taqman gene expression master mix  (Thermo Fisher)


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    Name:
    TaqMan Gene Expression Master Mix
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    Alternative Product Try TaqMan Fast Advanced Master Mix our highest performance probe based master mix With TaqMan Fast Advanced Master Mix we ve taken the best of TaqMan Gene Expression Master Mix and added additional capabilities for your gene expression analysis Get precise and reliable real time qPCR quantification over 9 logs of linear dynamic range with Applied Biosystems TaqMan Gene Expression Master Mix TaqMan Gene Expression Master Mix is an optimized 2X mix that contains all of the components excluding the template and primers for sensitive detection down to one copy of target Extended benchtop stability provides consistent results for automated liquid handling systems making it the choice for high throughput sample processing overnight or over the weekend TaqMan Gene Expression Master Mix has been validated with TaqMan Gene Expression Assays and Applied Biosystems real time systems providing precise quantification for a variety of gene expression based real time qPCR applications including • Rare transcript detection• Gene expression analysis• Pathogen detection and viral load quantification• Co amplification of two targets• Specific detection of gene family membersTaqMan Gene Expression Master Mix contains AmpliTaq Gold DNA Polymerase UP Ultra Pure for hot start activation and improved detection of bacterial targets a blend of dNTPs with dTTP dUTP and Uracil DNA Glycosylase UDG to minimize carry over PCR contamination and a passive internal reference based on proprietary ROX dye for superb precision on Applied Biosystems real time PCR instruments
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    4369016
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    Enzymes & Master Mixes for Real-Time PCR|Industrial & Applied Science|PCR & Real-Time PCR|PCR-Based Drug Discovery Assays|Pharma & Biopharma|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Target & Lead Identification & Validation|Gene Expression Analysis & Genotyping
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    Structured Review

    Thermo Fisher taqman gene expression master mix
    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by <t>TaqMan</t> <t>PCR</t> in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.
    Alternative Product Try TaqMan Fast Advanced Master Mix our highest performance probe based master mix With TaqMan Fast Advanced Master Mix we ve taken the best of TaqMan Gene Expression Master Mix and added additional capabilities for your gene expression analysis Get precise and reliable real time qPCR quantification over 9 logs of linear dynamic range with Applied Biosystems TaqMan Gene Expression Master Mix TaqMan Gene Expression Master Mix is an optimized 2X mix that contains all of the components excluding the template and primers for sensitive detection down to one copy of target Extended benchtop stability provides consistent results for automated liquid handling systems making it the choice for high throughput sample processing overnight or over the weekend TaqMan Gene Expression Master Mix has been validated with TaqMan Gene Expression Assays and Applied Biosystems real time systems providing precise quantification for a variety of gene expression based real time qPCR applications including • Rare transcript detection• Gene expression analysis• Pathogen detection and viral load quantification• Co amplification of two targets• Specific detection of gene family membersTaqMan Gene Expression Master Mix contains AmpliTaq Gold DNA Polymerase UP Ultra Pure for hot start activation and improved detection of bacterial targets a blend of dNTPs with dTTP dUTP and Uracil DNA Glycosylase UDG to minimize carry over PCR contamination and a passive internal reference based on proprietary ROX dye for superb precision on Applied Biosystems real time PCR instruments
    https://www.bioz.com/result/taqman gene expression master mix/product/Thermo Fisher
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    Images

    1) Product Images from "FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex"

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    Journal: eLife

    doi: 10.7554/eLife.35012

    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.
    Figure Legend Snippet: FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Polymerase Chain Reaction

    FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value
    Figure Legend Snippet: FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value

    Techniques Used: Activity Assay, Transfection, Western Blot, Staining, Polymerase Chain Reaction, Knock-Out, Immunostaining, Flow Cytometry, Cytometry, Fluorescence, Software, Incubation, MANN-WHITNEY

    2) Product Images from "FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex"

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    Journal: eLife

    doi: 10.7554/eLife.35012

    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (c trl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.
    Figure Legend Snippet: FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (c trl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Polymerase Chain Reaction

    FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value
    Figure Legend Snippet: FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value

    Techniques Used: Activity Assay, Transfection, Western Blot, Staining, Polymerase Chain Reaction, Knock-Out, Immunostaining, Flow Cytometry, Fluorescence, Software, Incubation, MANN-WHITNEY

    3) Product Images from "Intact piRNA pathway prevents L1 mobilization in male meiosis"

    Article Title: Intact piRNA pathway prevents L1 mobilization in male meiosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1701069114

    Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a TaqMan probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. Gapdh ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the SN1 transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.
    Figure Legend Snippet: Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a TaqMan probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. Gapdh ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the SN1 transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.

    Techniques Used: Derivative Assay, Methylation, RNA Expression, In Situ, Sequencing, Transgenic Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction

    4) Product Images from "Intact piRNA pathway prevents L1 mobilization in male meiosis"

    Article Title: Intact piRNA pathway prevents L1 mobilization in male meiosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1701069114

    Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a TaqMan probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. Gapdh ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the SN1 transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.
    Figure Legend Snippet: Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a TaqMan probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. Gapdh ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the SN1 transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.

    Techniques Used: Derivative Assay, Methylation, RNA Expression, In Situ, Sequencing, Transgenic Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction

    5) Product Images from "MicroRNA-mediated target mRNA cleavage and 3′-uridylation in human cells"

    Article Title: MicroRNA-mediated target mRNA cleavage and 3′-uridylation in human cells

    Journal: Scientific Reports

    doi: 10.1038/srep30242

    Effects of Target mRNA Structural and Positional Elements on Activities of miRNA-mediated Cleavage and Uridylation. ( a ) The plasmid construct of pLJ-T214 containing the EGFP reporter gene with two copies of predicted let-7a target sequences in its 3′UTR. ( b ) Cleaved mRNA 5′-fragments from let-7 targets T1 and T2 were detected by SLA–RT-PCR (upper gel panels) and SLA-qRT-PCR (lower panels). The predicted sizes of SLA–RT-PCR products derived from target sites T1 and T2 were 240-209 bp and 532-500 bp, respectively. Total RNAs were prepared from H1299 at 16, 40 and 72 h after pLJ-T214 transfection and RT reactions were performed using SLA-RT or 2U-SLA-RT primers. The corresponding target T2 cleavage and uridylation activities detected by SLA-qRT-PCR with T2 fragment specific PCR primers and TaqMan probe. ( c ) The plasmid construct of pLJ-T722. The 3′UTR of the EGFP reporter gene containing two copies of identical predicted let-7:target pairing sequences but with varied lengths and compositions of nt (underlined) at their 5′- and 3′-adjacent regions ( ST1 and ST2 ). ( d ) The effect of structural composition of target mRNA on let-7 miRNA–mediated mRNA cleavage activity as detected by SLA–RT-PCR assay (upper gel panels) and SLA-qRT-PCR (lower panels). Total RNAs were prepared from H1299 cells transfected with pLJ-T722 at 24 h, and SLA–RT-PCR was performed using SLA-RT primers. D50, a PCR primer specific to the pLJ-T722 transcript, was designed to bond at every 50 bases along the target sequences toward its 5′ end. The base numbers correspond to those shown in ( b ), and escaped bases are indicated by “•” between them. M: 0.03 μg of 1 kb DNA ladder. A Taqman probe-based qPCR assay was used to detect cleavage activity specific to target ST2 and a SYBR Green-based qPCR to detect both target ST1 and ST2 cleavage activities, respectively. All qPCR results were presented as relative fragment abundance (RFA). Each RFA value was represented as the mean of three independent experiments and error bars as standard errors to the mean. The target cleavage and uridylation activities in miRNA target regions were highlighted in red boxes.
    Figure Legend Snippet: Effects of Target mRNA Structural and Positional Elements on Activities of miRNA-mediated Cleavage and Uridylation. ( a ) The plasmid construct of pLJ-T214 containing the EGFP reporter gene with two copies of predicted let-7a target sequences in its 3′UTR. ( b ) Cleaved mRNA 5′-fragments from let-7 targets T1 and T2 were detected by SLA–RT-PCR (upper gel panels) and SLA-qRT-PCR (lower panels). The predicted sizes of SLA–RT-PCR products derived from target sites T1 and T2 were 240-209 bp and 532-500 bp, respectively. Total RNAs were prepared from H1299 at 16, 40 and 72 h after pLJ-T214 transfection and RT reactions were performed using SLA-RT or 2U-SLA-RT primers. The corresponding target T2 cleavage and uridylation activities detected by SLA-qRT-PCR with T2 fragment specific PCR primers and TaqMan probe. ( c ) The plasmid construct of pLJ-T722. The 3′UTR of the EGFP reporter gene containing two copies of identical predicted let-7:target pairing sequences but with varied lengths and compositions of nt (underlined) at their 5′- and 3′-adjacent regions ( ST1 and ST2 ). ( d ) The effect of structural composition of target mRNA on let-7 miRNA–mediated mRNA cleavage activity as detected by SLA–RT-PCR assay (upper gel panels) and SLA-qRT-PCR (lower panels). Total RNAs were prepared from H1299 cells transfected with pLJ-T722 at 24 h, and SLA–RT-PCR was performed using SLA-RT primers. D50, a PCR primer specific to the pLJ-T722 transcript, was designed to bond at every 50 bases along the target sequences toward its 5′ end. The base numbers correspond to those shown in ( b ), and escaped bases are indicated by “•” between them. M: 0.03 μg of 1 kb DNA ladder. A Taqman probe-based qPCR assay was used to detect cleavage activity specific to target ST2 and a SYBR Green-based qPCR to detect both target ST1 and ST2 cleavage activities, respectively. All qPCR results were presented as relative fragment abundance (RFA). Each RFA value was represented as the mean of three independent experiments and error bars as standard errors to the mean. The target cleavage and uridylation activities in miRNA target regions were highlighted in red boxes.

    Techniques Used: Plasmid Preparation, Construct, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Transfection, Polymerase Chain Reaction, Activity Assay, Real-time Polymerase Chain Reaction, SYBR Green Assay

    6) Product Images from "Central inhibition of granulocyte-macrophage colony-stimulating factor is analgesic in experimental neuropathic pain"

    Article Title: Central inhibition of granulocyte-macrophage colony-stimulating factor is analgesic in experimental neuropathic pain

    Journal: Pain

    doi: 10.1097/j.pain.0000000000001130

    Verification of GM-CSFR mRNA expression. Taqman profiles confirm expression of both GM-CSFR α-chain (A) and common β-chain (B) subunits in astrocytes and microglia. GM-CSFR, granulocyte-macrophage colony-stimulating factor receptor.
    Figure Legend Snippet: Verification of GM-CSFR mRNA expression. Taqman profiles confirm expression of both GM-CSFR α-chain (A) and common β-chain (B) subunits in astrocytes and microglia. GM-CSFR, granulocyte-macrophage colony-stimulating factor receptor.

    Techniques Used: Expressing

    7) Product Images from "1(OH) Vitamin D3 Supplementation Improves the Sensitivity of the Immune-Response during Peg-IFN/RBV Therapy in Chronic Hepatitis C Patients-Case Controlled Trial"

    Article Title: 1(OH) Vitamin D3 Supplementation Improves the Sensitivity of the Immune-Response during Peg-IFN/RBV Therapy in Chronic Hepatitis C Patients-Case Controlled Trial

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063672

    The effect of vitamin D3 on the expression of ISGs mRNA in the liver. The relative amount of target mRNA was obtained by using a comparative threshold cycle (CT) method. The expression levels of Mx, IFI44 or IFIT1 mRNA in an IL28B T/T patient treated without 1(OH) vitamin D3 are represented as 1.0 and the relative amounts of target mRNA in the other patients were calculated by the comparative Ct method [42] . Therefore, the standard amount of 3 ISGs (Mx, IFI44 and IFIT1) is 3. The relative amounts of the 3 kinds of ISGs were added and shown in the graph (A). Black circles indicate the data from IL28B (T/T) subjects treated without 1(OH) vitamin D3. White boxes indicate the data from IL28B (T/T) subjects treated with 1(OH) vitamin D3. Black triangles indicate the data from IL28B (T/G or G/G) subjects treated without 1(OH) vitamin D3. Black lines indicate the data from the subjects treated with 1(OH) vitamin D3 (A). The effect of vitamin D3 on the expression of ISGs mRNA in the hepatocyte cell culture are shown (B). Huh-7 cells were treated with ethanol (control), 1(OH) vitamin D3 (1.0 µM) or 1,25(OH) 2 vitamin D3 (1.0 µM) after transfection of poly IC (Sigma-Aldrich, St. Louis, MO) or in vitro transcribed JFH-1 full-length RNA. Cells were harvested 30 h after transfection, and the expression levels of Mx, IFI44 and IFIT1 mRNA were assessed by real-time PCR using TaqMan Gene Expression Master Mix (Applied Biosystems, Carlsbad, CA) and gene-specific primer and probe sets (TaqMan Gene Expression Assay; Applied Biosystems) in accordance with the manufacturer’s instructions. The expression levels of genes with or without vitamin D3 treatment were expressed by log fold increase of untreated Huh-7 cells.
    Figure Legend Snippet: The effect of vitamin D3 on the expression of ISGs mRNA in the liver. The relative amount of target mRNA was obtained by using a comparative threshold cycle (CT) method. The expression levels of Mx, IFI44 or IFIT1 mRNA in an IL28B T/T patient treated without 1(OH) vitamin D3 are represented as 1.0 and the relative amounts of target mRNA in the other patients were calculated by the comparative Ct method [42] . Therefore, the standard amount of 3 ISGs (Mx, IFI44 and IFIT1) is 3. The relative amounts of the 3 kinds of ISGs were added and shown in the graph (A). Black circles indicate the data from IL28B (T/T) subjects treated without 1(OH) vitamin D3. White boxes indicate the data from IL28B (T/T) subjects treated with 1(OH) vitamin D3. Black triangles indicate the data from IL28B (T/G or G/G) subjects treated without 1(OH) vitamin D3. Black lines indicate the data from the subjects treated with 1(OH) vitamin D3 (A). The effect of vitamin D3 on the expression of ISGs mRNA in the hepatocyte cell culture are shown (B). Huh-7 cells were treated with ethanol (control), 1(OH) vitamin D3 (1.0 µM) or 1,25(OH) 2 vitamin D3 (1.0 µM) after transfection of poly IC (Sigma-Aldrich, St. Louis, MO) or in vitro transcribed JFH-1 full-length RNA. Cells were harvested 30 h after transfection, and the expression levels of Mx, IFI44 and IFIT1 mRNA were assessed by real-time PCR using TaqMan Gene Expression Master Mix (Applied Biosystems, Carlsbad, CA) and gene-specific primer and probe sets (TaqMan Gene Expression Assay; Applied Biosystems) in accordance with the manufacturer’s instructions. The expression levels of genes with or without vitamin D3 treatment were expressed by log fold increase of untreated Huh-7 cells.

