real time pcr qpcr total rna  (Thermo Fisher)


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    Name:
    Total RNA Control Human
    Description:
    Human Total RNA is provided at a convenient reverse transcription ready concentration for all RT PCR reactions TaqMan Control Total RNA is from Raji cells Burkitt s lymphoma and is conveniently packaged in a PCR ready concentration for use as a control template in RT PCR reactions The Human total RNA is provided at a concentration of 50 ng µl
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    4307281
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    PCR & Real-Time PCR|Real Time PCR (qPCR)|Real-Time PCR Primers, Probes, Arrays & Controls
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    Thermo Fisher real time pcr qpcr total rna
    Overexpression of 14-3-3ε enhances HP-PRRSV infection. A Western blot analysis of 14-3-3β/ε overexpression cells. B Cell growth kinetics. Normal Marc-145 cells and Marc-145 wpxld , Marc-145 14-3-3β , and Marc-145 14-3-3ε cells were plated in 24-well plates. At 24, 48, 72, 96, 120, 144, and 168 h post-plating, the CCK-8 reagent was added to the cells. The cells were then incubated for 2 h at 37 °C. Cell counting was performed by measuring absorbance at 450 nm. Marc-145 14-3-3β , Marc-145 14-3-3ε , and Marc-145 wpxld cells were infected with TA-12 and CH-1R, respectively. The infected cells were harvested at 0, 6, 12, 24, 36, and 48 hpi for <t>RNA</t> extraction and at 12, 24, 36, and 48 hpi for protein extraction. Viral loads were evaluated by absolute <t>qPCR</t> targeting the N gene of TA-12 ( C ) and CH-1R ( D ). Cellular proteins were analyzed by Western blot analysis for detecting the viral N protein of TA-12 ( E ) and CH-1R ( F ). The GAPDH protein or GAPDH gene was used as the internal control. * P value
    Human Total RNA is provided at a convenient reverse transcription ready concentration for all RT PCR reactions TaqMan Control Total RNA is from Raji cells Burkitt s lymphoma and is conveniently packaged in a PCR ready concentration for use as a control template in RT PCR reactions The Human total RNA is provided at a concentration of 50 ng µl
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    real time pcr qpcr total rna - by Bioz Stars, 2020-08
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    1) Product Images from "14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection"

    Article Title: 14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection

    Journal: Veterinary Research

    doi: 10.1186/s13567-019-0636-0

    Overexpression of 14-3-3ε enhances HP-PRRSV infection. A Western blot analysis of 14-3-3β/ε overexpression cells. B Cell growth kinetics. Normal Marc-145 cells and Marc-145 wpxld , Marc-145 14-3-3β , and Marc-145 14-3-3ε cells were plated in 24-well plates. At 24, 48, 72, 96, 120, 144, and 168 h post-plating, the CCK-8 reagent was added to the cells. The cells were then incubated for 2 h at 37 °C. Cell counting was performed by measuring absorbance at 450 nm. Marc-145 14-3-3β , Marc-145 14-3-3ε , and Marc-145 wpxld cells were infected with TA-12 and CH-1R, respectively. The infected cells were harvested at 0, 6, 12, 24, 36, and 48 hpi for RNA extraction and at 12, 24, 36, and 48 hpi for protein extraction. Viral loads were evaluated by absolute qPCR targeting the N gene of TA-12 ( C ) and CH-1R ( D ). Cellular proteins were analyzed by Western blot analysis for detecting the viral N protein of TA-12 ( E ) and CH-1R ( F ). The GAPDH protein or GAPDH gene was used as the internal control. * P value
    Figure Legend Snippet: Overexpression of 14-3-3ε enhances HP-PRRSV infection. A Western blot analysis of 14-3-3β/ε overexpression cells. B Cell growth kinetics. Normal Marc-145 cells and Marc-145 wpxld , Marc-145 14-3-3β , and Marc-145 14-3-3ε cells were plated in 24-well plates. At 24, 48, 72, 96, 120, 144, and 168 h post-plating, the CCK-8 reagent was added to the cells. The cells were then incubated for 2 h at 37 °C. Cell counting was performed by measuring absorbance at 450 nm. Marc-145 14-3-3β , Marc-145 14-3-3ε , and Marc-145 wpxld cells were infected with TA-12 and CH-1R, respectively. The infected cells were harvested at 0, 6, 12, 24, 36, and 48 hpi for RNA extraction and at 12, 24, 36, and 48 hpi for protein extraction. Viral loads were evaluated by absolute qPCR targeting the N gene of TA-12 ( C ) and CH-1R ( D ). Cellular proteins were analyzed by Western blot analysis for detecting the viral N protein of TA-12 ( E ) and CH-1R ( F ). The GAPDH protein or GAPDH gene was used as the internal control. * P value

    Techniques Used: Over Expression, Infection, Western Blot, CCK-8 Assay, Incubation, Cell Counting, RNA Extraction, Protein Extraction, Real-time Polymerase Chain Reaction

