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  • 99
    Thermo Fisher taqman microrna reverse transcription kit
    Identification of QKI regulated <t>miRNAs</t> in U343 glioblastoma cells. (A) Cell extracts prepared from U343 cells <t>transfected</t> with siCTL or siQKI were separated by SDS-PAGE and immunoblotted with anti-pan-QKI antibodies and β-tubulin as a loading
    Taqman Microrna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 51673 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sirna transfection
    Identification of QKI regulated <t>miRNAs</t> in U343 glioblastoma cells. (A) Cell extracts prepared from U343 cells <t>transfected</t> with siCTL or siQKI were separated by SDS-PAGE and immunoblotted with anti-pan-QKI antibodies and β-tubulin as a loading
    Sirna Transfection, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1458 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna transfection/product/Millipore
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    94
    Horizon Discovery sirna transfection
    Restoration of STAG2 protein expression in UM-UC-3 bladder cancer cells. STAG2- deficient UM-UC-3 cells were transduced with lentiviral particles encoding no transgene (empty vector) or a 3xFLAG-tagged STAG2 transgene (FLAG-STAG2). Stable selected cell pools were used for further analysis. ( A ) Expression and nuclear localization of the transgenic FLAG-STAG2 protein was assayed by immunofluorescence microscopy. ( B ) Non-transduced UM-UC-3 cells, UM-UC-3 cells transduced with an empty vector and UM-UC-3 cells transduced with a FLAG-STAG2 transgene were transfected with NTC or STAG1 <t>siRNA</t> duplexes. Protein lysates prepared 72 hr after siRNA <t>transfection</t> were analyzed by immunoblotting. ( C ) Protein extracts prepared from UM-UC-3 cells expressing no transgene (empty vector) or a FLAG-STAG2 transgene were subjected to anti-FLAG immunoprecipitation. The input, unbound and precipitated fractions were analyzed by immunoblotting. Co-precipitation of cohesin subunits was only detected in FLAG-STAG2 expressing cells indicating successful incorporation of the transgenic STAG2 protein into the cohesin complex. DOI: http://dx.doi.org/10.7554/eLife.26980.015
    Sirna Transfection, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 94/100, based on 1560 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology sirna transfection reagent
    RSV reduced mtROS generation by stimulating Sirt3-mediated mitochondrial enzyme activities and SOD2 deacetylation. Cells were transfected with Sirt3 <t>siRNA</t> as described in the Materials and Methods section. At 24-h <t>post-transfection,</t> cells were pretreated with different concentrations of RSV (0.1, 1 and 10 μ M) for 2 h and then treated with or without t-BHP of 80 μ M for an additional 4 h. ( a – c ) The enzyme activities of IDH2 ( a ), GSH-Px ( b ) and SOD2 ( c ) were determined using the corresponding assay kits, according to the manufacturer's instructions. ( d ) After the indicated treatments, the cells were harvested and lysed to detect protein levels of ac-SOD2 by western blot analysis. All results are presented as means±S.E.M. of at least three independent experiments. a P
    Sirna Transfection Reagent, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1761 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology sirna transfection
    Expression of HMGB1 and IL-17A, and caspase 3 activity in microglial cells. After <t>transfection</t> with IL-17A or HMGB1 <t>siRNA</t> for 24 h, microglial cells were started in the OGD condition for 2 h and reperfusion for 24 h. HMGB1 and/or IL-17A expression as measured by Western blot analysis in EOC 2 culture supernatants (A) , mouse primary microglia culture supernatants (B) , and EOC 2 cells (C) . Protein concentrations of HMGB1 in EOC 2 (D) and mouse primary microglia (E) culture supernatants were determined by ELISA. (F) The activity of caspase-3 was monitored by the fluorometric method. * P
    Sirna Transfection, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology sirna transfection medium
    Effect of scrambled <t>siRNA</t> <t>transfection</t> on cardiomyocyte viability (a) and contractility (b). Control cells were transfected with scrambled siRNA. n = 4 heart isolations per group.
    Sirna Transfection Medium, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen sirna transfection
    Effect of scrambled <t>siRNA</t> <t>transfection</t> on cardiomyocyte viability (a) and contractility (b). Control cells were transfected with scrambled siRNA. n = 4 heart isolations per group.
    Sirna Transfection, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2147 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Identification of QKI regulated miRNAs in U343 glioblastoma cells. (A) Cell extracts prepared from U343 cells transfected with siCTL or siQKI were separated by SDS-PAGE and immunoblotted with anti-pan-QKI antibodies and β-tubulin as a loading

    Journal: Molecular and Cellular Biology

    Article Title: The QKI-5 and QKI-6 RNA Binding Proteins Regulate the Expression of MicroRNA 7 in Glial Cells

    doi: 10.1128/MCB.01604-12

    Figure Lengend Snippet: Identification of QKI regulated miRNAs in U343 glioblastoma cells. (A) Cell extracts prepared from U343 cells transfected with siCTL or siQKI were separated by SDS-PAGE and immunoblotted with anti-pan-QKI antibodies and β-tubulin as a loading

    Article Snippet: Plasmids were transfected with Lipofectamine 2000 (Invitrogen), and siRNAs, miRNA mimics, and miRNA inhibitors were transfected with Lipofectamine RNAi MAX (Invitrogen) according to the manufacturer's instructions.

    Techniques: Transfection, SDS Page

    LM-exosomes contain oncogenic miRNAs and mediate their transfer to promote intercellular communication. ( a ) The Venn diagram illustrates the overlapping result of differentially expressed miRNAs in whole cells and exosomes, including HOC313-P and HOC313-LM cell comparisons. Two miRNAs, miR-342-3p and miR-1246 , were upregulated in both whole cells and exosomes. (b and c) MiR-342-3p and miR-1246 were transferred to HOC313-P cells treated with LM-exosomes ( b ) or across a Transwell membrane through exosomes ( c ). ( d ) Validation of miR-342-3p and miR-1246 expression after each miRNA transfection in HOC313-P cells was measured by qRT-PCR. MicroRNA expression levels were normalized to RNU6B expression. MicroRNA expression levels of miR-342-3p (left) and miR-1246 (right) relative to the negative controls are shown. ( e ) Cell growth after 72 hours of transfection with miR-342-3p, miR-1246 and miR-NC in HOC313-P cells was evaluated by WST-8 assay. ( f and g ) Cell motility was assessed by Transwell migration assay ( f ) and cell invasion was assessed by Transwell invasion assay ( g ) in HOC313-P cells transfected with miR-342-3p or miR-1246 . Experiments were performed in triplicate. (Bars, SD). Student’s t -test was used for statistical analysis; asterisks represent P

    Journal: Scientific Reports

    Article Title: Exosomal microRNA miR-1246 induces cell motility and invasion through the regulation of DENND2D in oral squamous cell carcinoma

    doi: 10.1038/srep38750

    Figure Lengend Snippet: LM-exosomes contain oncogenic miRNAs and mediate their transfer to promote intercellular communication. ( a ) The Venn diagram illustrates the overlapping result of differentially expressed miRNAs in whole cells and exosomes, including HOC313-P and HOC313-LM cell comparisons. Two miRNAs, miR-342-3p and miR-1246 , were upregulated in both whole cells and exosomes. (b and c) MiR-342-3p and miR-1246 were transferred to HOC313-P cells treated with LM-exosomes ( b ) or across a Transwell membrane through exosomes ( c ). ( d ) Validation of miR-342-3p and miR-1246 expression after each miRNA transfection in HOC313-P cells was measured by qRT-PCR. MicroRNA expression levels were normalized to RNU6B expression. MicroRNA expression levels of miR-342-3p (left) and miR-1246 (right) relative to the negative controls are shown. ( e ) Cell growth after 72 hours of transfection with miR-342-3p, miR-1246 and miR-NC in HOC313-P cells was evaluated by WST-8 assay. ( f and g ) Cell motility was assessed by Transwell migration assay ( f ) and cell invasion was assessed by Transwell invasion assay ( g ) in HOC313-P cells transfected with miR-342-3p or miR-1246 . Experiments were performed in triplicate. (Bars, SD). Student’s t -test was used for statistical analysis; asterisks represent P

    Article Snippet: Gain-of-function was simulated by using miRNA mimics (miR-342 -3p [MC12328], miR-1246 [MC13182] and negative control [4464058]) purchased from Thermo Fisher Scientific.