    Techniques Used: Expressing, Cell Culture, Transfection, In Vitro, Real-time Polymerase Chain Reaction

    8) Product Images from "Many LINE1 elements contribute to the transcriptome of human somatic cells"

    Article Title: Many LINE1 elements contribute to the transcriptome of human somatic cells

    Journal: Genome Biology

    doi: 10.1186/gb-2009-10-9-r100

    Characterization of L1 at 4p15.32. (a) Diagram of L1 at 4p15.32 and the surrounding region. The arrow designates the L1 transcript. Blue boxes indicate exons of the CD38 gene, with exon number designated. Oligonucleotides CD38-a and CD38-b are indicated. Unmarked triangles indicate the positions of oligonucleotides used in L1 TaqMan qPCR assay. (b) Alignments of L1 at 4p15.32 3' end and related sequences. 'chr 4 short tag' - the major 3' expression tag cloned from this site. 'chr4 long tag' - longer 3' expression tag and 3' RACE sequence cloned from this site. 'chr6 transduction' - paralogous, transduced sequence downstream of L1 on chromosome 6. 'U35' - similar distinct 3' expression tag that cannot be mapped to the human reference genome. 3' end target site duplications are highlighted in blue. Single nucleotide differences in the chromosome 6 sequence are highlighted in dark red. (c) Diagram of the pedigree of the CEPH/UTAH individuals used in this study. (d) Relative expression of the L1 at 4p15.32 in lymphoblastoid cell lines from CEPH individuals. Expression is in arbitrary units normalized to HPRT1. Error bars indicate ± standard deviation from three replicates. (e) Expression of CEPH individuals of the L1 at 4p15.32 compared to flanking exons of CD38 , normalized to HPRT1. Expression is plotted on a logarithmic scale so that levels for both amplicons can be clearly visualized. Error bars represent ± standard deviations from three replicates. All data are representative of at least two biological replicates.
    Figure Legend Snippet: Characterization of L1 at 4p15.32. (a) Diagram of L1 at 4p15.32 and the surrounding region. The arrow designates the L1 transcript. Blue boxes indicate exons of the CD38 gene, with exon number designated. Oligonucleotides CD38-a and CD38-b are indicated. Unmarked triangles indicate the positions of oligonucleotides used in L1 TaqMan qPCR assay. (b) Alignments of L1 at 4p15.32 3' end and related sequences. 'chr 4 short tag' - the major 3' expression tag cloned from this site. 'chr4 long tag' - longer 3' expression tag and 3' RACE sequence cloned from this site. 'chr6 transduction' - paralogous, transduced sequence downstream of L1 on chromosome 6. 'U35' - similar distinct 3' expression tag that cannot be mapped to the human reference genome. 3' end target site duplications are highlighted in blue. Single nucleotide differences in the chromosome 6 sequence are highlighted in dark red. (c) Diagram of the pedigree of the CEPH/UTAH individuals used in this study. (d) Relative expression of the L1 at 4p15.32 in lymphoblastoid cell lines from CEPH individuals. Expression is in arbitrary units normalized to HPRT1. Error bars indicate ± standard deviation from three replicates. (e) Expression of CEPH individuals of the L1 at 4p15.32 compared to flanking exons of CD38 , normalized to HPRT1. Expression is plotted on a logarithmic scale so that levels for both amplicons can be clearly visualized. Error bars represent ± standard deviations from three replicates. All data are representative of at least two biological replicates.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Clone Assay, Sequencing, Transduction, Standard Deviation

    9) Product Images from "The Macrophage A2b Adenosine Receptor Regulates Tissue Insulin Sensitivity"

    Article Title: The Macrophage A2b Adenosine Receptor Regulates Tissue Insulin Sensitivity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098775

    Generation of transgenic mice expressing A2bAR in macrophages only. A . Genomic analysis by PCR of CD68-hA2bAR transgene in founder lines 1, 2, and 3 (Fo # 1, 2, 3) compared to A2bAR KO mice. Line 2 was used for the remainder of the studies based on expression analysis shown in panels c,d. B . Determination of primer efficiency. The amplification efficiency of human A2bAR (hA2bAR) and mouse A2bAR (mA2bAR) TaqMan primers was tested using the CT slope method. The target template was diluted over a log scale and CT values were determined by qPCR. A plot of CT versus log cDNA concentration is shown for hA2bAR and mA2bAR primers. Amplification efficiency (Ex) is calculated using the slope of the graph in the following equation: Ex = 10 (-1/slope) – 1. The calculated efficiencies are 1.18 and 1.03 for mA2bAR and hA2bAR, respectively. C . Human A2bAR and mouse A2bAR mRNA expression was measured by qPCR in Kupffer cells isolated from mice at 12 weeks of age (n = 5 WT, 6 A2bAR KO, 6 CD68-Tg; ns = not statistically different from WT). D . Visceral (epididymal) adipose tissue macrophages were sorted via flow cytometry-based markers (see Methods) and subjected to qPCR of A2bAR mRNA. Data are averages ± SD. Relative mRNA expression was determined using the ΔΔCT method and were normalized to 18s rRNA values. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value
    Figure Legend Snippet: Generation of transgenic mice expressing A2bAR in macrophages only. A . Genomic analysis by PCR of CD68-hA2bAR transgene in founder lines 1, 2, and 3 (Fo # 1, 2, 3) compared to A2bAR KO mice. Line 2 was used for the remainder of the studies based on expression analysis shown in panels c,d. B . Determination of primer efficiency. The amplification efficiency of human A2bAR (hA2bAR) and mouse A2bAR (mA2bAR) TaqMan primers was tested using the CT slope method. The target template was diluted over a log scale and CT values were determined by qPCR. A plot of CT versus log cDNA concentration is shown for hA2bAR and mA2bAR primers. Amplification efficiency (Ex) is calculated using the slope of the graph in the following equation: Ex = 10 (-1/slope) – 1. The calculated efficiencies are 1.18 and 1.03 for mA2bAR and hA2bAR, respectively. C . Human A2bAR and mouse A2bAR mRNA expression was measured by qPCR in Kupffer cells isolated from mice at 12 weeks of age (n = 5 WT, 6 A2bAR KO, 6 CD68-Tg; ns = not statistically different from WT). D . Visceral (epididymal) adipose tissue macrophages were sorted via flow cytometry-based markers (see Methods) and subjected to qPCR of A2bAR mRNA. Data are averages ± SD. Relative mRNA expression was determined using the ΔΔCT method and were normalized to 18s rRNA values. Data are averages ± SD. *Student two-tail t-test assuming equal variance was found significant only when p-value

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Concentration Assay, Isolation, Flow Cytometry, Cytometry

    10) Product Images from "BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer and confers sensitivity to BET inhibitors"

    Article Title: BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer and confers sensitivity to BET inhibitors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200826

    BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using TaqMan ® Copy-number Assay by qPCR. (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.
    Figure Legend Snippet: BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using TaqMan ® Copy-number Assay by qPCR. (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.

    Techniques Used: Expressing, Amplification, Derivative Assay, TaqMan Copy Number Assay, Real-time Polymerase Chain Reaction, Soft Agar Assay, Live Cell Imaging, Cell Cycle Assay

    11) Product Images from "Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD"

    Article Title: Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.08.005

    Analysis of miR-483 in DMD Patient Serum (A) Alignment of mouse and human miR-483 precursors. Differences are highlighted in yellow. (B) miRNA signature plots for mouse and human miR-483. Pooled mouse muscle library data are shown in red, and publically available miRBase data are shown in blue. Annotated and empirically derived miRNA seed regions are indicated. Serum from DMD patients (n = 28) and healthy controls (n = 16) were analyzed by sRNA TaqMan qRT-PCR for (C) miR-483-5p and miR-483-3p, and (D) the myomiRs: miR-1a-3p, miR-133a-3p, and miR-206-3p. Individual data points are shown and the mean ± SEM indicated. **p
    Figure Legend Snippet: Analysis of miR-483 in DMD Patient Serum (A) Alignment of mouse and human miR-483 precursors. Differences are highlighted in yellow. (B) miRNA signature plots for mouse and human miR-483. Pooled mouse muscle library data are shown in red, and publically available miRBase data are shown in blue. Annotated and empirically derived miRNA seed regions are indicated. Serum from DMD patients (n = 28) and healthy controls (n = 16) were analyzed by sRNA TaqMan qRT-PCR for (C) miR-483-5p and miR-483-3p, and (D) the myomiRs: miR-1a-3p, miR-133a-3p, and miR-206-3p. Individual data points are shown and the mean ± SEM indicated. **p

    Techniques Used: Derivative Assay, Quantitative RT-PCR

    sRNA Analysis in Dystrophic Muscle Mapped sRNA reads from muscle libraries were sorted into the following ncRNA classes: miRNA, tRNA, rRNA, snRNA, snoRA, scaRNA, mtRNA, and piRNA. (A) Pie chart showing percentage of reads mapping to each ncRNA category averaged across all muscle samples (pie charts for each individual muscle are shown in Figure S5 ). (B) Size distribution of sRNA reads after adaptor trimming in each set of muscle libraries. Differential expression of miRNAs in mdx diaphragm relative to wild-type controls as visualized by (C) volcano plot and (D) MA plot (volcano and MA plots for gastrocnemius, soleus, and TA muscles are shown in Figure S8 ). Statistically significant changes are highlighted in red and blue (for elevated and reduced levels in mdx serum, respectively). Labels are shown for miRNAs of interest. (E) Venn diagram showing overlap between differentially expressed miRNAs in dystrophic muscles. miRNAs that were commonly differentially expressed in all four muscle types are indicated. (F) Expression of miR-483-3p was validated by sRNA TaqMan qRT-PCR in each muscle using miR-16-5p as a control for normalization. Values are mean + SEM; n = 3. *p
    Figure Legend Snippet: sRNA Analysis in Dystrophic Muscle Mapped sRNA reads from muscle libraries were sorted into the following ncRNA classes: miRNA, tRNA, rRNA, snRNA, snoRA, scaRNA, mtRNA, and piRNA. (A) Pie chart showing percentage of reads mapping to each ncRNA category averaged across all muscle samples (pie charts for each individual muscle are shown in Figure S5 ). (B) Size distribution of sRNA reads after adaptor trimming in each set of muscle libraries. Differential expression of miRNAs in mdx diaphragm relative to wild-type controls as visualized by (C) volcano plot and (D) MA plot (volcano and MA plots for gastrocnemius, soleus, and TA muscles are shown in Figure S8 ). Statistically significant changes are highlighted in red and blue (for elevated and reduced levels in mdx serum, respectively). Labels are shown for miRNAs of interest. (E) Venn diagram showing overlap between differentially expressed miRNAs in dystrophic muscles. miRNAs that were commonly differentially expressed in all four muscle types are indicated. (F) Expression of miR-483-3p was validated by sRNA TaqMan qRT-PCR in each muscle using miR-16-5p as a control for normalization. Values are mean + SEM; n = 3. *p

    Techniques Used: Expressing, Quantitative RT-PCR

    12) Product Images from "miR-199a-5p Is Upregulated during Fibrogenic Response to Tissue Injury and Mediates TGFbeta-Induced Lung Fibroblast Activation by Targeting Caveolin-1"

    Article Title: miR-199a-5p Is Upregulated during Fibrogenic Response to Tissue Injury and Mediates TGFbeta-Induced Lung Fibroblast Activation by Targeting Caveolin-1

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003291

    TGFβ regulates CAV1 by increasing miR-199a-5p expression. MRC-5 lung fibroblasts were treated with 10 ng/mL TGFβ for 24 h and 48 h. MiR-199a-5p (A) and CAV1 expression levels (B) were determined by Taqman PCR. Data are expressed as mean ± SEM. ** p
    Figure Legend Snippet: TGFβ regulates CAV1 by increasing miR-199a-5p expression. MRC-5 lung fibroblasts were treated with 10 ng/mL TGFβ for 24 h and 48 h. MiR-199a-5p (A) and CAV1 expression levels (B) were determined by Taqman PCR. Data are expressed as mean ± SEM. ** p

    Techniques Used: Expressing, Polymerase Chain Reaction

    13) Product Images from "Functional Differences between Mitochondrial Haplogroup T and Haplogroup H in HEK293 Cybrid Cells"

    Article Title: Functional Differences between Mitochondrial Haplogroup T and Haplogroup H in HEK293 Cybrid Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052367

    Results of TaqMan qPCR analysis of HEK H and HEK T cybrid competitive co-cultures. After 10, 20 and 30 days (d10, d20, d30) of co-culture, isolated DNA of the cell mixtures was analyzed using TaqMan qPCR. ΔC t values were calculated by subtraction of the mean C t value of the FAM signal (probe recognizing haplogroup T) from the mean C t value of the VIC signal (probe recognizing haplogroup H). ΔΔC t values were calculated by subtraction of the mean ΔC t values of the original cell mixtures (n = 36; day zero) from the mean ΔC t values of all co-cultures at days 10, 20 or 30 (n = 36; except for d30 in galactose: n = 35). Dominance of haplogroup H results in a negative ΔΔC t value and is presented as gray bars, whereas dominance of haplogroup T results in a positive ΔΔC t value and is presented as black bars. (A) ΔΔC t values of competitive co-cultures cultivated in glucose medium, at days 10, 20 and 30. (B) ΔΔC t values of competitive co-cultures cultivated in galactose medium, at days 10, 20 and 30. Mean ΔΔC t values are given; error bars: standard deviation.
    Figure Legend Snippet: Results of TaqMan qPCR analysis of HEK H and HEK T cybrid competitive co-cultures. After 10, 20 and 30 days (d10, d20, d30) of co-culture, isolated DNA of the cell mixtures was analyzed using TaqMan qPCR. ΔC t values were calculated by subtraction of the mean C t value of the FAM signal (probe recognizing haplogroup T) from the mean C t value of the VIC signal (probe recognizing haplogroup H). ΔΔC t values were calculated by subtraction of the mean ΔC t values of the original cell mixtures (n = 36; day zero) from the mean ΔC t values of all co-cultures at days 10, 20 or 30 (n = 36; except for d30 in galactose: n = 35). Dominance of haplogroup H results in a negative ΔΔC t value and is presented as gray bars, whereas dominance of haplogroup T results in a positive ΔΔC t value and is presented as black bars. (A) ΔΔC t values of competitive co-cultures cultivated in glucose medium, at days 10, 20 and 30. (B) ΔΔC t values of competitive co-cultures cultivated in galactose medium, at days 10, 20 and 30. Mean ΔΔC t values are given; error bars: standard deviation.