    Difopein decreases HP-PRRSV infection in Marc-145 cells. A The cytotoxicity of difopein was determined by the CCK-8 assay. Monolayers of Marc-145 cells in 96-well plates were treated with difopein at different concentrations for 48 h, after which the CCK-8 reagent was added to each well. After incubation for 2 h, cell viability was evaluated by measuring absorbance at 450 nm. Marc-145 cells were inoculated with TA-12 and CH-1R and incubated for 1 h, after which the medium was replaced with maintenance medium containing 0.02 or 0.08 μg/mL difopein. The cells were collected 24 h later, and total RNA and cellular proteins were extracted. Viral loads were evaluated by absolute qPCR targeting the nucleocapsid ( N ) gene, and viral proteins were detected by Western blot analysis of TA-12- ( B ) and CH-1R- ( C ) infected cells. D CCID 50 analysis for titration of HP-PRRSV after difopein treatment. Marc-145 cells were seeded in 96-well plates. Virus supernatants were tenfold serially diluted (range 10 2 –10 10 ) and added to each well (at 100 μL per well) in eight repetitions. At 6 days post-infection, the numbers of cells in wells exhibiting cytopathic effects were counted, and CCID 50 was calculated by the Reed–Muench method. E Marc-145 cells were infected with TA-12 and treated with 0.08 or 0.1 μg/mL difopein at 24 hpi. After incubation for another 12 h, the cells were collected for RNA extraction and qPCR analysis. * P value
    Figure Legend Snippet: Difopein decreases HP-PRRSV infection in Marc-145 cells. A The cytotoxicity of difopein was determined by the CCK-8 assay. Monolayers of Marc-145 cells in 96-well plates were treated with difopein at different concentrations for 48 h, after which the CCK-8 reagent was added to each well. After incubation for 2 h, cell viability was evaluated by measuring absorbance at 450 nm. Marc-145 cells were inoculated with TA-12 and CH-1R and incubated for 1 h, after which the medium was replaced with maintenance medium containing 0.02 or 0.08 μg/mL difopein. The cells were collected 24 h later, and total RNA and cellular proteins were extracted. Viral loads were evaluated by absolute qPCR targeting the nucleocapsid ( N ) gene, and viral proteins were detected by Western blot analysis of TA-12- ( B ) and CH-1R- ( C ) infected cells. D CCID 50 analysis for titration of HP-PRRSV after difopein treatment. Marc-145 cells were seeded in 96-well plates. Virus supernatants were tenfold serially diluted (range 10 2 –10 10 ) and added to each well (at 100 μL per well) in eight repetitions. At 6 days post-infection, the numbers of cells in wells exhibiting cytopathic effects were counted, and CCID 50 was calculated by the Reed–Muench method. E Marc-145 cells were infected with TA-12 and treated with 0.08 or 0.1 μg/mL difopein at 24 hpi. After incubation for another 12 h, the cells were collected for RNA extraction and qPCR analysis. * P value

    Techniques Used: Infection, CCK-8 Assay, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Titration, Endpoint Dilution Assay, RNA Extraction

    14 - 3 - 3ε knockdown and difopein treatment decrease HP-PRRSV infection in PAMs. A Results of qPCR for detection of 14 - 3 - 3ε after knockdown. PAMs were transfected with siRNA. The cells were collected at 24 h post-transfection, and total RNA was prepared for detecting the mRNA levels of 14 - 3 - 3ε . B Results of qPCR for analysis of TA-12 replication after 14 - 3 - 3ε knockdown. PAMs were transfected with siRNA and inoculated with TA-12 at 24 h post-transfection. The cells were collected, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene of TA-12. C The cytotoxicity of difopein in PAMs was determined by the CCK-8 assay. PAMs were inoculated with TA-12 or CH-1R and incubated for 1 h, after which the medium was replaced with maintenance medium containing 0.02 or 0.08 μg/mL difopein. The cells were collected 24 h later, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene, and viral proteins were detected by Western blot analysis of TA-12- ( D ) CH-1R- ( E ) infected cells. F Therapeutic role of difopein in TA-12 infection. PAMs were infected with TA-12 and treated with 0.02 or 0.08 μg/mL difopein at 24 hpi. The cells were incubated for another 12 h and then harvested for RNA extraction and qPCR analysis.
    Figure Legend Snippet: 14 - 3 - 3ε knockdown and difopein treatment decrease HP-PRRSV infection in PAMs. A Results of qPCR for detection of 14 - 3 - 3ε after knockdown. PAMs were transfected with siRNA. The cells were collected at 24 h post-transfection, and total RNA was prepared for detecting the mRNA levels of 14 - 3 - 3ε . B Results of qPCR for analysis of TA-12 replication after 14 - 3 - 3ε knockdown. PAMs were transfected with siRNA and inoculated with TA-12 at 24 h post-transfection. The cells were collected, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene of TA-12. C The cytotoxicity of difopein in PAMs was determined by the CCK-8 assay. PAMs were inoculated with TA-12 or CH-1R and incubated for 1 h, after which the medium was replaced with maintenance medium containing 0.02 or 0.08 μg/mL difopein. The cells were collected 24 h later, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene, and viral proteins were detected by Western blot analysis of TA-12- ( D ) CH-1R- ( E ) infected cells. F Therapeutic role of difopein in TA-12 infection. PAMs were infected with TA-12 and treated with 0.02 or 0.08 μg/mL difopein at 24 hpi. The cells were incubated for another 12 h and then harvested for RNA extraction and qPCR analysis.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Transfection, CCK-8 Assay, Incubation, Western Blot, RNA Extraction