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Transwell Migration Assay, Transwell Invasion Assay

    Changes in mRNA levels in the miR-124 transfection time course of Wang and Wang (2006) can be modeled assuming a tri-exponential Ago-loading function. ( A ) Cartoon illustrating two models of miRNA transfection experiments and their parameters. Free, fitted parameters appear in black, fixed parameters from Figure 1 in gray. The bi-exponential model (in green) is the same as in Figure 1A . Also shown is a tri-exponential model of Ago loading (in red), which is identical to the bi-exponential model, except for the addition of an extra compartment ( V ) in which V 0 miRNAs are loaded at time t =0, and two additional rates: rate of miRNA decay in this compartment ( ) and rate of transfer to the Ago-accessible environment ( r ). ( B ) Log-likelihood profile of the clearance parameter given the mRNA profiling time-course data. The log-likelihood of the tri-exponential model (red line) is compared with that of the bi-exponential model (green line). ( C ) Cumulative distribution of the per-gene relative error between the model and the time-course data. The x -axis represents the per-gene relative error between the model prediction and the measurements. For any chosen cutoff on the relative error, the fraction of genes whose regulation following miRNA transfection could be predicted at the chosen error cutoff or less can be read on the y -axis. The dotted line marks a 20% error on the fold change typically observed in miRNA transfection experiments. ( D ) Boxplots of the model residual on log 2 fold changes for genes that fit the measured mRNA fold changes with less than a 20% error. Boxes span the interquartal range and whiskers extending up to 1.5 times the interquartal range. See also Supplementary Figure S2 .

    Journal: Molecular Systems Biology

    Article Title: Timescales and bottlenecks in miRNA-dependent gene regulation

    doi: 10.1038/msb.2013.68

    Figure Lengend Snippet: Changes in mRNA levels in the miR-124 transfection time course of Wang and Wang (2006) can be modeled assuming a tri-exponential Ago-loading function. ( A ) Cartoon illustrating two models of miRNA transfection experiments and their parameters. Free, fitted parameters appear in black, fixed parameters from Figure 1 in gray. The bi-exponential model (in green) is the same as in Figure 1A . Also shown is a tri-exponential model of Ago loading (in red), which is identical to the bi-exponential model, except for the addition of an extra compartment ( V ) in which V 0 miRNAs are loaded at time t =0, and two additional rates: rate of miRNA decay in this compartment ( ) and rate of transfer to the Ago-accessible environment ( r ). ( B ) Log-likelihood profile of the clearance parameter given the mRNA profiling time-course data. The log-likelihood of the tri-exponential model (red line) is compared with that of the bi-exponential model (green line). ( C ) Cumulative distribution of the per-gene relative error between the model and the time-course data. The x -axis represents the per-gene relative error between the model prediction and the measurements. For any chosen cutoff on the relative error, the fraction of genes whose regulation following miRNA transfection could be predicted at the chosen error cutoff or less can be read on the y -axis. The dotted line marks a 20% error on the fold change typically observed in miRNA transfection experiments. ( D ) Boxplots of the model residual on log 2 fold changes for genes that fit the measured mRNA fold changes with less than a 20% error. Boxes span the interquartal range and whiskers extending up to 1.5 times the interquartal range. See also Supplementary Figure S2 .

    Article Snippet: MiRNA transfections and luciferase assayKTN1 cells were split in 12-well plates for both transfection and induction experiments. hsa-miR-199a-3p mimic (c-300536-07-0005) and miRNA mimic negative control (CN-002000-01-05, Thermo Fisher Scientific) were transfected with a final concentration of 25 nM using lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer's protocol.

    Techniques: Transfection

    The kinetic model fits measured changes in mRNA and protein levels following miR-199a transfection ( A ) or induction ( B ) in HEK293 cells. The 3′ UTR of the KTN1 miR-199a target was cloned downstream of the stop codon of a luciferase reporter gene. Changes in mRNA expression following miR-199a transfection or induction were quantified by qPCR whereas changes in protein levels were determined by measuring luciferase activity. In the transfection experiment, changes in mRNA and protein levels were then fitted assuming the previously introduced three-exponential Ago-loading model. In the induction experiment, we assumed constant miRNA synthesis into an Ago-accessible environment. The best-fitted model appears as a continuous line and error bars represent 95% confidence intervals on the measured changes in mRNA and protein abundance. See also Supplementary Figure S3 .

    Journal: Molecular Systems Biology

    Article Title: Timescales and bottlenecks in miRNA-dependent gene regulation

    doi: 10.1038/msb.2013.68

    Figure Lengend Snippet: The kinetic model fits measured changes in mRNA and protein levels following miR-199a transfection ( A ) or induction ( B ) in HEK293 cells. The 3′ UTR of the KTN1 miR-199a target was cloned downstream of the stop codon of a luciferase reporter gene. Changes in mRNA expression following miR-199a transfection or induction were quantified by qPCR whereas changes in protein levels were determined by measuring luciferase activity. In the transfection experiment, changes in mRNA and protein levels were then fitted assuming the previously introduced three-exponential Ago-loading model. In the induction experiment, we assumed constant miRNA synthesis into an Ago-accessible environment. The best-fitted model appears as a continuous line and error bars represent 95% confidence intervals on the measured changes in mRNA and protein abundance. See also Supplementary Figure S3 .

    Article Snippet: MiRNA transfections and luciferase assayKTN1 cells were split in 12-well plates for both transfection and induction experiments. hsa-miR-199a-3p mimic (c-300536-07-0005) and miRNA mimic negative control (CN-002000-01-05, Thermo Fisher Scientific) were transfected with a final concentration of 25 nM using lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer's protocol.

    Techniques: Transfection, Clone Assay, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Activity Assay

    The kinetic model explains temporal changes in mRNA abundance, protein abundance and translation efficiency of most miRNA targets. Cumulative distributions of the relative error between the model prediction and the measurements in different transfection experiments. ( A ) Ribosome Protected Fragment (RPF) sequencing and mRNAseq experiments upon transfection of miR-155 and miR-1 by Guo et al (2010) . ( B ) SILAC and microarray experiments upon miRNA transfection of miR-124, miR-1 and miR-181a by Baek et al (2008) . ( C ) pSILAC and microarray experiments following the transfection of let-7b, miR-155, miR-16, miR-1 and miR-30a by Selbach et al (2008) . The x -axis of each panel represents the per-gene relative error between the model prediction and the measurements. For any chosen cutoff on the relative error, the fraction of genes whose regulation following miRNA transfection could be predicted at the chosen error cutoff or less can be read on the y -axis. The dotted, vertical bars mark a 20% error cutoff on the fold change. This error level is typically observed in miRNA transfection experiments. See also Supplementary Figure S4 .