    Techniques Used: Real-time Polymerase Chain Reaction, Co-Culture Assay, Isolation, Standard Deviation

    14) Product Images from "Recombinant Modified Vaccinia Virus Ankara Generating Excess Early Double-Stranded RNA Transiently Activates Protein Kinase R and Triggers Enhanced Innate Immune Responses"

    Article Title: Recombinant Modified Vaccinia Virus Ankara Generating Excess Early Double-Stranded RNA Transiently Activates Protein Kinase R and Triggers Enhanced Innate Immune Responses

    Journal: Journal of Virology

    doi: 10.1128/JVI.02082-14

    MVA recombinants expressing excess early dsRNA from neo or EGFP transgenes induce increased IFN-β expression. (A) Schematic representation of the two types of MVA recombinants generating excess early dsRNA either from two neo inserts (top) or from two EGFP inserts (bottom), each with the corresponding control and reference constructs. IGR, intergenic region. (B) Total RNA from murine BALB/3T3-A31 cells infected with the indicated viruses (MOI 10) or mock infected for 6 h was digested with RNase A/T1 (ssRNase digest) or RNase A/T1/V1 (ss+dsRNase digest) or not digested, and duplicate RT-qPCR quantification of total EGFP transcript (both sense and antisense) was performed as described in Materials and Methods. The mean of the fold induction values of EGFP or C7L transcripts over mock in undigested samples was set to 100%, and the mean percentage of the remaining qPCR signals after the indicated RNase digests was calculated for EGFP and C7L transcripts. Shown is one out two independent experiments. Where error bars are not visible, the standard error was negligible. (C) MEFs in 6-well plates were mock infected or infected with crude stocks of the indicated MVA recombinants at an MOI of 10 in duplicate. Fold induction of IFN-β mRNA over mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. using a commercially available TaqMan assay (Life Technologies) for the murine IFN-β gene. Poly(I·C) was transfected using Fugene HD at 2 μg/well. 18S rRNA served as the endogenous control in all RT-qPCR analyses. Where error bars are not visible, the standard error was negligible. (D) IFN-β amounts in supernatants of MEF cultures infected in parallel to those shown in panel C were determined at 14 h p.i. by ELISA. (E) Murine A31 cells were either preincubated with 40 μg/ml of AraC for 1 h or left untreated and infected in duplicate at an MOI of 10 with the indicated MVAs with either 40 μg/ml AraC throughout infection or without AraC. Cells were harvested at 6 h p.i. for isolation of total RNA. Messenger RNAs for murine IFN-β and the late F17R VACV gene were quantified by qRT-PCR analysis as described above.
    Figure Legend Snippet: MVA recombinants expressing excess early dsRNA from neo or EGFP transgenes induce increased IFN-β expression. (A) Schematic representation of the two types of MVA recombinants generating excess early dsRNA either from two neo inserts (top) or from two EGFP inserts (bottom), each with the corresponding control and reference constructs. IGR, intergenic region. (B) Total RNA from murine BALB/3T3-A31 cells infected with the indicated viruses (MOI 10) or mock infected for 6 h was digested with RNase A/T1 (ssRNase digest) or RNase A/T1/V1 (ss+dsRNase digest) or not digested, and duplicate RT-qPCR quantification of total EGFP transcript (both sense and antisense) was performed as described in Materials and Methods. The mean of the fold induction values of EGFP or C7L transcripts over mock in undigested samples was set to 100%, and the mean percentage of the remaining qPCR signals after the indicated RNase digests was calculated for EGFP and C7L transcripts. Shown is one out two independent experiments. Where error bars are not visible, the standard error was negligible. (C) MEFs in 6-well plates were mock infected or infected with crude stocks of the indicated MVA recombinants at an MOI of 10 in duplicate. Fold induction of IFN-β mRNA over mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. using a commercially available TaqMan assay (Life Technologies) for the murine IFN-β gene. Poly(I·C) was transfected using Fugene HD at 2 μg/well. 18S rRNA served as the endogenous control in all RT-qPCR analyses. Where error bars are not visible, the standard error was negligible. (D) IFN-β amounts in supernatants of MEF cultures infected in parallel to those shown in panel C were determined at 14 h p.i. by ELISA. (E) Murine A31 cells were either preincubated with 40 μg/ml of AraC for 1 h or left untreated and infected in duplicate at an MOI of 10 with the indicated MVAs with either 40 μg/ml AraC throughout infection or without AraC. Cells were harvested at 6 h p.i. for isolation of total RNA. Messenger RNAs for murine IFN-β and the late F17R VACV gene were quantified by qRT-PCR analysis as described above.

    Techniques Used: Expressing, Construct, Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Isolation, TaqMan Assay, Transfection, Enzyme-linked Immunosorbent Assay

    A CVA mutant expressing excess early dsRNA induces increased expression of IFN-β. (A) Schematic representation of a CVA mutant expressing sense and antisense mRNA (indicated by green and red wiggly lines) from two neo inserts and of control and reference constructs. Promoters driving transcription of the neo inserts are indicated, as well as flanking ORFs at the B15R locus. (B) Mouse embryo fibroblasts (MEFs) in 6-well plates were mock infected or infected with crude stocks of the indicated CVA recombinants at an MOI of 10 in duplicate. The relative amount of neo-rpsL antisense transcripts ( rpsL mRNA) compared to that of the mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. and custom TaqMan primers for the rpsL portion of the neo-rpsL antisense transcript. An 18S rRNA-specific probe served as the endogenous control in RT-qPCR analysis. (C) MEFs in 6-well plates were mock infected or infected with crude stocks of the indicated MVA recombinants in duplicate at an MOI of 10. Fold induction of IFN-β mRNA over mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. Poly(I·C) was transfected using Fugene HD at 2 μg/well. Where error bars are not visible, the standard error was negligible. (D) IFN-β amounts in supernatants of MEF cultures infected in parallel to those shown in panel C were determined at 14 h p.i. by ELISA. Human HeLa and murine BALB/3T12-3 cells (E) and MEFs from C57BL/6 mice (MEF-wt-B6) and from IFNAR −/− C57BL/6 mice (“MEF-IFNAR 0/0 -B6”) in 6-well plates (F) were infected in triplicate at an MOI of 0.025 of the indicated viruses. Cells and supernatants were harvested at the indicated time points, and infectious viral titers in lysates were determined by standard titration assays on CEF cells using the TCID 50 method. Total viral output at the indicated times is plotted, and each data point represents results from single titrations of three independent wells.
    Figure Legend Snippet: A CVA mutant expressing excess early dsRNA induces increased expression of IFN-β. (A) Schematic representation of a CVA mutant expressing sense and antisense mRNA (indicated by green and red wiggly lines) from two neo inserts and of control and reference constructs. Promoters driving transcription of the neo inserts are indicated, as well as flanking ORFs at the B15R locus. (B) Mouse embryo fibroblasts (MEFs) in 6-well plates were mock infected or infected with crude stocks of the indicated CVA recombinants at an MOI of 10 in duplicate. The relative amount of neo-rpsL antisense transcripts ( rpsL mRNA) compared to that of the mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. and custom TaqMan primers for the rpsL portion of the neo-rpsL antisense transcript. An 18S rRNA-specific probe served as the endogenous control in RT-qPCR analysis. (C) MEFs in 6-well plates were mock infected or infected with crude stocks of the indicated MVA recombinants in duplicate at an MOI of 10. Fold induction of IFN-β mRNA over mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. Poly(I·C) was transfected using Fugene HD at 2 μg/well. Where error bars are not visible, the standard error was negligible. (D) IFN-β amounts in supernatants of MEF cultures infected in parallel to those shown in panel C were determined at 14 h p.i. by ELISA. Human HeLa and murine BALB/3T12-3 cells (E) and MEFs from C57BL/6 mice (MEF-wt-B6) and from IFNAR −/− C57BL/6 mice (“MEF-IFNAR 0/0 -B6”) in 6-well plates (F) were infected in triplicate at an MOI of 0.025 of the indicated viruses. Cells and supernatants were harvested at the indicated time points, and infectious viral titers in lysates were determined by standard titration assays on CEF cells using the TCID 50 method. Total viral output at the indicated times is plotted, and each data point represents results from single titrations of three independent wells.

    Techniques Used: Mutagenesis, Expressing, Construct, Infection, Quantitative RT-PCR, Isolation, Transfection, Enzyme-linked Immunosorbent Assay, Mouse Assay, Titration

    15) Product Images from "Individual Variation of the Genetic Response to Bisphenol A in Human Foreskin Fibroblast Cells Derived from Cryptorchidism and Hypospadias Patients"

    Article Title: Individual Variation of the Genetic Response to Bisphenol A in Human Foreskin Fibroblast Cells Derived from Cryptorchidism and Hypospadias Patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052756

    Association between rs5000770 genotype and ARNT2 expression. ( A ) Boxplot of the ARNT2 mRNA level in hFFCs from child HS patients with different genotypes (6 AA and 15 AG/GG) relative to GAPDH measured using TaqMan® real-time PCR. Statistical significance was evaluated by the Mann-Whitney U test. ( B ) Scheme of the quantitative PCR strategy for screening splicing variants. No splicing variant was detected using intron-spanning RT-PCR in hFFCs either from HS or CO patients.
    Figure Legend Snippet: Association between rs5000770 genotype and ARNT2 expression. ( A ) Boxplot of the ARNT2 mRNA level in hFFCs from child HS patients with different genotypes (6 AA and 15 AG/GG) relative to GAPDH measured using TaqMan® real-time PCR. Statistical significance was evaluated by the Mann-Whitney U test. ( B ) Scheme of the quantitative PCR strategy for screening splicing variants. No splicing variant was detected using intron-spanning RT-PCR in hFFCs either from HS or CO patients.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Variant Assay, Reverse Transcription Polymerase Chain Reaction

    Effect of SNP rs5000770 genotype on the genetic response to low-dose BPA. Cells were treated with 10 nM BPA for 24 h, and then MMP11 and NTSR1 mRNA levels were measured by TaqMan® real-time PCR. Boxplot of the quantitative data comparing MMP11 ( A ) and NTSR1 ( B ) expression in child HS patients with different genotypes (6 AA and 15 AG/GG). Significance was evaluated by the Mann-Whitney U test.
    Figure Legend Snippet: Effect of SNP rs5000770 genotype on the genetic response to low-dose BPA. Cells were treated with 10 nM BPA for 24 h, and then MMP11 and NTSR1 mRNA levels were measured by TaqMan® real-time PCR. Boxplot of the quantitative data comparing MMP11 ( A ) and NTSR1 ( B ) expression in child HS patients with different genotypes (6 AA and 15 AG/GG). Significance was evaluated by the Mann-Whitney U test.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY

    ARNT2 mRNA levels in control children and child HS and CO patients. ARNT2 expression were measured in hFFCs derived from the control group ( n = 5), HS ( n = 21) group and CO ( n = 8) group by TaqMan® real-time PCR. ( A ) Boxplot and ( B ) summary of the quantitative data.
    Figure Legend Snippet: ARNT2 mRNA levels in control children and child HS and CO patients. ARNT2 expression were measured in hFFCs derived from the control group ( n = 5), HS ( n = 21) group and CO ( n = 8) group by TaqMan® real-time PCR. ( A ) Boxplot and ( B ) summary of the quantitative data.

    Techniques Used: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction

    Difference in the genetic response to low-dose BPA in hFFCs derived from control children and child HS and CO patients. Cells were treated with 10 nM BPA for 24 h, and then the expression of MMP11 ( A ) and NTSR1 ( B ) was measured by TaqMan® real-time PCR. Significance was evaluated by the Mann-Whitney U test or Wilcoxon test. The bottom numbers indicate the fold changes and P value induced by BPA compared with DMSO control.
    Figure Legend Snippet: Difference in the genetic response to low-dose BPA in hFFCs derived from control children and child HS and CO patients. Cells were treated with 10 nM BPA for 24 h, and then the expression of MMP11 ( A ) and NTSR1 ( B ) was measured by TaqMan® real-time PCR. Significance was evaluated by the Mann-Whitney U test or Wilcoxon test. The bottom numbers indicate the fold changes and P value induced by BPA compared with DMSO control.

    Techniques Used: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    16) Product Images from "Macrophage-Derived Extracellular Vesicles Induce Long-Lasting Immunity Against Hepatitis C Virus Which Is Blunted by Polyunsaturated Fatty Acids"

    Article Title: Macrophage-Derived Extracellular Vesicles Induce Long-Lasting Immunity Against Hepatitis C Virus Which Is Blunted by Polyunsaturated Fatty Acids

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00723

    Macrophage-derived extracellular vesicles (EVs) inhibit hepatitis C virus (HCV) replication. (A) Schematic outline of EV transfer experiments. Macrophages were treated with mock, IFN-α (500 IU/ml), or IFN-γ (25 ng/ml) for 24 h. EVs were isolated from conditioned supernatants of macrophages using differential centrifugation. EVs containing 100 µg of protein were transferred to Huh-7.5 hepatoma cells harboring subgenomic HCV replicons. (B,C) EVs from IFN-pulsed THP-1 cells (B) and MDMs (C) inhibit HCV replication. HCV mRNA levels (upper panel) were quantified by Taqman real-time PCR 24 or 96 h after transfer of EVs. Mean and SEM of three independent experiments are shown. Western blot analyses of HCV NS5A protein levels after exposure to THP-1 or MDM supernatants for 96 h (lower panel) (* P
    Figure Legend Snippet: Macrophage-derived extracellular vesicles (EVs) inhibit hepatitis C virus (HCV) replication. (A) Schematic outline of EV transfer experiments. Macrophages were treated with mock, IFN-α (500 IU/ml), or IFN-γ (25 ng/ml) for 24 h. EVs were isolated from conditioned supernatants of macrophages using differential centrifugation. EVs containing 100 µg of protein were transferred to Huh-7.5 hepatoma cells harboring subgenomic HCV replicons. (B,C) EVs from IFN-pulsed THP-1 cells (B) and MDMs (C) inhibit HCV replication. HCV mRNA levels (upper panel) were quantified by Taqman real-time PCR 24 or 96 h after transfer of EVs. Mean and SEM of three independent experiments are shown. Western blot analyses of HCV NS5A protein levels after exposure to THP-1 or MDM supernatants for 96 h (lower panel) (* P

    Techniques Used: Derivative Assay, Isolation, Centrifugation, Real-time Polymerase Chain Reaction, Western Blot

    Polyunsaturated fatty acids modulate extracellular vesicle (EV)-mediated innate antiviral immunity. (A) THP-1 macrophages were treated pretreated with mock (black bar), arachidonic acid (ARA) (gray bar), and eicosapentaenoic acid (EPA) (white bar) for 24 h (left) and 48 h (right), respectively. Then, EVs were isolated from conditioned supernatants 24 h after pulsation with mock, IFN-α (500 IU/ml), or IFN-γ (25 ng/ml) for 1 h, respectively. Purified EVs containing 100 µg of protein were transferred to Huh-7.5 hepatoma cells harboring subgenomic hepatitis C virus (HCV) replicons. HCV mRNA levels were quantified by Taqman real-time PCR (RT-PCR) after 96 h. Mean and SEM of three biological replicates are shown. (B) Huh-7.5 hepatoma cells harboring subgenomic HCV replicons were exposed to mock (black bar), ARA (5 µg/ml, gray bar), or EPA (5 µg/ml, white bar) for 24 and 48 h, and HCV mRNA levels were quantified by Taqman RT-PCR. Mean and SEM of three biological replicates are shown. (C) Western blot analysis of p-STAT1 and p-STAT2 in cell lysates of THP-1 cells were treated with mock (EtOH), ARA (5 µg/ml), or EPA (5 µg/ml) for 48 h before stimulation with IFN-α (500 IU/ml) for 120 min (* P
    Figure Legend Snippet: Polyunsaturated fatty acids modulate extracellular vesicle (EV)-mediated innate antiviral immunity. (A) THP-1 macrophages were treated pretreated with mock (black bar), arachidonic acid (ARA) (gray bar), and eicosapentaenoic acid (EPA) (white bar) for 24 h (left) and 48 h (right), respectively. Then, EVs were isolated from conditioned supernatants 24 h after pulsation with mock, IFN-α (500 IU/ml), or IFN-γ (25 ng/ml) for 1 h, respectively. Purified EVs containing 100 µg of protein were transferred to Huh-7.5 hepatoma cells harboring subgenomic hepatitis C virus (HCV) replicons. HCV mRNA levels were quantified by Taqman real-time PCR (RT-PCR) after 96 h. Mean and SEM of three biological replicates are shown. (B) Huh-7.5 hepatoma cells harboring subgenomic HCV replicons were exposed to mock (black bar), ARA (5 µg/ml, gray bar), or EPA (5 µg/ml, white bar) for 24 and 48 h, and HCV mRNA levels were quantified by Taqman RT-PCR. Mean and SEM of three biological replicates are shown. (C) Western blot analysis of p-STAT1 and p-STAT2 in cell lysates of THP-1 cells were treated with mock (EtOH), ARA (5 µg/ml), or EPA (5 µg/ml) for 48 h before stimulation with IFN-α (500 IU/ml) for 120 min (* P

    Techniques Used: Acetylene Reduction Assay, Isolation, Purification, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

    17) Product Images from "Identification of Novel Low-Dose Bisphenol A Targets in Human Foreskin Fibroblast Cells Derived from Hypospadias Patients"

    Article Title: Identification of Novel Low-Dose Bisphenol A Targets in Human Foreskin Fibroblast Cells Derived from Hypospadias Patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036711

    Reduced levels of MMP11 expression in hFFCs derived from child HS patients. Significantly lower MMP11 expression was observed in hFFCs derived from the HS ( n = 23) group compared with the CO ( n = 11) group by TaqMan real-time PCR. ( A ) Boxplot and ( B ) summary of the quantitative data comparing MMP11 expression levels in HS and CO groups.
    Figure Legend Snippet: Reduced levels of MMP11 expression in hFFCs derived from child HS patients. Significantly lower MMP11 expression was observed in hFFCs derived from the HS ( n = 23) group compared with the CO ( n = 11) group by TaqMan real-time PCR. ( A ) Boxplot and ( B ) summary of the quantitative data comparing MMP11 expression levels in HS and CO groups.