    Knockdown of 14 - 3 - 3ε decreases HP-PRRSV infection. A Results of qPCR for detection of 14 - 3 - 3β/ε after siRNA transfection. Marc-145 cells were transfected with siRNA. At 24 h post-transfection, the cells were collected, and total RNA was prepared for detecting the mRNA levels of 14 - 3 - 3β/ε . B Western blot analysis of 14-3-3β/ε expression after siRNA transfection. Marc-145 cells were transfected with siRNA. At 24 h post-transfection, the cells were collected, and cellular proteins were extracted for detecting 14-3-3β/ε proteins. C Marc-145 cells were transfected with siRNA and inoculated with TA-12 or mock infected with cell-culture medium at 24 h post-transfection. The mock-infected cells were collected at different time points after infection, and their viability was measured by the CCK-8 assay. D The TA-12-infected cells were collected at the same time points as the mock-infected cells and processed for total RNA extraction. Viral loads were evaluated by absolute qPCR targeting the nucleocapsid ( N ) gene of HP-PRRSV . E Viral proteins were evaluated by Western blot using a monoclonal antibody (6D10) targeting the PRRSV N protein. N.C: negative control. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. The GAPDH gene and protein were used as internal controls for qPCR and Western blot. The density of the protein bands—measured with a fusion analysis software by using the VILBER lourmat imaging system (Fusion FX7, France)—was determined after subtracting the density of the GAPDH bands. * P value
    Figure Legend Snippet: Knockdown of 14 - 3 - 3ε decreases HP-PRRSV infection. A Results of qPCR for detection of 14 - 3 - 3β/ε after siRNA transfection. Marc-145 cells were transfected with siRNA. At 24 h post-transfection, the cells were collected, and total RNA was prepared for detecting the mRNA levels of 14 - 3 - 3β/ε . B Western blot analysis of 14-3-3β/ε expression after siRNA transfection. Marc-145 cells were transfected with siRNA. At 24 h post-transfection, the cells were collected, and cellular proteins were extracted for detecting 14-3-3β/ε proteins. C Marc-145 cells were transfected with siRNA and inoculated with TA-12 or mock infected with cell-culture medium at 24 h post-transfection. The mock-infected cells were collected at different time points after infection, and their viability was measured by the CCK-8 assay. D The TA-12-infected cells were collected at the same time points as the mock-infected cells and processed for total RNA extraction. Viral loads were evaluated by absolute qPCR targeting the nucleocapsid ( N ) gene of HP-PRRSV . E Viral proteins were evaluated by Western blot using a monoclonal antibody (6D10) targeting the PRRSV N protein. N.C: negative control. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. The GAPDH gene and protein were used as internal controls for qPCR and Western blot. The density of the protein bands—measured with a fusion analysis software by using the VILBER lourmat imaging system (Fusion FX7, France)—was determined after subtracting the density of the GAPDH bands. * P value

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Expressing, Cell Culture, CCK-8 Assay, RNA Extraction, Negative Control, Software, Imaging

    2) Product Images from "Cis and Trans Acting Factors Involved in Human Cytomegalovirus Experimental and Natural Latent Infection of CD14 (+) Monocytes and CD34 (+) Cells"

    Article Title: Cis and Trans Acting Factors Involved in Human Cytomegalovirus Experimental and Natural Latent Infection of CD14 (+) Monocytes and CD34 (+) Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003366