    Journal: Molecular Systems Biology

    Article Title: Timescales and bottlenecks in miRNA-dependent gene regulation

    doi: 10.1038/msb.2013.68

    Figure Lengend Snippet: The kinetic model explains temporal changes in mRNA abundance, protein abundance and translation efficiency of most miRNA targets. Cumulative distributions of the relative error between the model prediction and the measurements in different transfection experiments. ( A ) Ribosome Protected Fragment (RPF) sequencing and mRNAseq experiments upon transfection of miR-155 and miR-1 by Guo et al (2010) . ( B ) SILAC and microarray experiments upon miRNA transfection of miR-124, miR-1 and miR-181a by Baek et al (2008) . ( C ) pSILAC and microarray experiments following the transfection of let-7b, miR-155, miR-16, miR-1 and miR-30a by Selbach et al (2008) . The x -axis of each panel represents the per-gene relative error between the model prediction and the measurements. For any chosen cutoff on the relative error, the fraction of genes whose regulation following miRNA transfection could be predicted at the chosen error cutoff or less can be read on the y -axis. The dotted, vertical bars mark a 20% error cutoff on the fold change. This error level is typically observed in miRNA transfection experiments. See also Supplementary Figure S4 .

    Article Snippet: MiRNA transfections and luciferase assayKTN1 cells were split in 12-well plates for both transfection and induction experiments. hsa-miR-199a-3p mimic (c-300536-07-0005) and miRNA mimic negative control (CN-002000-01-05, Thermo Fisher Scientific) were transfected with a final concentration of 25 nM using lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer's protocol.

    Techniques: Transfection, Sequencing, Microarray

    Restoration of STAG2 protein expression in UM-UC-3 bladder cancer cells. STAG2- deficient UM-UC-3 cells were transduced with lentiviral particles encoding no transgene (empty vector) or a 3xFLAG-tagged STAG2 transgene (FLAG-STAG2). Stable selected cell pools were used for further analysis. ( A ) Expression and nuclear localization of the transgenic FLAG-STAG2 protein was assayed by immunofluorescence microscopy. ( B ) Non-transduced UM-UC-3 cells, UM-UC-3 cells transduced with an empty vector and UM-UC-3 cells transduced with a FLAG-STAG2 transgene were transfected with NTC or STAG1 siRNA duplexes. Protein lysates prepared 72 hr after siRNA transfection were analyzed by immunoblotting. ( C ) Protein extracts prepared from UM-UC-3 cells expressing no transgene (empty vector) or a FLAG-STAG2 transgene were subjected to anti-FLAG immunoprecipitation. The input, unbound and precipitated fractions were analyzed by immunoblotting. Co-precipitation of cohesin subunits was only detected in FLAG-STAG2 expressing cells indicating successful incorporation of the transgenic STAG2 protein into the cohesin complex. DOI: http://dx.doi.org/10.7554/eLife.26980.015

    Journal: eLife

    Article Title: Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts

    doi: 10.7554/eLife.26980

    Figure Lengend Snippet: Restoration of STAG2 protein expression in UM-UC-3 bladder cancer cells. STAG2- deficient UM-UC-3 cells were transduced with lentiviral particles encoding no transgene (empty vector) or a 3xFLAG-tagged STAG2 transgene (FLAG-STAG2). Stable selected cell pools were used for further analysis. ( A ) Expression and nuclear localization of the transgenic FLAG-STAG2 protein was assayed by immunofluorescence microscopy. ( B ) Non-transduced UM-UC-3 cells, UM-UC-3 cells transduced with an empty vector and UM-UC-3 cells transduced with a FLAG-STAG2 transgene were transfected with NTC or STAG1 siRNA duplexes. Protein lysates prepared 72 hr after siRNA transfection were analyzed by immunoblotting. ( C ) Protein extracts prepared from UM-UC-3 cells expressing no transgene (empty vector) or a FLAG-STAG2 transgene were subjected to anti-FLAG immunoprecipitation. The input, unbound and precipitated fractions were analyzed by immunoblotting. Co-precipitation of cohesin subunits was only detected in FLAG-STAG2 expressing cells indicating successful incorporation of the transgenic STAG2 protein into the cohesin complex. DOI: http://dx.doi.org/10.7554/eLife.26980.015

    Article Snippet: siRNA transfection, cell viability, competition assay and apoptosis assay For knockdown experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes (Dharmacon, Lafayette, CO, US) and the Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, US).

    Techniques: Expressing, Transduction, Plasmid Preparation, Transgenic Assay, Immunofluorescence, Microscopy, Transfection, Immunoprecipitation

    Aberrant nuclear morphology in STAG2 -mutated but not parental HCT 116 cells upon depletion of STAG1. Parental and STAG2- 505c1 HCT 116 were transfected with NTC and STAG1 siRNA duplexes, fixed and stained with Hoechst 72 hr after transfection. Nuclei were scored for aberrant size ( > 20 μm) or morphology (multi-nucleated cells and cells with polylobed nuclei; n ≥ 612 nuclei, error bars denote standard deviation between two independent experiments). DOI: http://dx.doi.org/10.7554/eLife.26980.011

    Journal: eLife

    Article Title: Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts

    doi: 10.7554/eLife.26980

    Figure Lengend Snippet: Aberrant nuclear morphology in STAG2 -mutated but not parental HCT 116 cells upon depletion of STAG1. Parental and STAG2- 505c1 HCT 116 were transfected with NTC and STAG1 siRNA duplexes, fixed and stained with Hoechst 72 hr after transfection. Nuclei were scored for aberrant size ( > 20 μm) or morphology (multi-nucleated cells and cells with polylobed nuclei; n ≥ 612 nuclei, error bars denote standard deviation between two independent experiments). DOI: http://dx.doi.org/10.7554/eLife.26980.011

    Article Snippet: siRNA transfection, cell viability, competition assay and apoptosis assay For knockdown experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes (Dharmacon, Lafayette, CO, US) and the Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, US).

    Techniques: Transfection, Staining, Standard Deviation

    Double depletion experiment confirms STAG1-STAG2 synthetic lethality. ( A ) HCT 116 parental cells were co-transfected with NTC siRNA and siRNA duplexes targeting one of the following genes: NTC, PLK1, RAD21, CDCA5, SGOL1, STAG1 or STAG2. HCT 116 parental cells were also co-transfected with siRNA duplexes targeting STAG1 and STAG2. Cell viability was determined 8 days after transfection and is plotted normalized to cell viability of NTC siRNA-transfected cells (n = 3 biological repeats, error bars denote standard deviation). DOI: http://dx.doi.org/10.7554/eLife.26980.006

    Journal: eLife

    Article Title: Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts

    doi: 10.7554/eLife.26980

    Figure Lengend Snippet: Double depletion experiment confirms STAG1-STAG2 synthetic lethality. ( A ) HCT 116 parental cells were co-transfected with NTC siRNA and siRNA duplexes targeting one of the following genes: NTC, PLK1, RAD21, CDCA5, SGOL1, STAG1 or STAG2. HCT 116 parental cells were also co-transfected with siRNA duplexes targeting STAG1 and STAG2. Cell viability was determined 8 days after transfection and is plotted normalized to cell viability of NTC siRNA-transfected cells (n = 3 biological repeats, error bars denote standard deviation). DOI: http://dx.doi.org/10.7554/eLife.26980.006

    Article Snippet: siRNA transfection, cell viability, competition assay and apoptosis assay For knockdown experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes (Dharmacon, Lafayette, CO, US) and the Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, US).