    Techniques Used: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction

    18) Product Images from "Central inhibition of granulocyte-macrophage colony-stimulating factor is analgesic in experimental neuropathic pain"

    Article Title: Central inhibition of granulocyte-macrophage colony-stimulating factor is analgesic in experimental neuropathic pain

    Journal: Pain

    doi: 10.1097/j.pain.0000000000001130

    Verification of GM-CSFR mRNA expression. Taqman profiles confirm expression of both GM-CSFR α-chain (A) and common β-chain (B) subunits in astrocytes and microglia. GM-CSFR, granulocyte-macrophage colony-stimulating factor receptor.
    Figure Legend Snippet: Verification of GM-CSFR mRNA expression. Taqman profiles confirm expression of both GM-CSFR α-chain (A) and common β-chain (B) subunits in astrocytes and microglia. GM-CSFR, granulocyte-macrophage colony-stimulating factor receptor.

    Techniques Used: Expressing

    19) Product Images from "Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators"

    Article Title: Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08150-5

    E7107 induces BCL2A1-dependent apoptosis in melanoma cell lines. a RT quantitative PCR (RT-qPCR) analysis of total mRNA levels (pan), exonic mRNA levels covering the junction of exons (exon), and pre-mRNA levels detecting the intron (intron) of BCL2A1 . HT144 cells were pre-treated with 100 µg ml −1 cycloheximide (CHX) for 1 h, followed by addition of E7107 as indicated for 12 h to inhibit nonsense-mediated mRNA decay (NMD) before collecting RNA samples. Schematic representations of TaqMan primers and probes were shown. Boxes: exons; gray lines: introns; arrows: primers; and black lines: probes. b Box plots showing the distribution of E7107 Emax in the solid tumor cell lines separated by tissue/lineage: melanoma ( n = 24), breast ( n = 33), colon ( n = 27), kidney ( n = 10), lung ( n = 91), and pancreas ( n = 24). The box plots exhibit five number summary from bottom to top: minimum, first quartile, median, third quartile, and maximum/outliers. c Heatmap of BCL2A1 mRNA expression (based on CCLE RNA-seq, sorted from high to low in the solid tumor cell lines separated by tissue/lineage) and E7107 Emax. d RT-qPCR analysis of BCL2A1 mRNA levels (exon1/2 junction) in vector control (Vector) or stable BCL2A1 cDNA-expressing (BCL2A1) melanoma cell lines. Data represent means ± SD of biological triplicates. e Growth curves measured by CellTiter-Glo (CTG) and f apoptosis induction curves measured by IncuCyte Caspase-3/7 Green Apoptosis Assay in melanoma cell lines stably transduced with vector control (empty vector) or BCL2A1 cDNA (BCL2A1 cDNA) treated with E7107 for 72 h. Data represent means ± SD of biological triplicates
    Figure Legend Snippet: E7107 induces BCL2A1-dependent apoptosis in melanoma cell lines. a RT quantitative PCR (RT-qPCR) analysis of total mRNA levels (pan), exonic mRNA levels covering the junction of exons (exon), and pre-mRNA levels detecting the intron (intron) of BCL2A1 . HT144 cells were pre-treated with 100 µg ml −1 cycloheximide (CHX) for 1 h, followed by addition of E7107 as indicated for 12 h to inhibit nonsense-mediated mRNA decay (NMD) before collecting RNA samples. Schematic representations of TaqMan primers and probes were shown. Boxes: exons; gray lines: introns; arrows: primers; and black lines: probes. b Box plots showing the distribution of E7107 Emax in the solid tumor cell lines separated by tissue/lineage: melanoma ( n = 24), breast ( n = 33), colon ( n = 27), kidney ( n = 10), lung ( n = 91), and pancreas ( n = 24). The box plots exhibit five number summary from bottom to top: minimum, first quartile, median, third quartile, and maximum/outliers. c Heatmap of BCL2A1 mRNA expression (based on CCLE RNA-seq, sorted from high to low in the solid tumor cell lines separated by tissue/lineage) and E7107 Emax. d RT-qPCR analysis of BCL2A1 mRNA levels (exon1/2 junction) in vector control (Vector) or stable BCL2A1 cDNA-expressing (BCL2A1) melanoma cell lines. Data represent means ± SD of biological triplicates. e Growth curves measured by CellTiter-Glo (CTG) and f apoptosis induction curves measured by IncuCyte Caspase-3/7 Green Apoptosis Assay in melanoma cell lines stably transduced with vector control (empty vector) or BCL2A1 cDNA (BCL2A1 cDNA) treated with E7107 for 72 h. Data represent means ± SD of biological triplicates

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, RNA Sequencing Assay, Plasmid Preparation, CTG Assay, Apoptosis Assay, Stable Transfection, Transduction

    20) Product Images from "Kidney Androgen-Regulated Protein (KAP) Transgenic Mice Are Protected Against High-Fat Diet Induced Metabolic Syndrome"

    Article Title: Kidney Androgen-Regulated Protein (KAP) Transgenic Mice Are Protected Against High-Fat Diet Induced Metabolic Syndrome

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-16487-y

    Gene expression assays. Quantitative RT-PCR gene expression assays (qReal Time TaqMan probes PCR) in white adipose tissues of Tg and control littermate mice fed with chow or HFD, upon sacrifice at 7-month of age. Relative expression of MCP-1, Cd68, IL-6 and TNF-α pro-inflammatory genes are given in fold change (FC) 2 −ΔΔCt . Peptidyl-prolyl isomerase A (CypA) probe was used as endogenous control (n = 8 per group), #p
    Figure Legend Snippet: Gene expression assays. Quantitative RT-PCR gene expression assays (qReal Time TaqMan probes PCR) in white adipose tissues of Tg and control littermate mice fed with chow or HFD, upon sacrifice at 7-month of age. Relative expression of MCP-1, Cd68, IL-6 and TNF-α pro-inflammatory genes are given in fold change (FC) 2 −ΔΔCt . Peptidyl-prolyl isomerase A (CypA) probe was used as endogenous control (n = 8 per group), #p

    Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Mouse Assay

    KAP effects on IL-6 expression by TNF-α. KAP subcellular location and secretion. ( A ) KAP-HA expression (KAP) in the HK-2 cell line. Empty lentiviral vector transduced cells were used as a control (Control). KAP-HA expression by determined by Western blot analyses using anti-HA antibody (ROCHE). ( B ) IL-6 mRNA induction upon TNF-α stimulation in HK-2 cells. Control and KAP transduced HK-2 cells were treated with increasing doses of TNF-α (0,1,10 and 100 ng/μl) for 24 h and IL-6 expression performed by qRT-PCR using IL-6 TaqMan probes. CypA was used as endogen control. Results represented in this figure are the average of three independent biological replicas. *p
    Figure Legend Snippet: KAP effects on IL-6 expression by TNF-α. KAP subcellular location and secretion. ( A ) KAP-HA expression (KAP) in the HK-2 cell line. Empty lentiviral vector transduced cells were used as a control (Control). KAP-HA expression by determined by Western blot analyses using anti-HA antibody (ROCHE). ( B ) IL-6 mRNA induction upon TNF-α stimulation in HK-2 cells. Control and KAP transduced HK-2 cells were treated with increasing doses of TNF-α (0,1,10 and 100 ng/μl) for 24 h and IL-6 expression performed by qRT-PCR using IL-6 TaqMan probes. CypA was used as endogen control. Results represented in this figure are the average of three independent biological replicas. *p

    Techniques Used: Expressing, Plasmid Preparation, Western Blot, Quantitative RT-PCR

    21) Product Images from "BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer and confers sensitivity to BET inhibitors"

    Article Title: BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer and confers sensitivity to BET inhibitors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200826

    BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using TaqMan ® Copy-number Assay by qPCR. (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.
    Figure Legend Snippet: BRD4 expression is sufficient to transform immortalized ovarian surface epithelial cells. (A) Analysis of BRD4 copy-number from 1042 cell lines (CCLE). (B) BRD4 long and short-isoform expression in BRD4-transduced IOSE cells, and several BRD4-non-amplified (OV0419F, OV110F, OV2022F and OV0452F) and BRD4-amplified (HOXF062 and OV0857F) patient derived xenograft models. BRD4 copy-number (CN) in different PDX models were determined using TaqMan ® Copy-number Assay by qPCR. (C) Colony formation potential of IOSE cells expressing BRD4 -/+ 500 nM JQ1 was assessed using a soft-agar assay. (D) Proliferation of BRD4 expressing IOSE cells was examined by measuring cell confluence over time via live cell imaging. (E) Cell cycle analysis of long and short BRD4-isoform expressing IOSE cells.

    Techniques Used: Expressing, Amplification, Derivative Assay, TaqMan Copy Number Assay, Real-time Polymerase Chain Reaction, Soft Agar Assay, Live Cell Imaging, Cell Cycle Assay

    22) Product Images from "FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex"

    Article Title: FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

    Journal: eLife

    doi: 10.7554/eLife.35012

    FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.
    Figure Legend Snippet: FRMD8 stabilises endogenous iRhom2. ( A, B ) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. ( C ) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in ( B ) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Polymerase Chain Reaction

    FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value
    Figure Legend Snippet: FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity. ( A ) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. ( B, C ) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. ( D ) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. ( E, F ) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value > 0.05; *=p value

    Techniques Used: Activity Assay, Transfection, Western Blot, Staining, Polymerase Chain Reaction, Knock-Out, Immunostaining, Flow Cytometry, Fluorescence, Software, Incubation, MANN-WHITNEY

    23) Product Images from "Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure"

    Article Title: Targeting mir128-3p alleviates myocardial insulin resistance and prevents ischemia-induced heart failure

    Journal: eLife

    doi: 10.7554/eLife.54298

    MiRNA analysis in 4w-MI myocardium. ( A ) Mature mir144-3p and mir145-5p in 4w-MI myocardium were measured using TaqMan Advanced miRNA qPCR (N = 6–8 mice). Data were normalized against mature mir191-5p expression. ( B ) In situ hybridization with digoxigenin-labeled mir128-3p probes was quantified using ImageJ software (N = 3–5 mice). Groups were compared using the Mann-Whitney test. ( C ) Pri-mir128-1 and Pri-mir128-2 measurement in 4w-MI myocardium using TaqMan Gene expression (N = 6–8 mice). Data were normalised against 18S expression. Data presented as mean ± SEM. Groups were compared using two-tailed Student’s t-test. MiRNA analysis in 4w-MI myocardium.
    Figure Legend Snippet: MiRNA analysis in 4w-MI myocardium. ( A ) Mature mir144-3p and mir145-5p in 4w-MI myocardium were measured using TaqMan Advanced miRNA qPCR (N = 6–8 mice). Data were normalized against mature mir191-5p expression. ( B ) In situ hybridization with digoxigenin-labeled mir128-3p probes was quantified using ImageJ software (N = 3–5 mice). Groups were compared using the Mann-Whitney test. ( C ) Pri-mir128-1 and Pri-mir128-2 measurement in 4w-MI myocardium using TaqMan Gene expression (N = 6–8 mice). Data were normalised against 18S expression. Data presented as mean ± SEM. Groups were compared using two-tailed Student’s t-test. MiRNA analysis in 4w-MI myocardium.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, Expressing, In Situ Hybridization, Labeling, Software, MANN-WHITNEY, Two Tailed Test

    Mir128-3p overexpression and inhibition optimization. Validation of mir128-3p mimic and antagomir. ( A ) mir128-3p mimic and ( B ) antagomir validation was carried out on H9C2 cells at different concentrations. Mature miRNA was measured by TaqMan Advanced miRNA qPCR. Data were normalized against mature mir191-5p expression (N = 3 experiments). Data presented as mean ± SEM. Groups were compared using one-way ANOVA followed by Bonferroni post hoc tests. Mir128-3p overexpression and inhibition optimization.
    Figure Legend Snippet: Mir128-3p overexpression and inhibition optimization. Validation of mir128-3p mimic and antagomir. ( A ) mir128-3p mimic and ( B ) antagomir validation was carried out on H9C2 cells at different concentrations. Mature miRNA was measured by TaqMan Advanced miRNA qPCR. Data were normalized against mature mir191-5p expression (N = 3 experiments). Data presented as mean ± SEM. Groups were compared using one-way ANOVA followed by Bonferroni post hoc tests. Mir128-3p overexpression and inhibition optimization.

    Techniques Used: Over Expression, Inhibition, Real-time Polymerase Chain Reaction, Expressing

    Mir128-3p level in rat cardiomyocytes after hypoxia. Mature mir128-3p was measured using TaqMan Advanced miRNA qPCR (N = 6 experiments). Data were normalized against mature mir191-5p expression. Data presented as mean ± SEM. Groups were compared using one-way ANOVA followed by Bonferroni post hoc tests. Mir128-3p level in rat cardiomyocytes after hypoxia.
    Figure Legend Snippet: Mir128-3p level in rat cardiomyocytes after hypoxia. Mature mir128-3p was measured using TaqMan Advanced miRNA qPCR (N = 6 experiments). Data were normalized against mature mir191-5p expression. Data presented as mean ± SEM. Groups were compared using one-way ANOVA followed by Bonferroni post hoc tests. Mir128-3p level in rat cardiomyocytes after hypoxia.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    24) Product Images from "The Immune Response to Picornavirus Infection and the Effect of Immune Manipulation on Acute Seizures"

    Article Title: The Immune Response to Picornavirus Infection and the Effect of Immune Manipulation on Acute Seizures

    Journal: Journal of neurovirology

    doi: 10.1007/s13365-018-0636-2

    Gene expression analysis of IL-6 (a) and TNF-α (b) in the hippocampi of TMEV-infected mice treated with control liposomes or clodronate-containing liposomes. Mice were treated with control or clodronate-containing liposomes as described in the Methods and hippocampi were harvested at days 2 and 3 post infection (6 mice per group). Real time PCR for the detection of IL-6 (a) and TNF-α (b) was performed using TaqMan PCR as described in the Methods. Fold changes were calculated using the ΔΔCT method, in which fold change data are represented as 2−ΔΔCT. Error bars depict the SEM ΔCT values
    Figure Legend Snippet: Gene expression analysis of IL-6 (a) and TNF-α (b) in the hippocampi of TMEV-infected mice treated with control liposomes or clodronate-containing liposomes. Mice were treated with control or clodronate-containing liposomes as described in the Methods and hippocampi were harvested at days 2 and 3 post infection (6 mice per group). Real time PCR for the detection of IL-6 (a) and TNF-α (b) was performed using TaqMan PCR as described in the Methods. Fold changes were calculated using the ΔΔCT method, in which fold change data are represented as 2−ΔΔCT. Error bars depict the SEM ΔCT values

    Techniques Used: Expressing, Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    25) Product Images from "Analysis of monocyte infiltration in MPTP mice reveals that microglial CX3CR1 protects against neurotoxic over-induction of monocyte-attracting CCL2 by astrocytes"

    Article Title: Analysis of monocyte infiltration in MPTP mice reveals that microglial CX3CR1 protects against neurotoxic over-induction of monocyte-attracting CCL2 by astrocytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-017-0830-9