    Infected CD14 (+) cells express latency associated transcripts and stable DNA copy number after 14 days in culture. (A) RT-qPCR analysis for the expression of IE1/IE2, LUNA and UL138 mRNA in infected CD14 (+) monocytes. CD14 (+) cells were infected with FIX BAC virus and total cellular RNA was harvested various days post infection and subjected to RT-qPCR using TaqMan primers and probes specific for IE1 and IE2 mRNA. Inset figure: Detection of viral genomic DNA in infected CD14 (+) monocytes by PCR. Lanes: 1, uninfected; 2, 5-day post infection; 3, 18 day post infection. Primers used were specific for the TR region of the genome. (B) CD14 (+) monocytes infected cell viral genome copy number. Cells were harvested at various days post infection and subjected to qPCR using primers and probes specific for the HCMV viral chromosome. Absolute viral genome copy number was determined by comparing to a standard curve and calculated on a per cell basis. Error bars are the standard deviation of the mean for 4 separate wells.
    Figure Legend Snippet: Infected CD14 (+) cells express latency associated transcripts and stable DNA copy number after 14 days in culture. (A) RT-qPCR analysis for the expression of IE1/IE2, LUNA and UL138 mRNA in infected CD14 (+) monocytes. CD14 (+) cells were infected with FIX BAC virus and total cellular RNA was harvested various days post infection and subjected to RT-qPCR using TaqMan primers and probes specific for IE1 and IE2 mRNA. Inset figure: Detection of viral genomic DNA in infected CD14 (+) monocytes by PCR. Lanes: 1, uninfected; 2, 5-day post infection; 3, 18 day post infection. Primers used were specific for the TR region of the genome. (B) CD14 (+) monocytes infected cell viral genome copy number. Cells were harvested at various days post infection and subjected to qPCR using primers and probes specific for the HCMV viral chromosome. Absolute viral genome copy number was determined by comparing to a standard curve and calculated on a per cell basis. Error bars are the standard deviation of the mean for 4 separate wells.

    Techniques Used: Infection, Quantitative RT-PCR, Expressing, BAC Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Standard Deviation

    UV inactivation of virus abrogates accumulation of IE2 and latency associated transcripts in CD14 (+) monocytes. CD14 (+) monocytes were infected with either wt FIX BAC virus or UV inactivated virus. Total cellular RNA was harvested at 1 hr, 5 and 10 days post infection and qPCR was performed to detect transcripts IE2, UL44, UL84, LUNA, UL138, lncRNA 2.7 and lncRNA4.9. Transcript fold increase was determined using mock infected as reference. 3 separate experiments were preformed and error bars are the SD of the mean.
    Figure Legend Snippet: UV inactivation of virus abrogates accumulation of IE2 and latency associated transcripts in CD14 (+) monocytes. CD14 (+) monocytes were infected with either wt FIX BAC virus or UV inactivated virus. Total cellular RNA was harvested at 1 hr, 5 and 10 days post infection and qPCR was performed to detect transcripts IE2, UL44, UL84, LUNA, UL138, lncRNA 2.7 and lncRNA4.9. Transcript fold increase was determined using mock infected as reference. 3 separate experiments were preformed and error bars are the SD of the mean.

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

    RNA-Seq analysis of HCMV infected CD34 (+) cells. Latent transcriptome from CD34 (+) cells shares core transcripts with those observed from CD14 (+) latent infection. (A) qPCR evaluation of specific gene expression from CD34 (+) cells infected with FIX BAC virus at 3 and 10 days post infection. (B) RNA-Seq detection of transcripts from infected CD34 (+) cells during latent (11 days post infection), 3 days post infection and from cells reactivated after 11 days post infection. Peaks were calculated and determined based on 3 independent experiments and using a minimum of 20 read cut off and pValue of
    Figure Legend Snippet: RNA-Seq analysis of HCMV infected CD34 (+) cells. Latent transcriptome from CD34 (+) cells shares core transcripts with those observed from CD14 (+) latent infection. (A) qPCR evaluation of specific gene expression from CD34 (+) cells infected with FIX BAC virus at 3 and 10 days post infection. (B) RNA-Seq detection of transcripts from infected CD34 (+) cells during latent (11 days post infection), 3 days post infection and from cells reactivated after 11 days post infection. Peaks were calculated and determined based on 3 independent experiments and using a minimum of 20 read cut off and pValue of

    Techniques Used: RNA Sequencing Assay, Infection, Real-time Polymerase Chain Reaction, Expressing, BAC Assay

    UL44 and UL84 interact with the HCMV latent viral genome in CD14 (+) monocytes. (A) ChIP-Seq analysis peak map of UL44 and UL84 in HCMV latently infected CD14 (+) monocytes. UL44 or UL84 specific antibodies were used to immunoprecipitate protein-DNA complexes followed by next generation sequencing. Shown is the HCMV (Fix strain) virus genome and the location of DNA sequence reads, Red peaks = UL84 protein binding, Blue peaks = UL44 protein binding. (B) RNA4.9 interacts with components for the polycomb repressive complex. HCMV latently infected monocytes were fixed and RNA-protein complexes were immunoprecipitated with antibodies specific for EZH2, SUZ12 or UL84. RNA reverse-transcribed and cDNA was detected by PCR using primers specific for RNA4.9 or Cyclophilin A. Control immunoprecipitations were performed using an isotype control antibodies, mAb control (isotype control for SUZ12 and UL84), pAb control (isotype control for EZH2 and UL44). (C) SUZ12 and EZH2 interact with the MIEP region during latent infection. ChIP assays were performed from latently infected CD14 (+) cells using antibodies specific for SUZ12 or EZH2. Primers specific for the promoter of the MIE gene locus were used as well as control primers specific for the LUNA promoter.
    Figure Legend Snippet: UL44 and UL84 interact with the HCMV latent viral genome in CD14 (+) monocytes. (A) ChIP-Seq analysis peak map of UL44 and UL84 in HCMV latently infected CD14 (+) monocytes. UL44 or UL84 specific antibodies were used to immunoprecipitate protein-DNA complexes followed by next generation sequencing. Shown is the HCMV (Fix strain) virus genome and the location of DNA sequence reads, Red peaks = UL84 protein binding, Blue peaks = UL44 protein binding. (B) RNA4.9 interacts with components for the polycomb repressive complex. HCMV latently infected monocytes were fixed and RNA-protein complexes were immunoprecipitated with antibodies specific for EZH2, SUZ12 or UL84. RNA reverse-transcribed and cDNA was detected by PCR using primers specific for RNA4.9 or Cyclophilin A. Control immunoprecipitations were performed using an isotype control antibodies, mAb control (isotype control for SUZ12 and UL84), pAb control (isotype control for EZH2 and UL44). (C) SUZ12 and EZH2 interact with the MIEP region during latent infection. ChIP assays were performed from latently infected CD14 (+) cells using antibodies specific for SUZ12 or EZH2. Primers specific for the promoter of the MIE gene locus were used as well as control primers specific for the LUNA promoter.