    Techniques: Transfection, Standard Deviation

    The depletion of STAG1 prolongs mitosis in STAG2 mutated but not parental HCT 116 cells. Parental and STAG2- 505c1 HCT 116 were transfected with NTC and STAG1 siRNA duplexes. Cells were tracked from 0.5 to 72 hr (imaging interval 30 min) after transfection by bright-field live-cell imaging. The duration of mitosis (time elapsed from mitotic cell rounding to anaphase onset) and the fate of individual cells were determined (n ≥ 43 cells). The duration of mitosis of individual cells is plotted. Blue lines denote mean, red dots denote cells that died in mitosis. Significance levels were quantified using unpaired t test. DOI: http://dx.doi.org/10.7554/eLife.26980.010

    Journal: eLife

    Article Title: Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts

    doi: 10.7554/eLife.26980

    Figure Lengend Snippet: The depletion of STAG1 prolongs mitosis in STAG2 mutated but not parental HCT 116 cells. Parental and STAG2- 505c1 HCT 116 were transfected with NTC and STAG1 siRNA duplexes. Cells were tracked from 0.5 to 72 hr (imaging interval 30 min) after transfection by bright-field live-cell imaging. The duration of mitosis (time elapsed from mitotic cell rounding to anaphase onset) and the fate of individual cells were determined (n ≥ 43 cells). The duration of mitosis of individual cells is plotted. Blue lines denote mean, red dots denote cells that died in mitosis. Significance levels were quantified using unpaired t test. DOI: http://dx.doi.org/10.7554/eLife.26980.010

    Article Snippet: siRNA transfection, cell viability, competition assay and apoptosis assay For knockdown experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes (Dharmacon, Lafayette, CO, US) and the Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, US).

    Techniques: Transfection, Imaging, Live Cell Imaging

    Patient-derived STAG2 mutations cause STAG1 dependency in engineered isogenic HCT 116 cells. HCT 116 cell lines engineered to harbor the indicated deleterious patient-derived STAG2 mutations were transfected with NTC, STAG1 and SGOL1 siRNA duplexes. ( A ) Protein extracts were prepared 48 hr after transfection and analyzed by immunoblotting. ( B ) Cell viability was measured 7 days after siRNA transfection and plotted normalized to the viability of NTC-transfected cells (n = 4 independent experiments with 5 biological repeats each, error bars denote standard deviation). ( C ) Sister chromatid cohesion phenotypes were analyzed in Giemsa-stained mitotic chromosome spreads that were prepared 48 hr after siRNA transfection (n = 100 chromosome spreads, error bars denote standard deviation between two independently analyzed slides). DOI: http://dx.doi.org/10.7554/eLife.26980.013

    Journal: eLife

    Article Title: Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts

    doi: 10.7554/eLife.26980

    Figure Lengend Snippet: Patient-derived STAG2 mutations cause STAG1 dependency in engineered isogenic HCT 116 cells. HCT 116 cell lines engineered to harbor the indicated deleterious patient-derived STAG2 mutations were transfected with NTC, STAG1 and SGOL1 siRNA duplexes. ( A ) Protein extracts were prepared 48 hr after transfection and analyzed by immunoblotting. ( B ) Cell viability was measured 7 days after siRNA transfection and plotted normalized to the viability of NTC-transfected cells (n = 4 independent experiments with 5 biological repeats each, error bars denote standard deviation). ( C ) Sister chromatid cohesion phenotypes were analyzed in Giemsa-stained mitotic chromosome spreads that were prepared 48 hr after siRNA transfection (n = 100 chromosome spreads, error bars denote standard deviation between two independently analyzed slides). DOI: http://dx.doi.org/10.7554/eLife.26980.013

    Article Snippet: siRNA transfection, cell viability, competition assay and apoptosis assay For knockdown experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes (Dharmacon, Lafayette, CO, US) and the Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, US).

    Techniques: Derivative Assay, Transfection, Standard Deviation, Staining

    Depletion of STAG1 does not reduce viability in hTERT RPE-1 cells. Human telomerase-immortalized retinal pigment epithelial cells (hTERT RPE-1) were transfected with the indicated siRNA duplexes. Protein extracts were prepared 4 days after transfection and analyzed by immunoblotting (left panel). Cell viability was determined 5 days after transfection and is plotted normalized to the cell viability of NTC siRNA-transfected cells (n = 2 independent experiments with 3 biological replicates each, error bars denote standard deviation). DOI: http://dx.doi.org/10.7554/eLife.26980.008

    Journal: eLife

    Article Title: Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts

    doi: 10.7554/eLife.26980

    Figure Lengend Snippet: Depletion of STAG1 does not reduce viability in hTERT RPE-1 cells. Human telomerase-immortalized retinal pigment epithelial cells (hTERT RPE-1) were transfected with the indicated siRNA duplexes. Protein extracts were prepared 4 days after transfection and analyzed by immunoblotting (left panel). Cell viability was determined 5 days after transfection and is plotted normalized to the cell viability of NTC siRNA-transfected cells (n = 2 independent experiments with 3 biological replicates each, error bars denote standard deviation). DOI: http://dx.doi.org/10.7554/eLife.26980.008

    Article Snippet: siRNA transfection, cell viability, competition assay and apoptosis assay For knockdown experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes (Dharmacon, Lafayette, CO, US) and the Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, US).

    Techniques: Transfection, Standard Deviation

    Double depletion experiments indicate that the STAG1-STAG2 genetic interaction is independent of p53. Parental HCT 116 cells and STAG2- 505c1 cells were transfected with NTC (-) or TP53 (+) siRNA duplexes. Protein extracts were prepared 4 days after transfection and analyzed by immunoblotting (left panel). Parental HCT 116 cells and STAG2- 505c1 cells were co-transfected with the indicated siRNA duplexes (right panel). Cell viability was determined 8 days after transfection and is plotted normalized to the cell viability of NTC+NTC or NTC+TP53 siRNA-co-transfected cells (n = 3 biological repeats, error bars denote standard deviation). DOI: http://dx.doi.org/10.7554/eLife.26980.007

    Journal: eLife

    Article Title: Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts

    doi: 10.7554/eLife.26980

    Figure Lengend Snippet: Double depletion experiments indicate that the STAG1-STAG2 genetic interaction is independent of p53. Parental HCT 116 cells and STAG2- 505c1 cells were transfected with NTC (-) or TP53 (+) siRNA duplexes. Protein extracts were prepared 4 days after transfection and analyzed by immunoblotting (left panel). Parental HCT 116 cells and STAG2- 505c1 cells were co-transfected with the indicated siRNA duplexes (right panel). Cell viability was determined 8 days after transfection and is plotted normalized to the cell viability of NTC+NTC or NTC+TP53 siRNA-co-transfected cells (n = 3 biological repeats, error bars denote standard deviation). DOI: http://dx.doi.org/10.7554/eLife.26980.007

    Article Snippet: siRNA transfection, cell viability, competition assay and apoptosis assay For knockdown experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes (Dharmacon, Lafayette, CO, US) and the Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, US).

    Techniques: Transfection, Standard Deviation

    Rescue of the synthetic lethal interaction between STAG1 and STAG2 by expression of an siRNA-resistant FLAG-STAG1 transgene. HCT 116 parental cells, a STAG2 wild-type clone (502wt), and two STAG2- clones (505c1 and 502c4) were transduced with a lentivirus encoding no transgene (empty vector) or an siRNA-resistant and 3xFLAG-tagged STAG1 transgene (FLAG-STAG1). Stably selected cell pools were used for the analysis. ( A ) Immunofluorescence analysis of FLAG-STAG1 transgene expression and nuclear localization in HCT 116 STAG2- 505c1 cells. Scale bar, 20 μm. ( B ) Protein extracts prepared from HCT 116 STAG2- 505c1 cells expressing no transgene (empty vector) or a FLAG-STAG1 transgene were subjected to anti-FLAG immunoprecipitation. The input, unbound and precipitated fractions were analyzed by immunoblotting. Co-precipitation of cohesin subunits was only detected in FLAG-STAG1 expressing cells indicating specific incorporation of the transgenic protein into the cohesin complex. ( C ) Protein extracts prepared from the indicated cell lines that were transduced with an empty vector or a FLAG-STAG1 transgene were analyzed by immunoblotting (left panel) and transfected with NTC, STAG1 and SGOL1 siRNA duplexes (right panel). Cell viability was measured 7 days after transfection and is plotted normalized to the viability of NTC siRNA transfected cells (n ≥ 5 biological repeats, error bars denote standard deviation). DOI: http://dx.doi.org/10.7554/eLife.26980.005