    Chemokine RNA profiling in the substantia nigra of MPTP mice reveals early CCL2/7/12-CCR2 axis induction. a – b Laser microdissection (LMD) of Nissl-stained substantia nigra pars compacta (SNpc) from mouse midbrain tissue sections, used for TaqMan RT-qPCR profiling of the full chemokine family in MPTP mice. a Showing a SNpc before LMD and b showing multiple SNpcs collected by LMD. ( Scale bar ; b , 200 μm). c Selected results, showing fold change ( P
    Figure Legend Snippet: Chemokine RNA profiling in the substantia nigra of MPTP mice reveals early CCL2/7/12-CCR2 axis induction. a – b Laser microdissection (LMD) of Nissl-stained substantia nigra pars compacta (SNpc) from mouse midbrain tissue sections, used for TaqMan RT-qPCR profiling of the full chemokine family in MPTP mice. a Showing a SNpc before LMD and b showing multiple SNpcs collected by LMD. ( Scale bar ; b , 200 μm). c Selected results, showing fold change ( P

    Techniques Used: Mouse Assay, Laser Capture Microdissection, Staining, Quantitative RT-PCR

    26) Product Images from "Cleavage of BLOC1S1 mRNA by IRE1 Is Sequence Specific, Temporally Separate from XBP1 Splicing, and Dispensable for Cell Viability under Acute Endoplasmic Reticulum Stress"

    Article Title: Cleavage of BLOC1S1 mRNA by IRE1 Is Sequence Specific, Temporally Separate from XBP1 Splicing, and Dispensable for Cell Viability under Acute Endoplasmic Reticulum Stress

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00013-15

    BLOC1S1 degradation and XBP1 splicing are temporally separate. (A and C) Relative expression of BLOC1S1 in NCI-H929 or RPMI-8226 cells treated with the indicated stressors over time. BLOC1S1 mRNA was measured by SYBR green qPCR and normalized to the GAPDH control and is presented relative to time zero. (B and D) Western blots showing phospho-IRE1, IRE1, and β-actin in the cells treated for panel A. Also shown is agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site from the samples used in panel A. (E to L) TaqMan qPCR measurements of BLOC1S1 , XBP1u , and XBP1s for cells treated as indicated. The data were normalized to the GAPDH control and are presented relative to untreated cells. The data are representative of two independent experiments, and error bars show SEM from three technical replicates.
    Figure Legend Snippet: BLOC1S1 degradation and XBP1 splicing are temporally separate. (A and C) Relative expression of BLOC1S1 in NCI-H929 or RPMI-8226 cells treated with the indicated stressors over time. BLOC1S1 mRNA was measured by SYBR green qPCR and normalized to the GAPDH control and is presented relative to time zero. (B and D) Western blots showing phospho-IRE1, IRE1, and β-actin in the cells treated for panel A. Also shown is agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site from the samples used in panel A. (E to L) TaqMan qPCR measurements of BLOC1S1 , XBP1u , and XBP1s for cells treated as indicated. The data were normalized to the GAPDH control and are presented relative to untreated cells. The data are representative of two independent experiments, and error bars show SEM from three technical replicates.

    Techniques Used: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Western Blot, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    Inhibition of RIDD does not affect RPMI-8226 cell viability under acute ER stress. (A) Agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in samples from RPMI-8226 cells pretreated for the indicated times with the indicated concentration of 4μ8c and then treated with 2 mM DTT for 90 min. −, untreated cells; DTT, DTT alone. (B) SYBR green qPCR showing relative expression of BLOC1S1 mRNA in RPMI-8226 cells treated with 30 μM 4μ8c at −10 min ( t = −10) (pretreatment) or at 15 min of DTT (post-DTT) and treated with DTT at time zero ( t = 0) for 90 min before washout and recovery for the indicated times. DMSO, vehicle control-treated cells. (C and D) TaqMan qPCR quantification of XBP1u (C) and XBP1s (D) in the samples used for panel B. (E) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples from panel B. The arrows indicate the times of addition of the indicated treatments or washout. (F) SYBR green qPCR of relative CHOP expression in the samples used for panel B. (G) WST-1 cell viability assays of RPMI-8226 cells treated as for panel B or treated with 4μ8c or DMSO without DTT treatment. The data are relative to untreated cells; 0.5% Triton X-100 after DTT washout is shown as a control for maximal cell death. All the graphs show means ± SEM from four (B, F, and G) or three (C and D) independent experiments. n.s., P > 0.05 (unpaired t test). All qPCR measurements were normalized to GAPDH and are presented relative to time −10 for DMSO-treated cell samples.
    Figure Legend Snippet: Inhibition of RIDD does not affect RPMI-8226 cell viability under acute ER stress. (A) Agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in samples from RPMI-8226 cells pretreated for the indicated times with the indicated concentration of 4μ8c and then treated with 2 mM DTT for 90 min. −, untreated cells; DTT, DTT alone. (B) SYBR green qPCR showing relative expression of BLOC1S1 mRNA in RPMI-8226 cells treated with 30 μM 4μ8c at −10 min ( t = −10) (pretreatment) or at 15 min of DTT (post-DTT) and treated with DTT at time zero ( t = 0) for 90 min before washout and recovery for the indicated times. DMSO, vehicle control-treated cells. (C and D) TaqMan qPCR quantification of XBP1u (C) and XBP1s (D) in the samples used for panel B. (E) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples from panel B. The arrows indicate the times of addition of the indicated treatments or washout. (F) SYBR green qPCR of relative CHOP expression in the samples used for panel B. (G) WST-1 cell viability assays of RPMI-8226 cells treated as for panel B or treated with 4μ8c or DMSO without DTT treatment. The data are relative to untreated cells; 0.5% Triton X-100 after DTT washout is shown as a control for maximal cell death. All the graphs show means ± SEM from four (B, F, and G) or three (C and D) independent experiments. n.s., P > 0.05 (unpaired t test). All qPCR measurements were normalized to GAPDH and are presented relative to time −10 for DMSO-treated cell samples.

    Techniques Used: Inhibition, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, SYBR Green Assay, Real-time Polymerase Chain Reaction, Expressing

    RIDD is dispensable for cell viability under DTT treatment in HT-1080 fibrosarcoma cells. (A) TaqMan qPCR measuring BLOC1S1 , XBP1u , or XBP1s in HT-1080 cells treated with 2 mM DTT for the indicated times. The data are normalized to GAPDH and are presented relative to the maximum for each transcript. (B to G) SYBR green ( BLOC1S1 ) or TaqMan ( XBP1u and XBP1s ) qPCR measurements of the indicated transcripts over a time course of DTT (2 mM) treatment in HT-1080 cells that were treated with 4μ8c (30 μM) 10 min before (pretreatment) or 2 h or 4 h after (post-DTT) addition of DTT. The data are normalized to GAPDH and are presented relative to untreated cells. (H and I) WST-1 measurement of the viability of HT-1080 cells treated with 2 mM DTT or 30 μM 4μ8c, as indicated. Triton X-100 treatment is shown as a dead-cell control. The data show means ± SEM from three (A) or four (B to I) independent experiments. *, P
    Figure Legend Snippet: RIDD is dispensable for cell viability under DTT treatment in HT-1080 fibrosarcoma cells. (A) TaqMan qPCR measuring BLOC1S1 , XBP1u , or XBP1s in HT-1080 cells treated with 2 mM DTT for the indicated times. The data are normalized to GAPDH and are presented relative to the maximum for each transcript. (B to G) SYBR green ( BLOC1S1 ) or TaqMan ( XBP1u and XBP1s ) qPCR measurements of the indicated transcripts over a time course of DTT (2 mM) treatment in HT-1080 cells that were treated with 4μ8c (30 μM) 10 min before (pretreatment) or 2 h or 4 h after (post-DTT) addition of DTT. The data are normalized to GAPDH and are presented relative to untreated cells. (H and I) WST-1 measurement of the viability of HT-1080 cells treated with 2 mM DTT or 30 μM 4μ8c, as indicated. Triton X-100 treatment is shown as a dead-cell control. The data show means ± SEM from three (A) or four (B to I) independent experiments. *, P

    Techniques Used: Real-time Polymerase Chain Reaction, SYBR Green Assay

    BLOC1S1 is a RIDD target in myeloma cells. (A) TaqMan qPCR measuring expression of consistently identified RIDD targets containing a consensus IRE1 target sequence in stressed or unstressed myeloma cell lines with or without ActD. (B) Relative BLOC1S1 expression measured by SYBR green qPCR in the indicated myeloma cell lines treated with 2 mM DTT in the presence or absence of DMSO vehicle control or 4μ8c pretreatment. (C) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel B. The qPCR data are normalized to GAPDH mRNA and are expressed relative to untreated cells. (A and B) The data are means ± standard errors of the mean (SEM) from three independent experiments. *, P
    Figure Legend Snippet: BLOC1S1 is a RIDD target in myeloma cells. (A) TaqMan qPCR measuring expression of consistently identified RIDD targets containing a consensus IRE1 target sequence in stressed or unstressed myeloma cell lines with or without ActD. (B) Relative BLOC1S1 expression measured by SYBR green qPCR in the indicated myeloma cell lines treated with 2 mM DTT in the presence or absence of DMSO vehicle control or 4μ8c pretreatment. (C) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel B. The qPCR data are normalized to GAPDH mRNA and are expressed relative to untreated cells. (A and B) The data are means ± standard errors of the mean (SEM) from three independent experiments. *, P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Sequencing, SYBR Green Assay, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    BLOC1S1 degradation is dispensable for recovery from acute stress. (A) SYBR green qPCR quantification of relative expression of BLOC1S1 in RPMI-8226 cells transduced as indicated and then treated with 2 mM DTT for 90 min, followed by washout and recovery for the indicated times. (B and C) TaqMan qPCR quantification of XBP1u (B) and XBP1s (C) in the samples used for panel A. (D) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel A. (E) SYBR green qPCR quantification of relative expression of CHOP in the samples used for panel A. (F) Relative cell viability measured by WST-1 assay of cells transduced as indicated and then treated with 2 mM DTT for 90 min, followed by washout and recovery for 24 h. Triton X-100 (0.5%) treatment after DTT washout in untransduced cells is shown as a control for maximal cell death. The measurements are background-subtracted absorbances relative to the absorbance of unstressed cells for each transduced sample. All qPCR measurements were normalized to GAPDH and are presented relative to 0 min of DTT treatment in DMSO-treated cells. All the graphs show means ± SEM of three (A to C and E) or four (F) independent experiments.
    Figure Legend Snippet: BLOC1S1 degradation is dispensable for recovery from acute stress. (A) SYBR green qPCR quantification of relative expression of BLOC1S1 in RPMI-8226 cells transduced as indicated and then treated with 2 mM DTT for 90 min, followed by washout and recovery for the indicated times. (B and C) TaqMan qPCR quantification of XBP1u (B) and XBP1s (C) in the samples used for panel A. (D) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the samples used for panel A. (E) SYBR green qPCR quantification of relative expression of CHOP in the samples used for panel A. (F) Relative cell viability measured by WST-1 assay of cells transduced as indicated and then treated with 2 mM DTT for 90 min, followed by washout and recovery for 24 h. Triton X-100 (0.5%) treatment after DTT washout in untransduced cells is shown as a control for maximal cell death. The measurements are background-subtracted absorbances relative to the absorbance of unstressed cells for each transduced sample. All qPCR measurements were normalized to GAPDH and are presented relative to 0 min of DTT treatment in DMSO-treated cells. All the graphs show means ± SEM of three (A to C and E) or four (F) independent experiments.

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, WST-1 Assay

    Inhibition of RIDD does not affect NCI-H929 cell viability under acute ER stress. (A) WST-1 cell viability assays of NCI-H929 cells treated as indicated with 2 mM DTT for 60 min (from time zero) and either pretreated (at time −10 min) or posttreated (at time 15 min) with 30 μM 4μ8c and then washed and allowed to recover for 24 h. Cells treated with 0.5% Triton X-100 were included as a dead-cell control. (B) SYBR green qPCR quantification of BLOC1S1 in the DTT-treated samples from panel A. (C) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the DTT-treated samples from panel A. The timing of treatments is indicated below the gel images. (D and E) TaqMan qPCR quantification of XBP1u (D) or XBP1s (E) in the DTT-treated samples from panel A. (F) SYBR green qPCR of relative CHOP expression in the DTT-treated samples from panel A. All qPCR data are normalized to GAPDH and are presented relative to the DMSO control at time −10 min of DTT. All the graphs show means ± SEM from three independent experiments. n.s., P > 0.05 (unpaired t test).
    Figure Legend Snippet: Inhibition of RIDD does not affect NCI-H929 cell viability under acute ER stress. (A) WST-1 cell viability assays of NCI-H929 cells treated as indicated with 2 mM DTT for 60 min (from time zero) and either pretreated (at time −10 min) or posttreated (at time 15 min) with 30 μM 4μ8c and then washed and allowed to recover for 24 h. Cells treated with 0.5% Triton X-100 were included as a dead-cell control. (B) SYBR green qPCR quantification of BLOC1S1 in the DTT-treated samples from panel A. (C) Representative agarose gel electrophoresis of the RT-PCR product surrounding the XBP1 splice site in the DTT-treated samples from panel A. The timing of treatments is indicated below the gel images. (D and E) TaqMan qPCR quantification of XBP1u (D) or XBP1s (E) in the DTT-treated samples from panel A. (F) SYBR green qPCR of relative CHOP expression in the DTT-treated samples from panel A. All qPCR data are normalized to GAPDH and are presented relative to the DMSO control at time −10 min of DTT. All the graphs show means ± SEM from three independent experiments. n.s., P > 0.05 (unpaired t test).

    Techniques Used: Inhibition, SYBR Green Assay, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Expressing

    27) Product Images from "Differential expression and tumorigenic function of neurotensin receptor 1 in neuroendocrine tumor cells"

    Article Title: Differential expression and tumorigenic function of neurotensin receptor 1 in neuroendocrine tumor cells

    Journal: Oncotarget

    doi:

    Expression analysis of NTSRs in endogenous and 5-aza-CdR treated NET cell lines A. RT-PCR analysis of NTS , NTSR1, NTSR2, NTSR3 and β-actin expression in NET cells. B. RT-PCR analysis of NTSRs and β-actin expression in BON and QGP-1 cells treated with 0 (DMSO) or 10 μM 5-aza-CdR. The media containing 5-aza-CdR were replaced every 24 h for 4 d. C. Quantitative RT-PCR (qRT-PCR) analysis confirmed that treatment with 5-aza-CdR increased the expression of NTSR1 gene in BON and QGP-1 cells. The reaction was performed using a TaqMan Gene Expression Master Mix and TaqMan probes for human NTSR1 and GAPDH as internal control (Applied Biosystems). Expression levels were assessed by evaluating threshold cycle (Ct) values. The relative amount of mRNA expression was calculated by the comparative ΔΔCt method (* p
    Figure Legend Snippet: Expression analysis of NTSRs in endogenous and 5-aza-CdR treated NET cell lines A. RT-PCR analysis of NTS , NTSR1, NTSR2, NTSR3 and β-actin expression in NET cells. B. RT-PCR analysis of NTSRs and β-actin expression in BON and QGP-1 cells treated with 0 (DMSO) or 10 μM 5-aza-CdR. The media containing 5-aza-CdR were replaced every 24 h for 4 d. C. Quantitative RT-PCR (qRT-PCR) analysis confirmed that treatment with 5-aza-CdR increased the expression of NTSR1 gene in BON and QGP-1 cells. The reaction was performed using a TaqMan Gene Expression Master Mix and TaqMan probes for human NTSR1 and GAPDH as internal control (Applied Biosystems). Expression levels were assessed by evaluating threshold cycle (Ct) values. The relative amount of mRNA expression was calculated by the comparative ΔΔCt method (* p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    28) Product Images from "Repeat-induced gene silencing of L1 transgenes is correlated with differential promoter methylation"

    Article Title: Repeat-induced gene silencing of L1 transgenes is correlated with differential promoter methylation