    Techniques Used: Chromatin Immunoprecipitation, Infection, Next-Generation Sequencing, Sequencing, Protein Binding, Immunoprecipitation, Polymerase Chain Reaction

    3) Product Images from "Androgen Receptor Regulates Transcription of the ZEB1 Transcription Factor"

    Article Title: Androgen Receptor Regulates Transcription of the ZEB1 Transcription Factor

    Journal: International Journal of Endocrinology

    doi: 10.1155/2011/903918

    Endogenous tcf8 is not induced in the LNCaP cell lines . The four LNCaP-derived cell lines were each divided into two groups and were incubated with or without 5 nM DHT for 24 hrs. (a) For one group, RNA was isolated and subjected to qPCR for ZEB1 and GAPDH mRNA. ZEB1 mRNA levels were normalized to GAPDH. (b) For the second group, the cells were transfected with pBlueZEB974 at T = O, and β -galactosidase activity was determined at 24 hrs. * P
    Figure Legend Snippet: Endogenous tcf8 is not induced in the LNCaP cell lines . The four LNCaP-derived cell lines were each divided into two groups and were incubated with or without 5 nM DHT for 24 hrs. (a) For one group, RNA was isolated and subjected to qPCR for ZEB1 and GAPDH mRNA. ZEB1 mRNA levels were normalized to GAPDH. (b) For the second group, the cells were transfected with pBlueZEB974 at T = O, and β -galactosidase activity was determined at 24 hrs. * P

    Techniques Used: Derivative Assay, Incubation, Isolation, Real-time Polymerase Chain Reaction, Transfection, Activity Assay

    Endogenous ZEB1 mRNA expression is induced by DHT . PC-3/AR cells were treated with vehicle (0 nM) or increasing amounts of DHT as indicated. Twenty-four hours later, RNA was harvested and subjected to qPCR using the ZEB1 and RPL32 primers listed in Table 1 . ZEB1 mRNA levels were normalized to RPL32 and then plotted relative to the no DHT control. This experiment was repeated 6 times in triplicate. The errors bars represent the standard deviation from the mean average of all experiments. * P
    Figure Legend Snippet: Endogenous ZEB1 mRNA expression is induced by DHT . PC-3/AR cells were treated with vehicle (0 nM) or increasing amounts of DHT as indicated. Twenty-four hours later, RNA was harvested and subjected to qPCR using the ZEB1 and RPL32 primers listed in Table 1 . ZEB1 mRNA levels were normalized to RPL32 and then plotted relative to the no DHT control. This experiment was repeated 6 times in triplicate. The errors bars represent the standard deviation from the mean average of all experiments. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    4) Product Images from "Patterns of Gene Expression in Drosophila InsP3 Receptor Mutant Larvae Reveal a Role for InsP3 Signaling in Carbohydrate and Energy Metabolism"

    Article Title: Patterns of Gene Expression in Drosophila InsP3 Receptor Mutant Larvae Reveal a Role for InsP3 Signaling in Carbohydrate and Energy Metabolism

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024105

    Validation of candidate genes by quantitative real time PCR (qPCR). The Y-axis represents the log 2 of fold changes which were calculated by the ΔΔC t method in which the C t values of each gene were normalized to the level of a housekeeping gene ( rp49 ) in control RNA from CS larvae. Each value is the mean ± SEM of three independent experiments, obtained from three independent RNA samples. The rescued and suppressed values were tested for significant difference from the mutants by Students t-tests followed by a Bonferroni correction for multiple tests. Except for CG2650 and unc119 all other genes were significantly altered ( P
    Figure Legend Snippet: Validation of candidate genes by quantitative real time PCR (qPCR). The Y-axis represents the log 2 of fold changes which were calculated by the ΔΔC t method in which the C t values of each gene were normalized to the level of a housekeeping gene ( rp49 ) in control RNA from CS larvae. Each value is the mean ± SEM of three independent experiments, obtained from three independent RNA samples. The rescued and suppressed values were tested for significant difference from the mutants by Students t-tests followed by a Bonferroni correction for multiple tests. Except for CG2650 and unc119 all other genes were significantly altered ( P