    Journal: eLife

    Article Title: Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts

    doi: 10.7554/eLife.26980

    Figure Lengend Snippet: Rescue of the synthetic lethal interaction between STAG1 and STAG2 by expression of an siRNA-resistant FLAG-STAG1 transgene. HCT 116 parental cells, a STAG2 wild-type clone (502wt), and two STAG2- clones (505c1 and 502c4) were transduced with a lentivirus encoding no transgene (empty vector) or an siRNA-resistant and 3xFLAG-tagged STAG1 transgene (FLAG-STAG1). Stably selected cell pools were used for the analysis. ( A ) Immunofluorescence analysis of FLAG-STAG1 transgene expression and nuclear localization in HCT 116 STAG2- 505c1 cells. Scale bar, 20 μm. ( B ) Protein extracts prepared from HCT 116 STAG2- 505c1 cells expressing no transgene (empty vector) or a FLAG-STAG1 transgene were subjected to anti-FLAG immunoprecipitation. The input, unbound and precipitated fractions were analyzed by immunoblotting. Co-precipitation of cohesin subunits was only detected in FLAG-STAG1 expressing cells indicating specific incorporation of the transgenic protein into the cohesin complex. ( C ) Protein extracts prepared from the indicated cell lines that were transduced with an empty vector or a FLAG-STAG1 transgene were analyzed by immunoblotting (left panel) and transfected with NTC, STAG1 and SGOL1 siRNA duplexes (right panel). Cell viability was measured 7 days after transfection and is plotted normalized to the viability of NTC siRNA transfected cells (n ≥ 5 biological repeats, error bars denote standard deviation). DOI: http://dx.doi.org/10.7554/eLife.26980.005

    Article Snippet: siRNA transfection, cell viability, competition assay and apoptosis assay For knockdown experiments, cells were transfected with ON-TARGETplus SMARTpool siRNA duplexes (Dharmacon, Lafayette, CO, US) and the Lipofectamine RNAiMAX reagent according to the manufacturer’s instructions (Invitrogen, Waltham, MA, US).

    Techniques: Expressing, Clone Assay, Transduction, Plasmid Preparation, Stable Transfection, Immunofluorescence, Immunoprecipitation, Transgenic Assay, Transfection, Standard Deviation

    RSV reduced mtROS generation by stimulating Sirt3-mediated mitochondrial enzyme activities and SOD2 deacetylation. Cells were transfected with Sirt3 siRNA as described in the Materials and Methods section. At 24-h post-transfection, cells were pretreated with different concentrations of RSV (0.1, 1 and 10 μ M) for 2 h and then treated with or without t-BHP of 80 μ M for an additional 4 h. ( a – c ) The enzyme activities of IDH2 ( a ), GSH-Px ( b ) and SOD2 ( c ) were determined using the corresponding assay kits, according to the manufacturer's instructions. ( d ) After the indicated treatments, the cells were harvested and lysed to detect protein levels of ac-SOD2 by western blot analysis. All results are presented as means±S.E.M. of at least three independent experiments. a P

    Journal: Cell Death & Disease

    Article Title: Resveratrol regulates mitochondrial reactive oxygen species homeostasis through Sirt3 signaling pathway in human vascular endothelial cells

    doi: 10.1038/cddis.2014.530

    Figure Lengend Snippet: RSV reduced mtROS generation by stimulating Sirt3-mediated mitochondrial enzyme activities and SOD2 deacetylation. Cells were transfected with Sirt3 siRNA as described in the Materials and Methods section. At 24-h post-transfection, cells were pretreated with different concentrations of RSV (0.1, 1 and 10 μ M) for 2 h and then treated with or without t-BHP of 80 μ M for an additional 4 h. ( a – c ) The enzyme activities of IDH2 ( a ), GSH-Px ( b ) and SOD2 ( c ) were determined using the corresponding assay kits, according to the manufacturer's instructions. ( d ) After the indicated treatments, the cells were harvested and lysed to detect protein levels of ac-SOD2 by western blot analysis. All results are presented as means±S.E.M. of at least three independent experiments. a P

    Article Snippet: RNA interference The siRNAs for AMPK (human, sc-45312), PGC-1α (human, sc-38884), estrogen-related receptor α (ERRα ; human, sc-44706) and Sirt3 (human, sc-61555) along with control siRNA (human, sc-44230) and siRNA transfection reagent (human, sc-29528) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Transfection, Western Blot

    RSV reduced mtROS generation through the promotion of Sirt3-mediated mtDNA transcription. Cells were pretreated by RSV (10 μ M) for 2 h, then washed, and followed by treatment with or without t-BHP (80 μ M) for an additional 4 h. ( a ) Representative images show Sirt3 expression within mitochondria in HUVECs by immuofluorescence assay. Red fluorescence indicates mitochondria labeled with Mito-tracker Red. Green fluorescence originates from anti-Sirt3 labeling. And blue fluorescence indicates nuclei stained with DAPI. ( b ) The bar graph shows quantification of Sirt3 expression in the mitochondria. ( c ) The expressions of mtRNAPol, FoxO3A and Sirt3 in the mitochondria were determined by western blot. ( d ) The bar graph shows quantification of the indicated proteins. Sirt3 was knocked down by Sirt3 siRNA transfection as described in the Materials and Methods section. The cells were pretreated with RSV of 10 μ M for 2 h, then washed, and followed by incubation with fresh medium in the presence or absence of t-BHP of 80 μ M for an additional 4 h. ( e ) Cells were harvested for ChIP analysis and the recruitment of FoxO3A was measured by real-time PCR assay using primers targeted to Sirt3 . The results were plotted relative to those of the total input controls (untreated chromatin). ( f ) The expressions of mtDNA-encoded genes of ATP6, CO1, Cytb, ND2 and ND5 along with the housekeeping genes of β -actin, GAPDH and 18s rRNA were determined by real-time PCR assay. In order to generate a relative expression ratio, the expressions of the genes of interest were normalized to the geometric average of the housekeeping genes. ( g ) The enzyme activity of complex I was determined using the corresponding assay kit, according to the manufacturer's instructions. ( h ) ATP content was determined using an ATP determination kit. All results are presented as means±S.E.M. of at least three independent experiments. a P

    Journal: Cell Death & Disease

    Article Title: Resveratrol regulates mitochondrial reactive oxygen species homeostasis through Sirt3 signaling pathway in human vascular endothelial cells

    doi: 10.1038/cddis.2014.530

    Figure Lengend Snippet: RSV reduced mtROS generation through the promotion of Sirt3-mediated mtDNA transcription. Cells were pretreated by RSV (10 μ M) for 2 h, then washed, and followed by treatment with or without t-BHP (80 μ M) for an additional 4 h. ( a ) Representative images show Sirt3 expression within mitochondria in HUVECs by immuofluorescence assay. Red fluorescence indicates mitochondria labeled with Mito-tracker Red. Green fluorescence originates from anti-Sirt3 labeling. And blue fluorescence indicates nuclei stained with DAPI. ( b ) The bar graph shows quantification of Sirt3 expression in the mitochondria. ( c ) The expressions of mtRNAPol, FoxO3A and Sirt3 in the mitochondria were determined by western blot. ( d ) The bar graph shows quantification of the indicated proteins. Sirt3 was knocked down by Sirt3 siRNA transfection as described in the Materials and Methods section. The cells were pretreated with RSV of 10 μ M for 2 h, then washed, and followed by incubation with fresh medium in the presence or absence of t-BHP of 80 μ M for an additional 4 h. ( e ) Cells were harvested for ChIP analysis and the recruitment of FoxO3A was measured by real-time PCR assay using primers targeted to Sirt3 . The results were plotted relative to those of the total input controls (untreated chromatin). ( f ) The expressions of mtDNA-encoded genes of ATP6, CO1, Cytb, ND2 and ND5 along with the housekeeping genes of β -actin, GAPDH and 18s rRNA were determined by real-time PCR assay. In order to generate a relative expression ratio, the expressions of the genes of interest were normalized to the geometric average of the housekeeping genes. ( g ) The enzyme activity of complex I was determined using the corresponding assay kit, according to the manufacturer's instructions. ( h ) ATP content was determined using an ATP determination kit. All results are presented as means±S.E.M. of at least three independent experiments. a P