    Journal: Gene

    doi: 10.1016/j.gene.2010.02.005

    Generation of mice carrying reduced number of ORFeus L transgenes. (A) The parental mouse line carries a tandem array of ORFeus L ). The loxP site is illustrated as an open triangle. (B) After Cre-mediated recombination, intervening copies are excised, resulting in a transgene array with reduced copy number. A single-copy transgene is illustrated. Each copy is composed of a CAG promoter, followed by ORFeus coding sequences and a gfp based reporter cassette. The regions for DNA methylation analysis are indicated as blue lines above the vector diagram. (C) A 5′ truncated insertion resulting from ORFeus retrotransposition is illustrated. Note the insertion is intronless as the intronic sequence has been spliced out from ORFeus transcript prior to reverse transcription and integration. (D) The breeding scheme to reduce transgene copy number. (E) Determination of transgene copy number by donor-specific qPCR. The Taqman probe (purple line) and primers (black arrowheads) are specific to the intron that disrupts the gfp reporter (inset). The transgene copy number was reduced to either 1 or 2 copies in six G2 mice. Wild-type tail gDNA samples spiked with indicated number of pRSVGFPuvINT plasmid were used to measure assay sensitivity and dynamic range. Error bars represent standard deviations of normalized transgene copy number from three technical replicates for each sample. Values for individual samples were normalized to that of a sample known to contain a single-copy transgene (2G6).
    Figure Legend Snippet: Generation of mice carrying reduced number of ORFeus L transgenes. (A) The parental mouse line carries a tandem array of ORFeus L ). The loxP site is illustrated as an open triangle. (B) After Cre-mediated recombination, intervening copies are excised, resulting in a transgene array with reduced copy number. A single-copy transgene is illustrated. Each copy is composed of a CAG promoter, followed by ORFeus coding sequences and a gfp based reporter cassette. The regions for DNA methylation analysis are indicated as blue lines above the vector diagram. (C) A 5′ truncated insertion resulting from ORFeus retrotransposition is illustrated. Note the insertion is intronless as the intronic sequence has been spliced out from ORFeus transcript prior to reverse transcription and integration. (D) The breeding scheme to reduce transgene copy number. (E) Determination of transgene copy number by donor-specific qPCR. The Taqman probe (purple line) and primers (black arrowheads) are specific to the intron that disrupts the gfp reporter (inset). The transgene copy number was reduced to either 1 or 2 copies in six G2 mice. Wild-type tail gDNA samples spiked with indicated number of pRSVGFPuvINT plasmid were used to measure assay sensitivity and dynamic range. Error bars represent standard deviations of normalized transgene copy number from three technical replicates for each sample. Values for individual samples were normalized to that of a sample known to contain a single-copy transgene (2G6).

    Techniques Used: Mouse Assay, DNA Methylation Assay, Plasmid Preparation, Sequencing, Real-time Polymerase Chain Reaction

    29) Product Images from "The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly"

    Article Title: The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by recruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly

    Journal: Virology

    doi: 10.1016/j.virol.2017.03.002

    Development of a highly sensitive, strand-specific quantitative RT-PCR assay to detect expression of ASP RNA. Panel A: Scheme of RT-PCR assay for specific detection of the ASP transcript but not HIV-1 sense transcripts. The RT primer (AST-tag) introduces a tag at the 5′ end of the cDNA, which serves as a template for the reverse primer in the subsequence TaqMan PCR step. Panel B: Real time PCR traces show that amplification was obtained only with RNA from infected cells, and only when the RT reaction is carried out in the presence of the AST-tag RT primer and RT enzyme (cDNA was assayed in triplicate PCR reactions). Panel C: PCR traces obtained with 10°–10 5 copies of a linearized of pUC57-based plasmid carrying the ASP RNA amplicon (all dilutions run in triplicate samples).
    Figure Legend Snippet: Development of a highly sensitive, strand-specific quantitative RT-PCR assay to detect expression of ASP RNA. Panel A: Scheme of RT-PCR assay for specific detection of the ASP transcript but not HIV-1 sense transcripts. The RT primer (AST-tag) introduces a tag at the 5′ end of the cDNA, which serves as a template for the reverse primer in the subsequence TaqMan PCR step. Panel B: Real time PCR traces show that amplification was obtained only with RNA from infected cells, and only when the RT reaction is carried out in the presence of the AST-tag RT primer and RT enzyme (cDNA was assayed in triplicate PCR reactions). Panel C: PCR traces obtained with 10°–10 5 copies of a linearized of pUC57-based plasmid carrying the ASP RNA amplicon (all dilutions run in triplicate samples).

    Techniques Used: Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, AST Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Amplification, Infection, Plasmid Preparation

    30) Product Images from "Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method"

    Article Title: Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0231450

    HNRNPU regulates P/I-induced IL-6 mRNA expression. (A–C) HeLa cells were transfected with siRNAs, incubated for 72 hours, and then treated with P/I for 4 hours (B) or 24 hours (A, C) and harvested. HNRNPU protein expression was determined by Western blotting with actin as a loading control (A). IL-6 mRNA expression was examined using a TaqMan assay and normalized with RPLP0 (B). (C) IL-6 protein secreted in the condition medium was quantified by ELISA (R D Systems). Statistically significant differences are marked with *** ( p
    Figure Legend Snippet: HNRNPU regulates P/I-induced IL-6 mRNA expression. (A–C) HeLa cells were transfected with siRNAs, incubated for 72 hours, and then treated with P/I for 4 hours (B) or 24 hours (A, C) and harvested. HNRNPU protein expression was determined by Western blotting with actin as a loading control (A). IL-6 mRNA expression was examined using a TaqMan assay and normalized with RPLP0 (B). (C) IL-6 protein secreted in the condition medium was quantified by ELISA (R D Systems). Statistically significant differences are marked with *** ( p

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

    31) Product Images from "Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses"

    Article Title: Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses

    Journal: Communications Biology

    doi: 10.1038/s42003-020-0882-8

    HOIPIN-1 shows therapeutic effects on psoriasis. a Phenotypical presentation of mouse back skin after 5 days of treatment. BALB/c mice (8-week-old female) were treated daily with control vaseline or imiquimod cream, in the presence of clobetasol or 0.1% HOIPIN-1 in DMSO. b HOIPIN-1 alleviates imiquimod-induced psoriasis. Erythema, scaling, and thickness of the back skin were scored daily on a scale from 0 to 4, and the cumulative scores (means, n = 3) are shown. c HOIPIN-1 reduces the thickened epidermis induced by the imiquimod treatment. Mice were treated for 7 days with imiquimod, clobetasol, and/or HOIPIN-1, as indicated. H E staining of the back skin was performed. Bars, 50 μm. d The thickness of the epidermis, at 90–132 sites from three to four mice treated as indicated, was measured using the ImageJ software (National Institutes of Health, Bethesda, MD) and statistically analyzed. e HOIPIN-1 suppresses cytokine expression in the back skin of imiquimod-treated mice. The mRNA levels of Il17a , Il17f , Il22 , and Il23a in the back skin of the mice were examined by TaqMan PCR. Data are shown as means ± SEM. In d , e , NS not significant, * P
    Figure Legend Snippet: HOIPIN-1 shows therapeutic effects on psoriasis. a Phenotypical presentation of mouse back skin after 5 days of treatment. BALB/c mice (8-week-old female) were treated daily with control vaseline or imiquimod cream, in the presence of clobetasol or 0.1% HOIPIN-1 in DMSO. b HOIPIN-1 alleviates imiquimod-induced psoriasis. Erythema, scaling, and thickness of the back skin were scored daily on a scale from 0 to 4, and the cumulative scores (means, n = 3) are shown. c HOIPIN-1 reduces the thickened epidermis induced by the imiquimod treatment. Mice were treated for 7 days with imiquimod, clobetasol, and/or HOIPIN-1, as indicated. H E staining of the back skin was performed. Bars, 50 μm. d The thickness of the epidermis, at 90–132 sites from three to four mice treated as indicated, was measured using the ImageJ software (National Institutes of Health, Bethesda, MD) and statistically analyzed. e HOIPIN-1 suppresses cytokine expression in the back skin of imiquimod-treated mice. The mRNA levels of Il17a , Il17f , Il22 , and Il23a in the back skin of the mice were examined by TaqMan PCR. Data are shown as means ± SEM. In d , e , NS not significant, * P

    Techniques Used: Mouse Assay, Staining, Software, Expressing, Polymerase Chain Reaction

    32) Product Images from "Macrophage-Derived Extracellular Vesicles Induce Long-Lasting Immunity Against Hepatitis C Virus Which Is Blunted by Polyunsaturated Fatty Acids"

    Article Title: Macrophage-Derived Extracellular Vesicles Induce Long-Lasting Immunity Against Hepatitis C Virus Which Is Blunted by Polyunsaturated Fatty Acids

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00723

    Macrophage-derived extracellular vesicles (EVs) inhibit hepatitis C virus (HCV) replication. (A) Schematic outline of EV transfer experiments. Macrophages were treated with mock, IFN-α (500 IU/ml), or IFN-γ (25 ng/ml) for 24 h. EVs were isolated from conditioned supernatants of macrophages using differential centrifugation. EVs containing 100 µg of protein were transferred to Huh-7.5 hepatoma cells harboring subgenomic HCV replicons. (B,C) EVs from IFN-pulsed THP-1 cells (B) and MDMs (C) inhibit HCV replication. HCV mRNA levels (upper panel) were quantified by Taqman real-time PCR 24 or 96 h after transfer of EVs. Mean and SEM of three independent experiments are shown. Western blot analyses of HCV NS5A protein levels after exposure to THP-1 or MDM supernatants for 96 h (lower panel) (* P
    Figure Legend Snippet: Macrophage-derived extracellular vesicles (EVs) inhibit hepatitis C virus (HCV) replication. (A) Schematic outline of EV transfer experiments. Macrophages were treated with mock, IFN-α (500 IU/ml), or IFN-γ (25 ng/ml) for 24 h. EVs were isolated from conditioned supernatants of macrophages using differential centrifugation. EVs containing 100 µg of protein were transferred to Huh-7.5 hepatoma cells harboring subgenomic HCV replicons. (B,C) EVs from IFN-pulsed THP-1 cells (B) and MDMs (C) inhibit HCV replication. HCV mRNA levels (upper panel) were quantified by Taqman real-time PCR 24 or 96 h after transfer of EVs. Mean and SEM of three independent experiments are shown. Western blot analyses of HCV NS5A protein levels after exposure to THP-1 or MDM supernatants for 96 h (lower panel) (* P

    Techniques Used: Derivative Assay, Isolation, Centrifugation, Real-time Polymerase Chain Reaction, Western Blot

    Polyunsaturated fatty acids modulate extracellular vesicle (EV)-mediated innate antiviral immunity. (A) THP-1 macrophages were treated pretreated with mock (black bar), arachidonic acid (ARA) (gray bar), and eicosapentaenoic acid (EPA) (white bar) for 24 h (left) and 48 h (right), respectively. Then, EVs were isolated from conditioned supernatants 24 h after pulsation with mock, IFN-α (500 IU/ml), or IFN-γ (25 ng/ml) for 1 h, respectively. Purified EVs containing 100 µg of protein were transferred to Huh-7.5 hepatoma cells harboring subgenomic hepatitis C virus (HCV) replicons. HCV mRNA levels were quantified by Taqman real-time PCR (RT-PCR) after 96 h. Mean and SEM of three biological replicates are shown. (B) Huh-7.5 hepatoma cells harboring subgenomic HCV replicons were exposed to mock (black bar), ARA (5 µg/ml, gray bar), or EPA (5 µg/ml, white bar) for 24 and 48 h, and HCV mRNA levels were quantified by Taqman RT-PCR. Mean and SEM of three biological replicates are shown. (C) Western blot analysis of p-STAT1 and p-STAT2 in cell lysates of THP-1 cells were treated with mock (EtOH), ARA (5 µg/ml), or EPA (5 µg/ml) for 48 h before stimulation with IFN-α (500 IU/ml) for 120 min (* P
    Figure Legend Snippet: Polyunsaturated fatty acids modulate extracellular vesicle (EV)-mediated innate antiviral immunity. (A) THP-1 macrophages were treated pretreated with mock (black bar), arachidonic acid (ARA) (gray bar), and eicosapentaenoic acid (EPA) (white bar) for 24 h (left) and 48 h (right), respectively. Then, EVs were isolated from conditioned supernatants 24 h after pulsation with mock, IFN-α (500 IU/ml), or IFN-γ (25 ng/ml) for 1 h, respectively. Purified EVs containing 100 µg of protein were transferred to Huh-7.5 hepatoma cells harboring subgenomic hepatitis C virus (HCV) replicons. HCV mRNA levels were quantified by Taqman real-time PCR (RT-PCR) after 96 h. Mean and SEM of three biological replicates are shown. (B) Huh-7.5 hepatoma cells harboring subgenomic HCV replicons were exposed to mock (black bar), ARA (5 µg/ml, gray bar), or EPA (5 µg/ml, white bar) for 24 and 48 h, and HCV mRNA levels were quantified by Taqman RT-PCR. Mean and SEM of three biological replicates are shown. (C) Western blot analysis of p-STAT1 and p-STAT2 in cell lysates of THP-1 cells were treated with mock (EtOH), ARA (5 µg/ml), or EPA (5 µg/ml) for 48 h before stimulation with IFN-α (500 IU/ml) for 120 min (* P

    Techniques Used: Acetylene Reduction Assay, Isolation, Purification, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

    33) Product Images from "Sex-dependent effects of in utero cannabinoid exposure on cortical function"

    Article Title: Sex-dependent effects of in utero cannabinoid exposure on cortical function

    Journal: eLife

    doi: 10.7554/eLife.36234

    Prenatal cannabinoid treatment affects expression of endocannabinoid system components in a sex-dependent fashion. CB1, TRPV1, mGlu5, and DAGLα mRNA levels in medial prefrontal cortex at PND90 were determined by real-time PCR in male ( A, C, E, and G ) and female ( B, D, F, and H ) rats using Taqman probes and a QuantStudio7 thermocycler. Treatment of dams with WIN from GD5 to GD20 decreased TRPV1, mGlu5, and DAGLα mRNA in female offspring. However, the same treatment decreased only mGlu5 in male offspring. *p
    Figure Legend Snippet: Prenatal cannabinoid treatment affects expression of endocannabinoid system components in a sex-dependent fashion. CB1, TRPV1, mGlu5, and DAGLα mRNA levels in medial prefrontal cortex at PND90 were determined by real-time PCR in male ( A, C, E, and G ) and female ( B, D, F, and H ) rats using Taqman probes and a QuantStudio7 thermocycler. Treatment of dams with WIN from GD5 to GD20 decreased TRPV1, mGlu5, and DAGLα mRNA in female offspring. However, the same treatment decreased only mGlu5 in male offspring. *p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    34) Product Images from "Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD"

    Article Title: Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.08.005

    Analysis of miR-483 in DMD Patient Serum (A) Alignment of mouse and human miR-483 precursors. Differences are highlighted in yellow. (B) miRNA signature plots for mouse and human miR-483. Pooled mouse muscle library data are shown in red, and publically available miRBase data are shown in blue. Annotated and empirically derived miRNA seed regions are indicated. Serum from DMD patients (n = 28) and healthy controls (n = 16) were analyzed by sRNA TaqMan qRT-PCR for (C) miR-483-5p and miR-483-3p, and (D) the myomiRs: miR-1a-3p, miR-133a-3p, and miR-206-3p. Individual data points are shown and the mean ± SEM indicated. **p
    Figure Legend Snippet: Analysis of miR-483 in DMD Patient Serum (A) Alignment of mouse and human miR-483 precursors. Differences are highlighted in yellow. (B) miRNA signature plots for mouse and human miR-483. Pooled mouse muscle library data are shown in red, and publically available miRBase data are shown in blue. Annotated and empirically derived miRNA seed regions are indicated. Serum from DMD patients (n = 28) and healthy controls (n = 16) were analyzed by sRNA TaqMan qRT-PCR for (C) miR-483-5p and miR-483-3p, and (D) the myomiRs: miR-1a-3p, miR-133a-3p, and miR-206-3p. Individual data points are shown and the mean ± SEM indicated. **p