    Techniques Used: Real-time Polymerase Chain Reaction

    Expression of candidate genes in larval brains and fat bodies. Expression of candidate genes identified from the microarray analysis was tested in RNA from brains and fat bodies of third instar larvae by RT-PCR.
    Figure Legend Snippet: Expression of candidate genes in larval brains and fat bodies. Expression of candidate genes identified from the microarray analysis was tested in RNA from brains and fat bodies of third instar larvae by RT-PCR.

    Techniques Used: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction

    5) Product Images from "Skeletal muscle interleukin 15 promotes CD8+ T-cell function and autoimmune myositis"

    Article Title: Skeletal muscle interleukin 15 promotes CD8+ T-cell function and autoimmune myositis

    Journal: Skeletal Muscle

    doi: 10.1186/s13395-015-0058-2

    Skeletal muscle cells express IL-15/IL-15Rα protein complex in response to TNF-α and IFN-γ stimulation. a Expression of Il15 and Il15ra mRNA during C2C12 myoblast differentiation. Samples were collected before (0) and 2, 4, and 6 days after differentiation induction. b Expression of Il15 and Il15ra mRNA in C2C12 myotubes treated with TNF-α (10 ng/ml), IFN-γ (10 ng/ml), TNF-α + IFN-γ (TNF + IFN, 10 ng/ml each), or without cytokine ( con ) for 1, 2, 3, 6, 12, and 24 h. c Expression of Il15 and Il15ra mRNA in primary myotubes treated with TNF-α (5 ng/ml), IFN-γ (5 ng/ml), TNF + IFN (5 ng/ml each), or without cytokine ( con ) for 24 h. d Expression of IL-15/IL-15Rα complex protein in C2C12 myoblasts and myotubes treated with TNF-α (1 or 10 ng/ml), IFN-γ (1 or 10 ng/ml), TNF + IFN (1 or 10 ng/ml each), or without cytokine ( con ) for 24 h. e Expression of IL-15/IL-15Rα complex protein in primary myotubes treated with TNF + IFN (5 ng/ml each) or vehicle ( con ) for 24 h. f Expression of IL-15/IL-15Rα complex protein in skeletal muscle in vivo. TNF-α plus IFN-γ (1 mg each/injection) or PBS ( con ) was injected into the quadriceps muscles of mice three times at 4-h intervals. The injected muscles were collected 16 h after the last injection. Total RNA was isolated and analyzed by qPCR ( a–c ). Protein lysate and cell culture medium were collected and measured by ELISA ( d–f ). Data in ( a–c ) were triplicates and representative of two independent experiments with similar result. Data in ( d, e ) were pooled from three independent experiments. Data in ( f ) was pooled from three mice in each group. Data are mean ± SEM. * p
    Figure Legend Snippet: Skeletal muscle cells express IL-15/IL-15Rα protein complex in response to TNF-α and IFN-γ stimulation. a Expression of Il15 and Il15ra mRNA during C2C12 myoblast differentiation. Samples were collected before (0) and 2, 4, and 6 days after differentiation induction. b Expression of Il15 and Il15ra mRNA in C2C12 myotubes treated with TNF-α (10 ng/ml), IFN-γ (10 ng/ml), TNF-α + IFN-γ (TNF + IFN, 10 ng/ml each), or without cytokine ( con ) for 1, 2, 3, 6, 12, and 24 h. c Expression of Il15 and Il15ra mRNA in primary myotubes treated with TNF-α (5 ng/ml), IFN-γ (5 ng/ml), TNF + IFN (5 ng/ml each), or without cytokine ( con ) for 24 h. d Expression of IL-15/IL-15Rα complex protein in C2C12 myoblasts and myotubes treated with TNF-α (1 or 10 ng/ml), IFN-γ (1 or 10 ng/ml), TNF + IFN (1 or 10 ng/ml each), or without cytokine ( con ) for 24 h. e Expression of IL-15/IL-15Rα complex protein in primary myotubes treated with TNF + IFN (5 ng/ml each) or vehicle ( con ) for 24 h. f Expression of IL-15/IL-15Rα complex protein in skeletal muscle in vivo. TNF-α plus IFN-γ (1 mg each/injection) or PBS ( con ) was injected into the quadriceps muscles of mice three times at 4-h intervals. The injected muscles were collected 16 h after the last injection. Total RNA was isolated and analyzed by qPCR ( a–c ). Protein lysate and cell culture medium were collected and measured by ELISA ( d–f ). Data in ( a–c ) were triplicates and representative of two independent experiments with similar result. Data in ( d, e ) were pooled from three independent experiments. Data in ( f ) was pooled from three mice in each group. Data are mean ± SEM. * p