    Article Snippet: RNA interference The siRNAs for AMPK (human, sc-45312), PGC-1α (human, sc-38884), estrogen-related receptor α (ERRα ; human, sc-44706) and Sirt3 (human, sc-61555) along with control siRNA (human, sc-44230) and siRNA transfection reagent (human, sc-29528) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Expressing, Fluorescence, Labeling, Staining, Western Blot, Transfection, Incubation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activity Assay

    RSV suppressed t-BHP-induced collapse of mitochondrial membrane potential in HUVECs. Sirt3 was knocked down by Sirt3 siRNA transfection as described in the Materials and Methods section. At 24-h post-transfection, cells were pretreated with RSV of 10 μ M for 2 h, washed, and then treated with or without t-BHP of 80 μ M for an additional 4 h. ( a ) Determination of Δ ψ m was carried out using CLSM. Red fluorescence was emitted by JC-1 aggregates in healthy mitochondria with polarized inner mitochondrial membranes, whereas green fluorescence was emitted by cytosolic JC-1 monomers, indicating Δ ψ m dissipation. Merged images indicate co-localization of JC-1 aggregates and monomers. ( b ) Δ ψ m in each group was calculated as the ratio of red to green fluorescence. All results are presented as mean±S.E.M. of at least three independent experiments. b P

    Journal: Cell Death & Disease

    Article Title: Resveratrol regulates mitochondrial reactive oxygen species homeostasis through Sirt3 signaling pathway in human vascular endothelial cells

    doi: 10.1038/cddis.2014.530

    Figure Lengend Snippet: RSV suppressed t-BHP-induced collapse of mitochondrial membrane potential in HUVECs. Sirt3 was knocked down by Sirt3 siRNA transfection as described in the Materials and Methods section. At 24-h post-transfection, cells were pretreated with RSV of 10 μ M for 2 h, washed, and then treated with or without t-BHP of 80 μ M for an additional 4 h. ( a ) Determination of Δ ψ m was carried out using CLSM. Red fluorescence was emitted by JC-1 aggregates in healthy mitochondria with polarized inner mitochondrial membranes, whereas green fluorescence was emitted by cytosolic JC-1 monomers, indicating Δ ψ m dissipation. Merged images indicate co-localization of JC-1 aggregates and monomers. ( b ) Δ ψ m in each group was calculated as the ratio of red to green fluorescence. All results are presented as mean±S.E.M. of at least three independent experiments. b P

    Article Snippet: RNA interference The siRNAs for AMPK (human, sc-45312), PGC-1α (human, sc-38884), estrogen-related receptor α (ERRα ; human, sc-44706) and Sirt3 (human, sc-61555) along with control siRNA (human, sc-44230) and siRNA transfection reagent (human, sc-29528) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Transfection, Confocal Laser Scanning Microscopy, Fluorescence

    RSV attenuated t-BHP-induced mitochondrial dysfunction by inhibiting mtROS generation in HUVECs. Cells were transfected with Sirt3 siRNA as described in the Materials and Methods section. At 24-h post-transfection, cells were pretreated with RSV (0.1, 1 and 10 μ M) for 2 h, washed, and then incubated with fresh medium in the presence or absence of t-BHP (80 μ M) for an additional 4 h. ( a ) The mitochondrial O 2 •− levels were estimated using MitoSOX Red and the fluorescence values were read at an excitation wavelength of 510 nm and emission wavelength of 579 nm. ( b ) The bar charts show quantification of the mitochondrial O 2 •− levels expressed as the fold change relative to the control group. All results are presented as means±S.E.M. of at least three independent experiments. b P

    Journal: Cell Death & Disease

    Article Title: Resveratrol regulates mitochondrial reactive oxygen species homeostasis through Sirt3 signaling pathway in human vascular endothelial cells

    doi: 10.1038/cddis.2014.530

    Figure Lengend Snippet: RSV attenuated t-BHP-induced mitochondrial dysfunction by inhibiting mtROS generation in HUVECs. Cells were transfected with Sirt3 siRNA as described in the Materials and Methods section. At 24-h post-transfection, cells were pretreated with RSV (0.1, 1 and 10 μ M) for 2 h, washed, and then incubated with fresh medium in the presence or absence of t-BHP (80 μ M) for an additional 4 h. ( a ) The mitochondrial O 2 •− levels were estimated using MitoSOX Red and the fluorescence values were read at an excitation wavelength of 510 nm and emission wavelength of 579 nm. ( b ) The bar charts show quantification of the mitochondrial O 2 •− levels expressed as the fold change relative to the control group. All results are presented as means±S.E.M. of at least three independent experiments. b P

    Article Snippet: RNA interference The siRNAs for AMPK (human, sc-45312), PGC-1α (human, sc-38884), estrogen-related receptor α (ERRα ; human, sc-44706) and Sirt3 (human, sc-61555) along with control siRNA (human, sc-44230) and siRNA transfection reagent (human, sc-29528) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Transfection, Incubation, Fluorescence

    RSV stimulated AMPK-PGC-1 α -ERR α -Sirt3 signaling pathway in HUVECs. ( a ) Cells were pretreated with RSV (0.1, 1 and 10 μ M) for 2 h and then treated with or without t-BHP of 80 μ M for an additional 4 h. The cells were harvested and lysed to detect protein levels of AMPK, p-AMPK, PGC-1 α and Sirt3 by western blot assay. ( b ) The bar charts show quantification of the indicated proteins. Cells were transfected with PGC-1α siRNA as described in the Materials and Methods section. ( c ) The mRNA levels of the Sirt3 genes were determined by real-time PCR assay. ( d ) The cells were collected and lysed, and Sirt3 protein levels were detected by western blot analysis. Cells were preincubated with compound C (10 μ M), AMPK siRNA or AICAR (500 μ M) as described in the Materials and Methods section. Then cells were pretreated with RSV of 10 μ M for 2 h, followed by incubation in the presence or absence of t-BHP of 80 μ M for an additional 4 h. ( e ) The expressions of AMPK, p-AMPK and PGC-1 α were measured by western blot. ( f ) The bar charts show quantification of the indicated proteins. Cells were transfencted with PGC-1α siRNA as described in the Materials and Methods section. At 24-h post-transfection, the cells were pretreated with RSV of 10 μ M for 2 h and then treated with or without t-BHP of 80 μ M for an additional 4 h. ( g ) Cells were harvested using ChIP assay, and real-time PCR assay was carried out using primers specific to the Sirt3 promoter. The results were determined relative to those of the total input controls (untreated chromatin). ( h ) Cells were co-transfected with pRL-TK reporter plasmid supplemented with ERRE-luc or NEG-PG04 (vehicle control), respectively. ERR α was knocked down by ERRα siRNA transfection, as described in the Materials and Methods section. And then cells were pretreated with RSV of 10 μ M for 2 h, followed by treatment with or without t-BHP of 80 μ M for an additional 4 h. Both firefly and Renilla luciferase activities were measured sequentially using the Dual-Glo luciferase reporter assay system. All results are presented as means±S.E.M. of at least three independent experiments. a P