    Techniques Used: Derivative Assay, Quantitative RT-PCR

    sRNA Analysis in Dystrophic Muscle ). (B) Size distribution of sRNA reads after adaptor trimming in each set of muscle libraries. Differential expression of miRNAs in mdx ). Statistically significant changes are highlighted in red and blue (for elevated and reduced levels in mdx serum, respectively). Labels are shown for miRNAs of interest. (E) Venn diagram showing overlap between differentially expressed miRNAs in dystrophic muscles. miRNAs that were commonly differentially expressed in all four muscle types are indicated. (F) Expression of miR-483-3p was validated by sRNA TaqMan qRT-PCR in each muscle using miR-16-5p as a control for normalization. Values are mean + SEM; n = 3. *p
    Figure Legend Snippet: sRNA Analysis in Dystrophic Muscle ). (B) Size distribution of sRNA reads after adaptor trimming in each set of muscle libraries. Differential expression of miRNAs in mdx ). Statistically significant changes are highlighted in red and blue (for elevated and reduced levels in mdx serum, respectively). Labels are shown for miRNAs of interest. (E) Venn diagram showing overlap between differentially expressed miRNAs in dystrophic muscles. miRNAs that were commonly differentially expressed in all four muscle types are indicated. (F) Expression of miR-483-3p was validated by sRNA TaqMan qRT-PCR in each muscle using miR-16-5p as a control for normalization. Values are mean + SEM; n = 3. *p

    Techniques Used: Expressing, Quantitative RT-PCR

    35) Product Images from "Sucrose non-fermenting related kinase enzyme is essential for cardiac metabolism"

    Article Title: Sucrose non-fermenting related kinase enzyme is essential for cardiac metabolism

    Journal: Biology Open

    doi: 10.1242/bio.20149811

    Gene ontology data and microarray validation of metabolism genes. (A) Gene ontology (GO) terms for biological processes that over represented in SNRK WT and SNRK KO microarray data sets. Fold change analysis obtained by TaqMan qPCR analysis from mRNA isolated from E17.5 (B) and Neonate (C) SNRK WT and KO hearts (n = 3 for each genotype) and (D) SNRK knockdown in hESC-derived CMs infected with empty vector shRNA control (Control) lentivirus and SNRK shRNA lentivirus (shSNRK) (n = 3 from three independent cardiomyocyte infections). The results are the mean of the fold change ± SEM. * p-value
    Figure Legend Snippet: Gene ontology data and microarray validation of metabolism genes. (A) Gene ontology (GO) terms for biological processes that over represented in SNRK WT and SNRK KO microarray data sets. Fold change analysis obtained by TaqMan qPCR analysis from mRNA isolated from E17.5 (B) and Neonate (C) SNRK WT and KO hearts (n = 3 for each genotype) and (D) SNRK knockdown in hESC-derived CMs infected with empty vector shRNA control (Control) lentivirus and SNRK shRNA lentivirus (shSNRK) (n = 3 from three independent cardiomyocyte infections). The results are the mean of the fold change ± SEM. * p-value

    Techniques Used: Microarray, Real-time Polymerase Chain Reaction, Isolation, Derivative Assay, Infection, Plasmid Preparation, shRNA

    36) Product Images from "MiRNA-615-5p Functions as a Tumor Suppressor in Pancreatic Ductal Adenocarcinoma by Targeting AKT2"

    Article Title: MiRNA-615-5p Functions as a Tumor Suppressor in Pancreatic Ductal Adenocarcinoma by Targeting AKT2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119783

    MiR-615 overexpression inhibits tumor growth in vivo . MIA PaCa-2 cells transduced with lentivirus pLV-miR-615 (LV-miR-615-MIA) or pLV-miR-mock (LV-control-MIA) (6 × 10 6 cells) were subcutaneously inoculated into the dorsal flanks of BALB/c nude mice. (A) Relative miR-615-5p expression was determined by TaqMan microRNA qRT-PCR assay. (B) Tumor volume (cm 3 ) was assessed by calipers every week. (C) Tumor weights (g) measured at the end of the experiment were significantly reduced in the LV-miR-615-5p-MIA group. (D) The extent of apoptosis was determined in tumor tissue by TUNEL staining (scale bar: 600um). (E) Determination of Ki-67 in tumor tissues by immunofluorescence (Magnification:×100). Red: Rhodamine; Blue: DAPI. The numbers of apoptotic cells (D) and the percentages of Ki-67-positive cells (E) were determined from 10 randomly selected fields in a single sample. Two independent reviewers were blinded to the grouping when measuring the numbers of apoptotic cells and the percentages of Ki-67-positive cells. The data are shown as mean±standard deviation (SD) (n = 8 in LV-control-MIA group; n = 6 in LV-miR-615-MIA group). * P
    Figure Legend Snippet: MiR-615 overexpression inhibits tumor growth in vivo . MIA PaCa-2 cells transduced with lentivirus pLV-miR-615 (LV-miR-615-MIA) or pLV-miR-mock (LV-control-MIA) (6 × 10 6 cells) were subcutaneously inoculated into the dorsal flanks of BALB/c nude mice. (A) Relative miR-615-5p expression was determined by TaqMan microRNA qRT-PCR assay. (B) Tumor volume (cm 3 ) was assessed by calipers every week. (C) Tumor weights (g) measured at the end of the experiment were significantly reduced in the LV-miR-615-5p-MIA group. (D) The extent of apoptosis was determined in tumor tissue by TUNEL staining (scale bar: 600um). (E) Determination of Ki-67 in tumor tissues by immunofluorescence (Magnification:×100). Red: Rhodamine; Blue: DAPI. The numbers of apoptotic cells (D) and the percentages of Ki-67-positive cells (E) were determined from 10 randomly selected fields in a single sample. Two independent reviewers were blinded to the grouping when measuring the numbers of apoptotic cells and the percentages of Ki-67-positive cells. The data are shown as mean±standard deviation (SD) (n = 8 in LV-control-MIA group; n = 6 in LV-miR-615-MIA group). * P

    Techniques Used: Over Expression, In Vivo, Transduction, Mouse Assay, Expressing, Quantitative RT-PCR, TUNEL Assay, Staining, Immunofluorescence, Standard Deviation

    37) Product Images from "Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis"

    Article Title: Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070714

    Fixation and storage introduces major gene-to-gene variations in qRT-PCR. Aliquots of a human liver sample were cryopreserved or fixed in formalin and paraffin embedded. (A) RNA was extracted from samples at different timepoints including technical replicates. (B) Comparison of qRT-PCR data for 92 genes from cryopreserved and FFPE human liver samples reveals an average difference of the ct values ranging from 4 cycles (6 months) to 8 cycles (1 year) increasing with storage time at room temperature. Extraction from the FFPE sample which had been stored for one year was done in duplicates. cDNA generation was performed in duplicates from the same RNA, and qRT-PCR was performed twice from the same cDNA. Data was generated with the TaqMan “Human Molecular Mechanisms of Cancer” assay, individual ct values are shown.
    Figure Legend Snippet: Fixation and storage introduces major gene-to-gene variations in qRT-PCR. Aliquots of a human liver sample were cryopreserved or fixed in formalin and paraffin embedded. (A) RNA was extracted from samples at different timepoints including technical replicates. (B) Comparison of qRT-PCR data for 92 genes from cryopreserved and FFPE human liver samples reveals an average difference of the ct values ranging from 4 cycles (6 months) to 8 cycles (1 year) increasing with storage time at room temperature. Extraction from the FFPE sample which had been stored for one year was done in duplicates. cDNA generation was performed in duplicates from the same RNA, and qRT-PCR was performed twice from the same cDNA. Data was generated with the TaqMan “Human Molecular Mechanisms of Cancer” assay, individual ct values are shown.

    Techniques Used: Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded, Generated

    38) Product Images from "Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators"

    Article Title: Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08150-5

    E7107 induces BCL2A1-dependent apoptosis in melanoma cell lines. a RT quantitative PCR (RT-qPCR) analysis of total mRNA levels (pan), exonic mRNA levels covering the junction of exons (exon), and pre-mRNA levels detecting the intron (intron) of BCL2A1 . HT144 cells were pre-treated with 100 µg ml −1 cycloheximide (CHX) for 1 h, followed by addition of E7107 as indicated for 12 h to inhibit nonsense-mediated mRNA decay (NMD) before collecting RNA samples. Schematic representations of TaqMan primers and probes were shown. Boxes: exons; gray lines: introns; arrows: primers; and black lines: probes. b Box plots showing the distribution of E7107 Emax in the solid tumor cell lines separated by tissue/lineage: melanoma ( n = 24), breast ( n = 33), colon ( n = 27), kidney ( n = 10), lung ( n = 91), and pancreas ( n = 24). The box plots exhibit five number summary from bottom to top: minimum, first quartile, median, third quartile, and maximum/outliers. c Heatmap of BCL2A1 mRNA expression (based on CCLE RNA-seq, sorted from high to low in the solid tumor cell lines separated by tissue/lineage) and E7107 Emax. d RT-qPCR analysis of BCL2A1 mRNA levels (exon1/2 junction) in vector control (Vector) or stable BCL2A1 cDNA-expressing (BCL2A1) melanoma cell lines. Data represent means ± SD of biological triplicates. e Growth curves measured by CellTiter-Glo (CTG) and f apoptosis induction curves measured by IncuCyte Caspase-3/7 Green Apoptosis Assay in melanoma cell lines stably transduced with vector control (empty vector) or BCL2A1 cDNA (BCL2A1 cDNA) treated with E7107 for 72 h. Data represent means ± SD of biological triplicates
    Figure Legend Snippet: E7107 induces BCL2A1-dependent apoptosis in melanoma cell lines. a RT quantitative PCR (RT-qPCR) analysis of total mRNA levels (pan), exonic mRNA levels covering the junction of exons (exon), and pre-mRNA levels detecting the intron (intron) of BCL2A1 . HT144 cells were pre-treated with 100 µg ml −1 cycloheximide (CHX) for 1 h, followed by addition of E7107 as indicated for 12 h to inhibit nonsense-mediated mRNA decay (NMD) before collecting RNA samples. Schematic representations of TaqMan primers and probes were shown. Boxes: exons; gray lines: introns; arrows: primers; and black lines: probes. b Box plots showing the distribution of E7107 Emax in the solid tumor cell lines separated by tissue/lineage: melanoma ( n = 24), breast ( n = 33), colon ( n = 27), kidney ( n = 10), lung ( n = 91), and pancreas ( n = 24). The box plots exhibit five number summary from bottom to top: minimum, first quartile, median, third quartile, and maximum/outliers. c Heatmap of BCL2A1 mRNA expression (based on CCLE RNA-seq, sorted from high to low in the solid tumor cell lines separated by tissue/lineage) and E7107 Emax. d RT-qPCR analysis of BCL2A1 mRNA levels (exon1/2 junction) in vector control (Vector) or stable BCL2A1 cDNA-expressing (BCL2A1) melanoma cell lines. Data represent means ± SD of biological triplicates. e Growth curves measured by CellTiter-Glo (CTG) and f apoptosis induction curves measured by IncuCyte Caspase-3/7 Green Apoptosis Assay in melanoma cell lines stably transduced with vector control (empty vector) or BCL2A1 cDNA (BCL2A1 cDNA) treated with E7107 for 72 h. Data represent means ± SD of biological triplicates

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, RNA Sequencing Assay, Plasmid Preparation, CTG Assay, Apoptosis Assay, Stable Transfection, Transduction

    39) Product Images from "A variably imprinted epiallele impacts seed development"

    Article Title: A variably imprinted epiallele impacts seed development

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007469

    HDG3 is imprinted in Cvi HDG3 IR lines. (A) HDG3 in situ hybridization for indicated genotypes. Arrowheads indicate regions of in situ signal. Right panels shows magnification of chalazal region. Scale bars, 50 μm. (B) RT-qPCR of relative HDG3 transcript abundance in F 1 endosperm at 6–7 DAP. Dashed line separates experiments done at different times. Left, avg of 3 technical replicates. Right, avg of biological duplicates. Bars show upper and lower range. (C) % of HDG3 from Cvi allele in endosperm by TaqMan RT-qPCR assay.
    Figure Legend Snippet: HDG3 is imprinted in Cvi HDG3 IR lines. (A) HDG3 in situ hybridization for indicated genotypes. Arrowheads indicate regions of in situ signal. Right panels shows magnification of chalazal region. Scale bars, 50 μm. (B) RT-qPCR of relative HDG3 transcript abundance in F 1 endosperm at 6–7 DAP. Dashed line separates experiments done at different times. Left, avg of 3 technical replicates. Right, avg of biological duplicates. Bars show upper and lower range. (C) % of HDG3 from Cvi allele in endosperm by TaqMan RT-qPCR assay.

    Techniques Used: In Situ Hybridization, In Situ, Quantitative RT-PCR

    40) Product Images from "Functional evidence implicating chromosome 7q22 haploinsufficiency in myelodysplastic syndrome pathogenesis"

    Article Title: Functional evidence implicating chromosome 7q22 haploinsufficiency in myelodysplastic syndrome pathogenesis

