific demethylase 1 in human breast cancer

    doi: 10.1038/oncsis.2017.38

    Figure Lengend Snippet: LSD1 acts cooperatively with Slug to inactivate ESR1 promoters by demethylating H3K4me2. ( a , b ) Western blot ( a ) and RT–PCR ( b ) analyze effects of LSD1 and Slug on ERα expression via Slug overexpression and LSD1 knockdown in MCF-7 cells. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of effects of LSD1 and Slug on ERα expression via Slug and LSD1 knockdown in MDA-MB-231 cells. ( e ) MDA-MB-231 cells expressing pHAGE-CMV-Flag-HA-LSD1 and pcDNA3.1-Slug-Flag or pcDNA3.1-ΔN-Slug-Flag was analyzed by IP. Cell lysates were immunoprecipitated with anti-HA antibody. Immunocomplexes were then immunoblotted using antibodies against Flag and HA. ( f ) A dual-luciferase reporter assay is used to investigate ESR1 promoter 2 activity in MDA-MB-231 cells by overexpressing wild-type or mutant Slug. ( g ) ChIP-qPCR analysis of LSD1 recruitment on ESR1 promoter in MDA-MB-231shSlug cells and their control cells. ( h ) ChIP-qPCR analysis of H3K4me2 recruitment on ESR1 promoter in MDA-MB-231shLSD1 cells and their control cells. All the ChIP-qPCR results represent % of input chromatin. ( i ) A dual-luciferase reporter assay is used to investigate ESR1 promoter activity in MDA-MB-231 cells after LSD1 knockdown. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Article Snippet: All the statistical analyses were performed by GraphPad Prism version 7.0.

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Multiple Displacement Amplification, Immunoprecipitation, Luciferase, Reporter Assay, Activity Assay, Mutagenesis, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    LSD1 acts cooperatively with Slug to promote proliferation, migration, invasion and colony formation abilities in MDA-MB-231 cell line. ( a ) A wound healing assay with the indicated siRNAs. Representative images are shown. Magnification, × 100. ( b ) Migration and invasion assays with the indicated siRNAs. Representative images of migrated (upper) and invaded (lower) cells are presented. Magnification, × 400. ( c ) Comparison of proliferative ability with the indicated siRNAs. ( d ) Colony formation ability with the indicated siRNAs. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Journal: Oncogenesis

    Article Title: The zinc-finger transcriptional factor Slug transcriptionally downregulates ERα by recruiting lysine-specific demethylase 1 in human breast cancer

    doi: 10.1038/oncsis.2017.38

    Figure Lengend Snippet: LSD1 acts cooperatively with Slug to promote proliferation, migration, invasion and colony formation abilities in MDA-MB-231 cell line. ( a ) A wound healing assay with the indicated siRNAs. Representative images are shown. Magnification, × 100. ( b ) Migration and invasion assays with the indicated siRNAs. Representative images of migrated (upper) and invaded (lower) cells are presented. Magnification, × 400. ( c ) Comparison of proliferative ability with the indicated siRNAs. ( d ) Colony formation ability with the indicated siRNAs. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Article Snippet: All the statistical analyses were performed by GraphPad Prism version 7.0.

    Techniques: Migration, Multiple Displacement Amplification, Wound Healing Assay

    Negative relationship between Slug and ERα in human breast cancer cells. ( a , b ) Analysis of Slug and ERα expression levels in MCF-7, T47D, SKBR3, MDA-MB-231 and BT549 by western blot and RT–PCR. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of protein and mRNA levels as indicated in MCF-7 cells transfected with increasing concentrations (2, 4 and 8 μg, respectively) of Slug. ( e , f ) Western blot ( e ) and RT–PCR ( f ) analysis of protein and mRNA levels in MDA-MB-231 cells transfected with two different interference sequences of Slug (40 n m , separately). ( g ) Western blot analysis of Slug knockdown level in stable cell lines of MDA-MB231shSlug. ( h ) Immunofluorescence micrographs of Slug (red) and ERα (green) expression in MDA-MB-231shNC cells (upper) and MDA-MB-231shSlug (lower). DAPI is for nuclei (blue). Magnification, × 400. Protein and mRNA expression was normalized to β-actin. In figure 2d, NC means transfecting with control pcDNA3.1. In figure 2e and f, siNC is scrambled control siRNA used for transient transfection. ShNC without shRNA is used as a control by infecting pLKO.1 lentivirus in MDA-MB-231 cell line. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Journal: Oncogenesis