    Journal: Cell Death & Disease

    Article Title: Resveratrol regulates mitochondrial reactive oxygen species homeostasis through Sirt3 signaling pathway in human vascular endothelial cells

    doi: 10.1038/cddis.2014.530

    Figure Lengend Snippet: RSV stimulated AMPK-PGC-1 α -ERR α -Sirt3 signaling pathway in HUVECs. ( a ) Cells were pretreated with RSV (0.1, 1 and 10 μ M) for 2 h and then treated with or without t-BHP of 80 μ M for an additional 4 h. The cells were harvested and lysed to detect protein levels of AMPK, p-AMPK, PGC-1 α and Sirt3 by western blot assay. ( b ) The bar charts show quantification of the indicated proteins. Cells were transfected with PGC-1α siRNA as described in the Materials and Methods section. ( c ) The mRNA levels of the Sirt3 genes were determined by real-time PCR assay. ( d ) The cells were collected and lysed, and Sirt3 protein levels were detected by western blot analysis. Cells were preincubated with compound C (10 μ M), AMPK siRNA or AICAR (500 μ M) as described in the Materials and Methods section. Then cells were pretreated with RSV of 10 μ M for 2 h, followed by incubation in the presence or absence of t-BHP of 80 μ M for an additional 4 h. ( e ) The expressions of AMPK, p-AMPK and PGC-1 α were measured by western blot. ( f ) The bar charts show quantification of the indicated proteins. Cells were transfencted with PGC-1α siRNA as described in the Materials and Methods section. At 24-h post-transfection, the cells were pretreated with RSV of 10 μ M for 2 h and then treated with or without t-BHP of 80 μ M for an additional 4 h. ( g ) Cells were harvested using ChIP assay, and real-time PCR assay was carried out using primers specific to the Sirt3 promoter. The results were determined relative to those of the total input controls (untreated chromatin). ( h ) Cells were co-transfected with pRL-TK reporter plasmid supplemented with ERRE-luc or NEG-PG04 (vehicle control), respectively. ERR α was knocked down by ERRα siRNA transfection, as described in the Materials and Methods section. And then cells were pretreated with RSV of 10 μ M for 2 h, followed by treatment with or without t-BHP of 80 μ M for an additional 4 h. Both firefly and Renilla luciferase activities were measured sequentially using the Dual-Glo luciferase reporter assay system. All results are presented as means±S.E.M. of at least three independent experiments. a P

    Article Snippet: RNA interference The siRNAs for AMPK (human, sc-45312), PGC-1α (human, sc-38884), estrogen-related receptor α (ERRα ; human, sc-44706) and Sirt3 (human, sc-61555) along with control siRNA (human, sc-44230) and siRNA transfection reagent (human, sc-29528) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Pyrolysis Gas Chromatography, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Incubation, Chromatin Immunoprecipitation, Plasmid Preparation, Luciferase, Reporter Assay

    Expression of HMGB1 and IL-17A, and caspase 3 activity in microglial cells. After transfection with IL-17A or HMGB1 siRNA for 24 h, microglial cells were started in the OGD condition for 2 h and reperfusion for 24 h. HMGB1 and/or IL-17A expression as measured by Western blot analysis in EOC 2 culture supernatants (A) , mouse primary microglia culture supernatants (B) , and EOC 2 cells (C) . Protein concentrations of HMGB1 in EOC 2 (D) and mouse primary microglia (E) culture supernatants were determined by ELISA. (F) The activity of caspase-3 was monitored by the fluorometric method. * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: IL-17A Enhances Microglial Response to OGD by Regulating p53 and PI3K/Akt Pathways with Involvement of ROS/HMGB1

    doi: 10.3389/fnmol.2017.00271

    Figure Lengend Snippet: Expression of HMGB1 and IL-17A, and caspase 3 activity in microglial cells. After transfection with IL-17A or HMGB1 siRNA for 24 h, microglial cells were started in the OGD condition for 2 h and reperfusion for 24 h. HMGB1 and/or IL-17A expression as measured by Western blot analysis in EOC 2 culture supernatants (A) , mouse primary microglia culture supernatants (B) , and EOC 2 cells (C) . Protein concentrations of HMGB1 in EOC 2 (D) and mouse primary microglia (E) culture supernatants were determined by ELISA. (F) The activity of caspase-3 was monitored by the fluorometric method. * P

    Article Snippet: After siRNA transfection, Tenovin-6 (10 μM; SantaCruz, USA) and MK-2206 (5 μM; Selleck, China) were added or not.

    Techniques: Expressing, Activity Assay, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    IL-17A enhances microglia apoptosis in OGD model through regulation of p53 and PI3K/Akt pathways. After transfection with IL-17A siRNA for 24 h, EOC 2 cells (A) and mouse primary microglia (B) were started in the OGD condition for 2 h and reperfusion for 24 h. Tenovin-6 (10 μM) or MK-2206 (5 μM) were added or not after microglial cells reperfusion for 2 h. Then, apoptosis was examined by a TUNEL-DAPI co-staining assay. Green fluorescence indicates the DNA fragmentation, and blue fluorescence indicates the cell nucleus.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: IL-17A Enhances Microglial Response to OGD by Regulating p53 and PI3K/Akt Pathways with Involvement of ROS/HMGB1

    doi: 10.3389/fnmol.2017.00271

    Figure Lengend Snippet: IL-17A enhances microglia apoptosis in OGD model through regulation of p53 and PI3K/Akt pathways. After transfection with IL-17A siRNA for 24 h, EOC 2 cells (A) and mouse primary microglia (B) were started in the OGD condition for 2 h and reperfusion for 24 h. Tenovin-6 (10 μM) or MK-2206 (5 μM) were added or not after microglial cells reperfusion for 2 h. Then, apoptosis was examined by a TUNEL-DAPI co-staining assay. Green fluorescence indicates the DNA fragmentation, and blue fluorescence indicates the cell nucleus.

    Article Snippet: After siRNA transfection, Tenovin-6 (10 μM; SantaCruz, USA) and MK-2206 (5 μM; Selleck, China) were added or not.

    Techniques: Transfection, TUNEL Assay, Staining, Fluorescence

    Expression of p-p53 and p-Akt, caspase 3 activity in microglial cells. After transfection with IL-17A siRNA for 24 h, EOC 2 cells were started in the OGD condition for 2 h and reperfusion for 24 h. P-p53 (A) and p-Akt (B) expression as measured by Western blot analysis. (C) Data are presented as the means ± SD from three independent experiments, * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: IL-17A Enhances Microglial Response to OGD by Regulating p53 and PI3K/Akt Pathways with Involvement of ROS/HMGB1

    doi: 10.3389/fnmol.2017.00271

    Figure Lengend Snippet: Expression of p-p53 and p-Akt, caspase 3 activity in microglial cells. After transfection with IL-17A siRNA for 24 h, EOC 2 cells were started in the OGD condition for 2 h and reperfusion for 24 h. P-p53 (A) and p-Akt (B) expression as measured by Western blot analysis. (C) Data are presented as the means ± SD from three independent experiments, * P

    Article Snippet: After siRNA transfection, Tenovin-6 (10 μM; SantaCruz, USA) and MK-2206 (5 μM; Selleck, China) were added or not.