    Journal: eLife

    doi: 10.7554/eLife.07839

    Changes in gene expression and oxidative phosphorylation in 5A3 +/del HSC and MP. ( A ) Relative mRNA abundances for genes within the deleted 5A3 interval expressed at detectable levels in sorted HSC populations were determined by TaqMan reverse transcriptase PCR (n = 3 per genotype). ( B ) Expression levels of genes located within and flanking the deleted interval measured by RNA-Seq in sorted CD150 hi HSC and CD150 lo HSC from 5 mice of each genotype. Each column presents data from an individual mouse, and genes within the 5A3 deleted region are delimited with a black box. Three non-coding RNAs (6030443J06Rik, AC112688.1 and 5031425E22Rik) are located within the 5A3 deletion. Two of these (6030443J06Rik and AC112688.1) are expressed at extremely low levels in HSC, and the other (5031425E22Rik) showed ∼50% lower expression in 5A3 +/del HSC. 5031425E22Rik is homologous to the human KMT2E (a.k.a. MLL5 ) antisense RNA1. Expression levels of the flanking Fbxl13 and Srpk2 genes are modestly up-regulated in 5A3 +/del HSC, which is consistent with the targeting strategy used to generate the segmental deletion. ( C ) Gene Set Enrichment Analysis of 5A3 +/del CD150 hi HSCs revealed negative enrichment for genes associated with oxidative phosphorylation (OXPHOS). False discovery rate (FDR) q-val, nominal p-value (NOM p-value), and normalized enrichment scores (NESs) are indicated. ( D ) ATP levels in HSC and MPP from 8- to 12-week-old WT (n = 6) and 5A3 +/del (n = 5) mice. Data shown are mean values ±SEM of results from two independent experiments. ( E ) Fold change in the mean MitoTracker Orange fluorescence levels in 5A3 +/del cells normalized to values in WT cells analyzed in the same experiment. ( F ) Fold change in the mean fluorescence level (MFI) of 5A3 +/del cells that are CellROX Orange positive normalized to values in WT cells analyzed in the same experiment. For the MitoTracker and CellROX experiments, n = 13 for WT and n = 12 for 5A3 +/del young mice, three independent experiments; n = 5 for WT and n = 6 for 5A3 +/del aged mice, two independent experiments. Data shown are mean values ±SEM of results from independent experiments. ( G and H ) Oxygen consumption rate (OCR) was assessed basally and in response to the mitochondrial inhibitors oligomycin (oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A and rotenone (A/R) for ( G ) KLS and ( H ) MP cells. Data are shown as mean ±SEM of n = 5 mice of each genotype from two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.07839.008
    Figure Legend Snippet: Changes in gene expression and oxidative phosphorylation in 5A3 +/del HSC and MP. ( A ) Relative mRNA abundances for genes within the deleted 5A3 interval expressed at detectable levels in sorted HSC populations were determined by TaqMan reverse transcriptase PCR (n = 3 per genotype). ( B ) Expression levels of genes located within and flanking the deleted interval measured by RNA-Seq in sorted CD150 hi HSC and CD150 lo HSC from 5 mice of each genotype. Each column presents data from an individual mouse, and genes within the 5A3 deleted region are delimited with a black box. Three non-coding RNAs (6030443J06Rik, AC112688.1 and 5031425E22Rik) are located within the 5A3 deletion. Two of these (6030443J06Rik and AC112688.1) are expressed at extremely low levels in HSC, and the other (5031425E22Rik) showed ∼50% lower expression in 5A3 +/del HSC. 5031425E22Rik is homologous to the human KMT2E (a.k.a. MLL5 ) antisense RNA1. Expression levels of the flanking Fbxl13 and Srpk2 genes are modestly up-regulated in 5A3 +/del HSC, which is consistent with the targeting strategy used to generate the segmental deletion. ( C ) Gene Set Enrichment Analysis of 5A3 +/del CD150 hi HSCs revealed negative enrichment for genes associated with oxidative phosphorylation (OXPHOS). False discovery rate (FDR) q-val, nominal p-value (NOM p-value), and normalized enrichment scores (NESs) are indicated. ( D ) ATP levels in HSC and MPP from 8- to 12-week-old WT (n = 6) and 5A3 +/del (n = 5) mice. Data shown are mean values ±SEM of results from two independent experiments. ( E ) Fold change in the mean MitoTracker Orange fluorescence levels in 5A3 +/del cells normalized to values in WT cells analyzed in the same experiment. ( F ) Fold change in the mean fluorescence level (MFI) of 5A3 +/del cells that are CellROX Orange positive normalized to values in WT cells analyzed in the same experiment. For the MitoTracker and CellROX experiments, n = 13 for WT and n = 12 for 5A3 +/del young mice, three independent experiments; n = 5 for WT and n = 6 for 5A3 +/del aged mice, two independent experiments. Data shown are mean values ±SEM of results from independent experiments. ( G and H ) Oxygen consumption rate (OCR) was assessed basally and in response to the mitochondrial inhibitors oligomycin (oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A and rotenone (A/R) for ( G ) KLS and ( H ) MP cells. Data are shown as mean ±SEM of n = 5 mice of each genotype from two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.07839.008

    Techniques Used: Expressing, Polymerase Chain Reaction, RNA Sequencing Assay, Mouse Assay, Fluorescence

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    Expressing:

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  • 99
    Thermo Fisher gene exp lcn2 mm01324470 m1
    <t>LCN2</t> protects against UTI and causes loss of fitness in mutants of UPEC unable to use salmochelin, aerobactin, or yersiniabactin. Infection of C57BL/6 wild-type and Lcn2- deficient mice with nonpathogenic and LCN2-sensitive E. coli H9049 and competition
    Gene Exp Lcn2 Mm01324470 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a <t>TaqMan</t> probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. Gapdh ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the <t>SN1</t> transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.
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    Thermo Fisher taqman gene expression cells to ct kit
    Dose-dependent viral RNA amplification signal curve. Vero cells (2 × 10 4 /well) were cultured in a 96-well plate for 24 h in the absence of compounds. After incubation, the cells were infected with the indicated amount of STFSV and further incubated. After three days, the amount of intracellular viral RNA was determined by the real-time <t>RT-PCR</t> method using <t>TaqMan</t> Gene Expression Cells-to-CT™ Kit. The experiment was carried out in triplicate and means ± SD are shown.
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    LCN2 protects against UTI and causes loss of fitness in mutants of UPEC unable to use salmochelin, aerobactin, or yersiniabactin. Infection of C57BL/6 wild-type and Lcn2- deficient mice with nonpathogenic and LCN2-sensitive E. coli H9049 and competition

    Journal: The Journal of Immunology Author Choice

    Article Title: Lipocalin 2 Imparts Selective Pressure on Bacterial Growth in the Bladder and Is Elevated in Women with Urinary Tract Infection

    doi: 10.4049/jimmunol.1401528

    Figure Lengend Snippet: LCN2 protects against UTI and causes loss of fitness in mutants of UPEC unable to use salmochelin, aerobactin, or yersiniabactin. Infection of C57BL/6 wild-type and Lcn2- deficient mice with nonpathogenic and LCN2-sensitive E. coli H9049 and competition

    Article Snippet: After cDNA synthesis (high-capacity reverse transcription kit, Applied Biosystems), quantitative real-time PCR was performed on a StepOnePlus PCR system using TaqMan fast advanced master mix and TaqMan gene expression assays (Applied Biosystems: LCN2 [Mm01324470_m1], LF [Mm00434787_m1], CXCL1 [Mm00433859_m1], CCL2 [Mm00441242_m1], IL-1β [Mm01336189_m1], IL-6 [Mm00446190_m1], TNF [Mm00443258_m1], IL-10 [Mm00439614_m1], IL-17a [Mm00439618_m1], and IL-22 (Mm01226722_g1]).

    Techniques: Infection, Mouse Assay

    LCN2 is expressed in epithelial cells and granulocytes of infected bladders. C57BL/6 wild-type mice were inoculated transurethrally with UPEC strain CFT073 for 1 d before bladders, ureters, and kidneys were analyzed. ( A ) H E and LCN2 immunohistological

    Journal: The Journal of Immunology Author Choice

    Article Title: Lipocalin 2 Imparts Selective Pressure on Bacterial Growth in the Bladder and Is Elevated in Women with Urinary Tract Infection

    doi: 10.4049/jimmunol.1401528

    Figure Lengend Snippet: LCN2 is expressed in epithelial cells and granulocytes of infected bladders. C57BL/6 wild-type mice were inoculated transurethrally with UPEC strain CFT073 for 1 d before bladders, ureters, and kidneys were analyzed. ( A ) H E and LCN2 immunohistological

    Article Snippet: After cDNA synthesis (high-capacity reverse transcription kit, Applied Biosystems), quantitative real-time PCR was performed on a StepOnePlus PCR system using TaqMan fast advanced master mix and TaqMan gene expression assays (Applied Biosystems: LCN2 [Mm01324470_m1], LF [Mm00434787_m1], CXCL1 [Mm00433859_m1], CCL2 [Mm00441242_m1], IL-1β [Mm01336189_m1], IL-6 [Mm00446190_m1], TNF [Mm00443258_m1], IL-10 [Mm00439614_m1], IL-17a [Mm00439618_m1], and IL-22 (Mm01226722_g1]).

    Techniques: Infection, Mouse Assay

    Expression of LCN2 evasive siderophores is associated with higher E. coli CFUs during cystitis episodes

    Journal: The Journal of Immunology Author Choice

    Article Title: Lipocalin 2 Imparts Selective Pressure on Bacterial Growth in the Bladder and Is Elevated in Women with Urinary Tract Infection

    doi: 10.4049/jimmunol.1401528

    Figure Lengend Snippet: Expression of LCN2 evasive siderophores is associated with higher E. coli CFUs during cystitis episodes

    Article Snippet: After cDNA synthesis (high-capacity reverse transcription kit, Applied Biosystems), quantitative real-time PCR was performed on a StepOnePlus PCR system using TaqMan fast advanced master mix and TaqMan gene expression assays (Applied Biosystems: LCN2 [Mm01324470_m1], LF [Mm00434787_m1], CXCL1 [Mm00433859_m1], CCL2 [Mm00441242_m1], IL-1β [Mm01336189_m1], IL-6 [Mm00446190_m1], TNF [Mm00443258_m1], IL-10 [Mm00439614_m1], IL-17a [Mm00439618_m1], and IL-22 (Mm01226722_g1]).

    Techniques: Expressing

    Onset of UTI is preceded by a rapid increase in urine LCN2 levels . Pairwise comparison is shown of LCN2 protein levels in urine samples from 14 patients taken 3 d prior to onset of UTI and at cystitis episodes (enrollment UTI or recurrent UTI during

    Journal: The Journal of Immunology Author Choice

    Article Title: Lipocalin 2 Imparts Selective Pressure on Bacterial Growth in the Bladder and Is Elevated in Women with Urinary Tract Infection

    doi: 10.4049/jimmunol.1401528

    Figure Lengend Snippet: Onset of UTI is preceded by a rapid increase in urine LCN2 levels . Pairwise comparison is shown of LCN2 protein levels in urine samples from 14 patients taken 3 d prior to onset of UTI and at cystitis episodes (enrollment UTI or recurrent UTI during

    Article Snippet: After cDNA synthesis (high-capacity reverse transcription kit, Applied Biosystems), quantitative real-time PCR was performed on a StepOnePlus PCR system using TaqMan fast advanced master mix and TaqMan gene expression assays (Applied Biosystems: LCN2 [Mm01324470_m1], LF [Mm00434787_m1], CXCL1 [Mm00433859_m1], CCL2 [Mm00441242_m1], IL-1β [Mm01336189_m1], IL-6 [Mm00446190_m1], TNF [Mm00443258_m1], IL-10 [Mm00439614_m1], IL-17a [Mm00439618_m1], and IL-22 (Mm01226722_g1]).

    Techniques:

    LCN2 is expressed in the urinary tract during UTI. C57BL/6 wild-type and Lcn2- deficient mice were transurethrally inoculated with UPEC strain CFT073 for the indicated period of time before bladders and kidneys were harvested and analyzed for transcript

    Journal: The Journal of Immunology Author Choice

    Article Title: Lipocalin 2 Imparts Selective Pressure on Bacterial Growth in the Bladder and Is Elevated in Women with Urinary Tract Infection

    doi: 10.4049/jimmunol.1401528

    Figure Lengend Snippet: LCN2 is expressed in the urinary tract during UTI. C57BL/6 wild-type and Lcn2- deficient mice were transurethrally inoculated with UPEC strain CFT073 for the indicated period of time before bladders and kidneys were harvested and analyzed for transcript

    Article Snippet: After cDNA synthesis (high-capacity reverse transcription kit, Applied Biosystems), quantitative real-time PCR was performed on a StepOnePlus PCR system using TaqMan fast advanced master mix and TaqMan gene expression assays (Applied Biosystems: LCN2 [Mm01324470_m1], LF [Mm00434787_m1], CXCL1 [Mm00433859_m1], CCL2 [Mm00441242_m1], IL-1β [Mm01336189_m1], IL-6 [Mm00446190_m1], TNF [Mm00443258_m1], IL-10 [Mm00439614_m1], IL-17a [Mm00439618_m1], and IL-22 (Mm01226722_g1]).

    Techniques: Mouse Assay

    LCN2 levels rise during cystitis episodes

    Journal: The Journal of Immunology Author Choice

    Article Title: Lipocalin 2 Imparts Selective Pressure on Bacterial Growth in the Bladder and Is Elevated in Women with Urinary Tract Infection

    doi: 10.4049/jimmunol.1401528

    Figure Lengend Snippet: LCN2 levels rise during cystitis episodes

    Article Snippet: After cDNA synthesis (high-capacity reverse transcription kit, Applied Biosystems), quantitative real-time PCR was performed on a StepOnePlus PCR system using TaqMan fast advanced master mix and TaqMan gene expression assays (Applied Biosystems: LCN2 [Mm01324470_m1], LF [Mm00434787_m1], CXCL1 [Mm00433859_m1], CCL2 [Mm00441242_m1], IL-1β [Mm01336189_m1], IL-6 [Mm00446190_m1], TNF [Mm00443258_m1], IL-10 [Mm00439614_m1], IL-17a [Mm00439618_m1], and IL-22 (Mm01226722_g1]).

    Techniques:

    Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a TaqMan probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. Gapdh ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the SN1 transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Intact piRNA pathway prevents L1 mobilization in male meiosis

    doi: 10.1073/pnas.1701069114

    Figure Lengend Snippet: Characterization of the 5′UTR-ORFeus transgene. ( A ) The donor 5′UTR-ORFeus transgene is driven by an endogenous mouse L1 5′UTR promoter. The protein-coding domains ORF1 and ORF2 are derived from the codon-optimized mouse L1 ORFeus-Mm. An introndisrupted EGFP cassette is embedded in the antisense orientation relative to L1 transcription in the 3′UTR of the transgene. The EGFP cassette is flanked by the CMV immediate early promoter and the HSV thymidine kinase polyadenylation signal (both on antisense strand; not depicted). The donor transgene is terminated by the SV40 late polyadenylation signal (not depicted). Methylation status of the transgene promoter is interrogated by a bisulfite primer pair targeting the 5′UTR/ORF1 junction (shown as two converging arrowheads). RNA expression of the transgene is examined in situ by RNAscope with probes specific to the codon-optimized ORF1 sequence (shown as the letter ZZ). Retrotransposition is quantified by ddPCR with a TaqMan probe spanning the splicing junction of EGFP (shown as a starred bar). As depicted, the majority of insertions are 5′ truncated. ( B ) Location of primers for bisulfite analysis of endogenous mouse L1 promoters. The same forward primer is used for donor transgene but the reverse primer is located in the tether region between 5′UTR monomers and ORF1. Note that this primer pair is expected to amplify both endogenous and transgenic 5′UTRs. However, the signal from the transgene is negligible because of the overwhelming abundance of endogenous sequences. ( C ), against the human γ-globin intron, which is contained in the EGFP-based retrotransposition indicator cassette and shared by all transgenic lines. Gapdh ). Genomic DNA spiked with a dilution series of plasmid pRSVGFPuvINT was used to measure qPCR dynamic range and plotted on the x axis. A power regression trendline is shown for the plasmid spike-in series (power). ( D ) Genomic location of the SN1 transgene. The transgene is positioned in the middle of the 213 kb intron 1 of Tnr . The 3′ flanking genomic sequence is shown.

    Article Snippet: The SN1 transgene expression was quantified using TaqMan Gene Expression Master Mix (Thermo Fisher) and SN1 and Gapdh probes in duplex.

    Techniques: Derivative Assay, Methylation, RNA Expression, In Situ, Sequencing, Transgenic Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Dose-dependent viral RNA amplification signal curve. Vero cells (2 × 10 4 /well) were cultured in a 96-well plate for 24 h in the absence of compounds. After incubation, the cells were infected with the indicated amount of STFSV and further incubated. After three days, the amount of intracellular viral RNA was determined by the real-time RT-PCR method using TaqMan Gene Expression Cells-to-CT™ Kit. The experiment was carried out in triplicate and means ± SD are shown.

    Journal: Antiviral Chemistry & Chemotherapy

    Article Title: Establishment of an antiviral assay system and identification of severe fever with thrombocytopenia syndrome virus inhibitors

    doi: 10.1177/2040206617740303

    Figure Lengend Snippet: Dose-dependent viral RNA amplification signal curve. Vero cells (2 × 10 4 /well) were cultured in a 96-well plate for 24 h in the absence of compounds. After incubation, the cells were infected with the indicated amount of STFSV and further incubated. After three days, the amount of intracellular viral RNA was determined by the real-time RT-PCR method using TaqMan Gene Expression Cells-to-CT™ Kit. The experiment was carried out in triplicate and means ± SD are shown.

    Article Snippet: The infected cells exposed to various concentrations of test compounds can be treated with lysis buffer after washing with PBS and subjected to real-time RT-PCR without extracting RNA, when TaqMan Gene Expression Cells-to-CT™ Kit is used for the assay.

    Techniques: Amplification, Cell Culture, Incubation, Infection, Quantitative RT-PCR, Expressing