    Techniques Used: Expressing, In Vivo, Injection, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging"

    Article Title: Disruption of the Cx43/miR21 pathway leads to osteocyte apoptosis and increased osteoclastogenesis with aging

    Journal: Aging Cell

    doi: 10.1111/acel.12586

    Cx43 deficiency and old age results in decreased miR21 expression. (A) Profile of 48 apoptosis‐associated mi RNA assessed in MLO ‐Y4 osteocytic cells expressing or not for Cx43 using an mi RNA plate array and corrected by U6 RNA ( n = 3). (B) Changes in the expression of miR21 and miR218 in MLO ‐Y4 osteocytic cells were validated by qPCR and corrected by miR135 ( n = 3). (C) Expression of miR21 and miR218 corrected by miR135 in Ob‐6 osteoblastic cells ( n = 3). (D) mi RNA expression in calvaria from 4‐ to 24‐month‐old mice measured by qPCR and corrected by miR135 ( n = 5–9). Bars represent mean ± SD. # P
    Figure Legend Snippet: Cx43 deficiency and old age results in decreased miR21 expression. (A) Profile of 48 apoptosis‐associated mi RNA assessed in MLO ‐Y4 osteocytic cells expressing or not for Cx43 using an mi RNA plate array and corrected by U6 RNA ( n = 3). (B) Changes in the expression of miR21 and miR218 in MLO ‐Y4 osteocytic cells were validated by qPCR and corrected by miR135 ( n = 3). (C) Expression of miR21 and miR218 corrected by miR135 in Ob‐6 osteoblastic cells ( n = 3). (D) mi RNA expression in calvaria from 4‐ to 24‐month‐old mice measured by qPCR and corrected by miR135 ( n = 5–9). Bars represent mean ± SD. # P

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

    7) Product Images from "Kindlin 2 Regulates Myogenic Related Factor Myogenin via a Canonical Wnt Signaling in Myogenic Differentiation"

    Article Title: Kindlin 2 Regulates Myogenic Related Factor Myogenin via a Canonical Wnt Signaling in Myogenic Differentiation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063490

    Kindlin 2 is activated during muscle cell differentiation. (A) C2C12 cells were cultured in growth medium (GM) or differentiation medium (DM) for 0, 1, 3, 5 days. Proteins were extracted from the cells at different stages, and Western blot (WB) assays were performed using the indicated antibodies. β-actin was used as a loading control. (B) Protein bands in A were scanned, and relative band intensities were normalized for each β-actin band. The column diagrams represent average relative band intensity with standard error from three independent experiments. (C–D) Total RNA was extracted from the cells at different stages. The mRNA levels of Kindlin 2 and MyHC were examined by qPCR. Error bars indicate s.d. values, n = 3; * indicates p
    Figure Legend Snippet: Kindlin 2 is activated during muscle cell differentiation. (A) C2C12 cells were cultured in growth medium (GM) or differentiation medium (DM) for 0, 1, 3, 5 days. Proteins were extracted from the cells at different stages, and Western blot (WB) assays were performed using the indicated antibodies. β-actin was used as a loading control. (B) Protein bands in A were scanned, and relative band intensities were normalized for each β-actin band. The column diagrams represent average relative band intensity with standard error from three independent experiments. (C–D) Total RNA was extracted from the cells at different stages. The mRNA levels of Kindlin 2 and MyHC were examined by qPCR. Error bars indicate s.d. values, n = 3; * indicates p

    Techniques Used: Cell Differentiation, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction

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    Transfection:

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    Article Snippet: .. Stealth™ siRNAs targeting ZNF217 (siRNA‐ZNF217‐A, leading to intermediate knock‐down of ZNF217 and siRNA‐ZNF217‐B, leading to total ZNF217 knock‐down) , ESR1 and scrambled control RNA (Life Technologies, Paisley, UK) were transfected (5 × 10−9 M) as previously described ( ). .. Briefly, 2 × 106 cells were lysed using the Subcellular Protein Fractionation Kit (Thermo Scientific, Waltham, MA, USA).

    Article Title: The novel transcriptional factor HP1BP3 negatively regulates Hsp70 transcription in Crassostrea hongkongensis
    Article Snippet: .. Quantitative real-time PCR Total RNA was extracted from hemocytes transfected with either siRNA or control RNA as well as from the gills of oysters that were either non-stressed or stressed by heat, CdCl2 , or NP at various time points using the Trizol reagent (Cat. No. 15596-026; Invitrogen). .. Two micrograms of total RNA was used as template to synthesize the first-strand cDNA with MLV Reverse transcriptase (Promega) and oligo (dT) primer.

    Article Title: PAR-1 is a novel mechano-sensor transducing laminar flow-mediated endothelial signaling
    Article Snippet: .. For PAR-1 silencing, HUVECs were transiently transfected with 100 pmol/L of control RNA or siRNA targeting human PAR-1 using Lipofectamine 2000 reagent (#11668-019, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. .. A non-specific control siRNA from Bioneer was used as a negative control.