    Article Title: The zinc-finger transcriptional factor Slug transcriptionally downregulates ERα by recruiting lysine-specific demethylase 1 in human breast cancer

    doi: 10.1038/oncsis.2017.38

    Figure Lengend Snippet: Negative relationship between Slug and ERα in human breast cancer cells. ( a , b ) Analysis of Slug and ERα expression levels in MCF-7, T47D, SKBR3, MDA-MB-231 and BT549 by western blot and RT–PCR. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of protein and mRNA levels as indicated in MCF-7 cells transfected with increasing concentrations (2, 4 and 8 μg, respectively) of Slug. ( e , f ) Western blot ( e ) and RT–PCR ( f ) analysis of protein and mRNA levels in MDA-MB-231 cells transfected with two different interference sequences of Slug (40 n m , separately). ( g ) Western blot analysis of Slug knockdown level in stable cell lines of MDA-MB231shSlug. ( h ) Immunofluorescence micrographs of Slug (red) and ERα (green) expression in MDA-MB-231shNC cells (upper) and MDA-MB-231shSlug (lower). DAPI is for nuclei (blue). Magnification, × 400. Protein and mRNA expression was normalized to β-actin. In figure 2d, NC means transfecting with control pcDNA3.1. In figure 2e and f, siNC is scrambled control siRNA used for transient transfection. ShNC without shRNA is used as a control by infecting pLKO.1 lentivirus in MDA-MB-231 cell line. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Article Snippet: All the statistical analyses were performed by GraphPad Prism version 7.0.

    Techniques: Expressing, Multiple Displacement Amplification, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Stable Transfection, Immunofluorescence, shRNA

    ERα expression is directly regulated by Slug. ( a ) A schematic representation of the ESR1 promoter region in which the promoter is divided into three subregions including eight E-box-binding sites. ( b ) Different concentration of Slug siRNA (20, 40 or 80 n m , respectively) was co-transfected with the ESR1 promoter-luciferase plasmid in MDA-MB-231 cells. Variation in transfection efficiency was normalized by Renilla luciferase activity. ( c ) Different concentration of Slug (0.1, 0.3 or 0.9 μg, respectively) was co-expressed with the ESR1 promoter-luciferase construct in MCF-7 cells. After 24 h, Renilla luciferase activity was used to normalize variation in transfection efficiency. ( d ) Normal IgG or anti-Slug antibodies were used in a ChIP assay to determine which E-box-binding site Slug binds in the ESR1 promoter in MDA-MB-231 cells. Seven primer pairs covering different binding sites within promoter 1 and 2 are designed. ( e , f ) After ChIP, qPCR was used to analyse Slug recruitment on CDH1 ( e ) and ESR1 ( f ) promoter in MDA-MB-231shSlug cells and their control cells. The results represent % of input chromatin. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Journal: Oncogenesis