    Techniques: Expressing, Activity Assay, Transfection, Western Blot

    Expression of IL-17A in microglial cells. After transfection with IL-17A siRNA for 24 h, microglial cells were placed in an oxygen deprived incubator at 37°C for 2 h, and then moved to normal conditions to terminate the OGD and start reperfusion for 24 h. IL-17A expression as measured by Western blot analysis in EOC 2 cells (A) , EOC 2 culture supernatants (B) , and mouse primary microglia culture supernatants (C) . Protein concentrations of IL-17A in EOC 2 (D) and mouse primary microglia (E) culture supernatants were determined by ELISA. Experiments were carried out at least in triplicate and the results were expressed as the mean values, * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: IL-17A Enhances Microglial Response to OGD by Regulating p53 and PI3K/Akt Pathways with Involvement of ROS/HMGB1

    doi: 10.3389/fnmol.2017.00271

    Figure Lengend Snippet: Expression of IL-17A in microglial cells. After transfection with IL-17A siRNA for 24 h, microglial cells were placed in an oxygen deprived incubator at 37°C for 2 h, and then moved to normal conditions to terminate the OGD and start reperfusion for 24 h. IL-17A expression as measured by Western blot analysis in EOC 2 cells (A) , EOC 2 culture supernatants (B) , and mouse primary microglia culture supernatants (C) . Protein concentrations of IL-17A in EOC 2 (D) and mouse primary microglia (E) culture supernatants were determined by ELISA. Experiments were carried out at least in triplicate and the results were expressed as the mean values, * P

    Article Snippet: After siRNA transfection, Tenovin-6 (10 μM; SantaCruz, USA) and MK-2206 (5 μM; Selleck, China) were added or not.

    Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay

    Effect of scrambled siRNA transfection on cardiomyocyte viability (a) and contractility (b). Control cells were transfected with scrambled siRNA. n = 4 heart isolations per group.

    Journal: BioMed Research International

    Article Title: Inhibition of MMP-2 Expression with siRNA Increases Baseline Cardiomyocyte Contractility and Protects against Simulated Ischemic Reperfusion Injury

    doi: 10.1155/2014/810371

    Figure Lengend Snippet: Effect of scrambled siRNA transfection on cardiomyocyte viability (a) and contractility (b). Control cells were transfected with scrambled siRNA. n = 4 heart isolations per group.

    Article Snippet: Following the initial 7-hour stabilization period in DMEM and 10% FBS, cardiomyocytes were washed with siRNA transfection medium (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated for 24 hours at 37°C in 200 μ L of transfection medium containing 0.8 μ M of MMP-2 siRNA or scrambled siRNA ( ) according to the manufacturer's protocol.

    Techniques: Transfection

    Effect of MMP-2 siRNA transfection on the level of the contractile proteins MLC1 and MLC2 (a), the formation of the complex between MMP-2 and MLC1 or MLC2 (b), and cardiomyocyte contractility (c). As a protein loading control the tubulin level was measured. Control cells were transfected with scrambled siRNA. n = 4–6 heart preparation isolations per group. n = 11 per group for contractility measurement and n = 4–6 for measurement of protein levels. * P

    Journal: BioMed Research International

    Article Title: Inhibition of MMP-2 Expression with siRNA Increases Baseline Cardiomyocyte Contractility and Protects against Simulated Ischemic Reperfusion Injury

    doi: 10.1155/2014/810371

    Figure Lengend Snippet: Effect of MMP-2 siRNA transfection on the level of the contractile proteins MLC1 and MLC2 (a), the formation of the complex between MMP-2 and MLC1 or MLC2 (b), and cardiomyocyte contractility (c). As a protein loading control the tubulin level was measured. Control cells were transfected with scrambled siRNA. n = 4–6 heart preparation isolations per group. n = 11 per group for contractility measurement and n = 4–6 for measurement of protein levels. * P

    Article Snippet: Following the initial 7-hour stabilization period in DMEM and 10% FBS, cardiomyocytes were washed with siRNA transfection medium (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated for 24 hours at 37°C in 200 μ L of transfection medium containing 0.8 μ M of MMP-2 siRNA or scrambled siRNA ( ) according to the manufacturer's protocol.

    Techniques: Transfection

    Effect of MMP-2 siRNA transfection on MMP-2 expression in isolated cardiomyocytes. (a) Efficiency of siRNA transfection and MMP-2 protein levels measured by immunocytochemistry. Scale bar, 50 μ m. (b) Measurement of MMP-2 gelatinolytic activity by zymography. (c) MMP-2 protein level. (a) n = 25 cells from 3 different hearts per group; n = 9-10 heart isolates per group for MMP-2 activity (b), in (c) n = 4 heart isolates per group, * P

    Journal: BioMed Research International

    Article Title: Inhibition of MMP-2 Expression with siRNA Increases Baseline Cardiomyocyte Contractility and Protects against Simulated Ischemic Reperfusion Injury

    doi: 10.1155/2014/810371

    Figure Lengend Snippet: Effect of MMP-2 siRNA transfection on MMP-2 expression in isolated cardiomyocytes. (a) Efficiency of siRNA transfection and MMP-2 protein levels measured by immunocytochemistry. Scale bar, 50 μ m. (b) Measurement of MMP-2 gelatinolytic activity by zymography. (c) MMP-2 protein level. (a) n = 25 cells from 3 different hearts per group; n = 9-10 heart isolates per group for MMP-2 activity (b), in (c) n = 4 heart isolates per group, * P

    Article Snippet: Following the initial 7-hour stabilization period in DMEM and 10% FBS, cardiomyocytes were washed with siRNA transfection medium (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated for 24 hours at 37°C in 200 μ L of transfection medium containing 0.8 μ M of MMP-2 siRNA or scrambled siRNA ( ) according to the manufacturer's protocol.

    Techniques: Transfection, Expressing, Isolation, Immunocytochemistry, Activity Assay, Zymography

    Schematic representation of the perfusion protocol for isolated cardiomyocytes. Scrambled siRNA was used as a control of MMP-2 siRNA. Arrows indicate when cell contractility was measured: (1) before siRNA transfection, (2) before ischemia, and (3) at the end of reperfusion.

    Journal: BioMed Research International

    Article Title: Inhibition of MMP-2 Expression with siRNA Increases Baseline Cardiomyocyte Contractility and Protects against Simulated Ischemic Reperfusion Injury

    doi: 10.1155/2014/810371

    Figure Lengend Snippet: Schematic representation of the perfusion protocol for isolated cardiomyocytes. Scrambled siRNA was used as a control of MMP-2 siRNA. Arrows indicate when cell contractility was measured: (1) before siRNA transfection, (2) before ischemia, and (3) at the end of reperfusion.

    Article Snippet: Following the initial 7-hour stabilization period in DMEM and 10% FBS, cardiomyocytes were washed with siRNA transfection medium (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated for 24 hours at 37°C in 200 μ L of transfection medium containing 0.8 μ M of MMP-2 siRNA or scrambled siRNA ( ) according to the manufacturer's protocol.

    Techniques: Isolation, Transfection

    Effect of MMP-2 siRNA transfection on cardiomyocyte contractility (a) and MLC1 and MLC2 in cardiomyocytes subjected to I/R. As a protein loading control the tubulin level was measured. Control cells were transfected with scrambled siRNA. n = 11 per group for contractility measurement and n = 4 for measurement of protein levels. * P

    Journal: BioMed Research International

    Article Title: Inhibition of MMP-2 Expression with siRNA Increases Baseline Cardiomyocyte Contractility and Protects against Simulated Ischemic Reperfusion Injury

    doi: 10.1155/2014/810371

    Figure Lengend Snippet: Effect of MMP-2 siRNA transfection on cardiomyocyte contractility (a) and MLC1 and MLC2 in cardiomyocytes subjected to I/R. As a protein loading control the tubulin level was measured. Control cells were transfected with scrambled siRNA. n = 11 per group for contractility measurement and n = 4 for measurement of protein levels. * P

    Article Snippet: Following the initial 7-hour stabilization period in DMEM and 10% FBS, cardiomyocytes were washed with siRNA transfection medium (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated for 24 hours at 37°C in 200 μ L of transfection medium containing 0.8 μ M of MMP-2 siRNA or scrambled siRNA ( ) according to the manufacturer's protocol.

    Techniques: Transfection