    Article Title: The zinc-finger transcriptional factor Slug transcriptionally downregulates ERα by recruiting lysine-specific demethylase 1 in human breast cancer

    doi: 10.1038/oncsis.2017.38

    Figure Lengend Snippet: ERα expression is directly regulated by Slug. ( a ) A schematic representation of the ESR1 promoter region in which the promoter is divided into three subregions including eight E-box-binding sites. ( b ) Different concentration of Slug siRNA (20, 40 or 80 n m , respectively) was co-transfected with the ESR1 promoter-luciferase plasmid in MDA-MB-231 cells. Variation in transfection efficiency was normalized by Renilla luciferase activity. ( c ) Different concentration of Slug (0.1, 0.3 or 0.9 μg, respectively) was co-expressed with the ESR1 promoter-luciferase construct in MCF-7 cells. After 24 h, Renilla luciferase activity was used to normalize variation in transfection efficiency. ( d ) Normal IgG or anti-Slug antibodies were used in a ChIP assay to determine which E-box-binding site Slug binds in the ESR1 promoter in MDA-MB-231 cells. Seven primer pairs covering different binding sites within promoter 1 and 2 are designed. ( e , f ) After ChIP, qPCR was used to analyse Slug recruitment on CDH1 ( e ) and ESR1 ( f ) promoter in MDA-MB-231shSlug cells and their control cells. The results represent % of input chromatin. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Article Snippet: All the statistical analyses were performed by GraphPad Prism version 7.0.

    Techniques: Expressing, Binding Assay, Concentration Assay, Transfection, Luciferase, Plasmid Preparation, Multiple Displacement Amplification, Activity Assay, Construct, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Negative correlation between Slug and ERα expression exists in human breast cancer tissues, and high Slug mRNA expression is correlated to poorer RFS in ERα-negative breast cancer patients. ( a , b ) Correlation of Slug mRNA ( a ) and protein ( b ) levels in ERα-negative and -positive breast cancer patients with z -score analysis. ( c , d ) Slug and ERα correlation analysis in z -scores of mRNA level ( c ) and protein level ( d ) is performed by two-tailed Pearson’s R tests. Z -scores of Slug and ERα are directly obtained from the online data ( www.cbioportal.org ). The formula is: z =(expression in tumor sample−mean expression in reference sample)/s.d. of expression in reference sample. ( e – g ) The prognostic effect of Slug mRNA is obtained from the website: www.kmplot.com . Survival curves are plotted for all patients ( n =3951) ( e ) for ERα-positive cancers ( n =1802) ( f ) and for ERα-negative cases ( n =671) ( g ). Statistical analysis is performed using GraphPad Prism version 7.0 (GraphPad Software Inc., San Diego, CA, USA). * P

    Journal: Oncogenesis

    Article Title: The zinc-finger transcriptional factor Slug transcriptionally downregulates ERα by recruiting lysine-specific demethylase 1 in human breast cancer

    doi: 10.1038/oncsis.2017.38

    Figure Lengend Snippet: Negative correlation between Slug and ERα expression exists in human breast cancer tissues, and high Slug mRNA expression is correlated to poorer RFS in ERα-negative breast cancer patients. ( a , b ) Correlation of Slug mRNA ( a ) and protein ( b ) levels in ERα-negative and -positive breast cancer patients with z -score analysis. ( c , d ) Slug and ERα correlation analysis in z -scores of mRNA level ( c ) and protein level ( d ) is performed by two-tailed Pearson’s R tests. Z -scores of Slug and ERα are directly obtained from the online data ( www.cbioportal.org ). The formula is: z =(expression in tumor sample−mean expression in reference sample)/s.d. of expression in reference sample. ( e – g ) The prognostic effect of Slug mRNA is obtained from the website: www.kmplot.com . Survival curves are plotted for all patients ( n =3951) ( e ) for ERα-positive cancers ( n =1802) ( f ) and for ERα-negative cases ( n =671) ( g ). Statistical analysis is performed using GraphPad Prism version 7.0 (GraphPad Software Inc., San Diego, CA, USA). * P

    Article Snippet: All the statistical analyses were performed by GraphPad Prism version 7.0.

    Techniques: Expressing, Two Tailed Test, Software

    Elevated Mcl-1 expression protects Fbw7-deficient T-ALL cell lines from ABT-737-induced apoptosis a , Cell viability assays showing that Fbw7-deficient T-ALL cell lines were more sensitive to sorafenib, but resistant to ABT-737 treatment. T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib or ABT-737 for 48 hours before performing the cell viability assays. Data was shown as mean ± SD for three independent experiments. b , Immunoblot analysis of the indicated human T-ALL cell lines with or without ABT-737 (0.8 µM) treatment. c , Specific depletion of endogenous Mcl-1 expression restored ABT-737 sensitivity in the indicated Fbw7-deficient T-ALL cell lines. Various T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of ABT-737 for 48 hours before performing the cell viability assays, or with or without ABT-737 (0.8 µM) treatment for 24 hours before collecting whole cell lysates for immunoblot analysis with the indicated antibodies. For cell viability assays, data was shown as mean ± SD for three independent experiments. d , 7-Amino-Actinomycin D (7-AAD)/Annexin V double-staining FACS analysis to detect the percentage of ABT-737-induced apoptosis in the indicated Fbw7-deficient T-ALL cell lines where the endogenous Mcl-1 was depleted by lentiviral shRNA treatment (lentiviral shGFP was used as a negative control). Various T-ALL cells were cultured in 10% FBS-containing medium with or without ABT-737 (0.8 µM) treatment for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells. e , 7-AAD/Annexin V double-staining FACS analysis to demonstrate that sorafenib treatment restores ABT-737 sensitivity to Fbw7-deficient HPB-ALL cells. HPB-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib and/or ABT-737 for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells.

    Journal: Nature

    Article Title: SCFFbw7 Regulates Cellular Apoptosis By Targeting Mcl-1 for Ubiquitination and Destruction

    doi: 10.1038/nature09732

    Figure Lengend Snippet: Elevated Mcl-1 expression protects Fbw7-deficient T-ALL cell lines from ABT-737-induced apoptosis a , Cell viability assays showing that Fbw7-deficient T-ALL cell lines were more sensitive to sorafenib, but resistant to ABT-737 treatment. T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib or ABT-737 for 48 hours before performing the cell viability assays. Data was shown as mean ± SD for three independent experiments. b , Immunoblot analysis of the indicated human T-ALL cell lines with or without ABT-737 (0.8 µM) treatment. c , Specific depletion of endogenous Mcl-1 expression restored ABT-737 sensitivity in the indicated Fbw7-deficient T-ALL cell lines. Various T-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of ABT-737 for 48 hours before performing the cell viability assays, or with or without ABT-737 (0.8 µM) treatment for 24 hours before collecting whole cell lysates for immunoblot analysis with the indicated antibodies. For cell viability assays, data was shown as mean ± SD for three independent experiments. d , 7-Amino-Actinomycin D (7-AAD)/Annexin V double-staining FACS analysis to detect the percentage of ABT-737-induced apoptosis in the indicated Fbw7-deficient T-ALL cell lines where the endogenous Mcl-1 was depleted by lentiviral shRNA treatment (lentiviral shGFP was used as a negative control). Various T-ALL cells were cultured in 10% FBS-containing medium with or without ABT-737 (0.8 µM) treatment for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells. e , 7-AAD/Annexin V double-staining FACS analysis to demonstrate that sorafenib treatment restores ABT-737 sensitivity to Fbw7-deficient HPB-ALL cells. HPB-ALL cells were cultured in 10% FBS-containing medium with the indicated concentrations of sorafenib and/or ABT-737 for 48 hours before the FACS analysis. Numbers indicate the percentage of apoptotic cells.

    Article Snippet: For detection of apoptosis, cells treated with various drugs were stained with propidium iodide (Roche), or co-stained with Annexin V-PE and 7-amino-actinomycin D (Annexin V-PE Apoptosis Detection Kit I, BD Bioscience) according to the manufacturer’s instructions.

    Techniques: Expressing, Cell Culture, Double Staining, FACS, shRNA, Negative Control