    Article Title: miR-16 and miR-26a target checkpoint kinases Wee1 and Chk1 in response to p53 activation by genotoxic stress
    Article Snippet: .. For luciferase reporter assays, cells were cultured in six-well plates, and each well was transfected with 0.5 μ g of firefly luciferase reporter plasmid, 0.5 μ g of β -galactosidase expression plasmid (Ambion), and equal amounts of scrambled negative control RNA, pre-miR-26a, pre-miR-16, or anti-miR-16 using Lipofectamine 2000 (Invitrogen). .. The β -galactosidase plasmid was used as a transfection control.

    Luciferase:

    Article Title: miR-16 and miR-26a target checkpoint kinases Wee1 and Chk1 in response to p53 activation by genotoxic stress
    Article Snippet: .. For luciferase reporter assays, cells were cultured in six-well plates, and each well was transfected with 0.5 μ g of firefly luciferase reporter plasmid, 0.5 μ g of β -galactosidase expression plasmid (Ambion), and equal amounts of scrambled negative control RNA, pre-miR-26a, pre-miR-16, or anti-miR-16 using Lipofectamine 2000 (Invitrogen). .. The β -galactosidase plasmid was used as a transfection control.

    Negative Control:

    Article Title: miR-16 and miR-26a target checkpoint kinases Wee1 and Chk1 in response to p53 activation by genotoxic stress
    Article Snippet: .. For luciferase reporter assays, cells were cultured in six-well plates, and each well was transfected with 0.5 μ g of firefly luciferase reporter plasmid, 0.5 μ g of β -galactosidase expression plasmid (Ambion), and equal amounts of scrambled negative control RNA, pre-miR-26a, pre-miR-16, or anti-miR-16 using Lipofectamine 2000 (Invitrogen). .. The β -galactosidase plasmid was used as a transfection control.

    Article Title: LUC7L3/CROP inhibits replication of hepatitis B virus via suppressing enhancer II/basal core promoter activity
    Article Snippet: .. ACIN1-specific siRNA, BCLAF1-specific siRNA, CNBP1-specific siRNA, CTCF-specific siRNA, HDGFRP2-specific siRNA, HMGN1-specific siRNA, LUC7L3-specific siRNA (siLUC7) and the negative control RNA (siNC) was provided by Bonac (Fukuoka, Japan). siRNAs for HNF4α (siHNF4a), C/EBPα (siC/EBPa) and the negative control RNA (siNC-2) were purchased from Ambion (CA, USA). .. The synthetic siRNAs (50 pmol) were transfected into cells using ScreenFect A (Wako).

    Cell Culture:

    Article Title: miR-16 and miR-26a target checkpoint kinases Wee1 and Chk1 in response to p53 activation by genotoxic stress
    Article Snippet: .. For luciferase reporter assays, cells were cultured in six-well plates, and each well was transfected with 0.5 μ g of firefly luciferase reporter plasmid, 0.5 μ g of β -galactosidase expression plasmid (Ambion), and equal amounts of scrambled negative control RNA, pre-miR-26a, pre-miR-16, or anti-miR-16 using Lipofectamine 2000 (Invitrogen). .. The β -galactosidase plasmid was used as a transfection control.

    Real-time Polymerase Chain Reaction:

    Article Title: The novel transcriptional factor HP1BP3 negatively regulates Hsp70 transcription in Crassostrea hongkongensis
    Article Snippet: .. Quantitative real-time PCR Total RNA was extracted from hemocytes transfected with either siRNA or control RNA as well as from the gills of oysters that were either non-stressed or stressed by heat, CdCl2 , or NP at various time points using the Trizol reagent (Cat. No. 15596-026; Invitrogen). .. Two micrograms of total RNA was used as template to synthesize the first-strand cDNA with MLV Reverse transcriptase (Promega) and oligo (dT) primer.

    Small Interfering RNA:

    Article Title: Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes
    Article Snippet: .. For RNA interference, pre-designed GC-B short interfering RNA (siRNA) or scrambled RNA control (Ambion Biosciences, Invitrogen) were introduced into GH3 cells by the lipofectin siRNAmax reverse-transfection method (Invitrogen). .. For MAPK phosphatase-1 (MKP1) over-expression studies, 5 μg/well MKP1 or pcDNA expression vectors were reverse-transfected into GH3 cells, prior to serum starvation and subsequent stimulation.

    Expressing:

    Article Title: miR-16 and miR-26a target checkpoint kinases Wee1 and Chk1 in response to p53 activation by genotoxic stress
    Article Snippet: .. For luciferase reporter assays, cells were cultured in six-well plates, and each well was transfected with 0.5 μ g of firefly luciferase reporter plasmid, 0.5 μ g of β -galactosidase expression plasmid (Ambion), and equal amounts of scrambled negative control RNA, pre-miR-26a, pre-miR-16, or anti-miR-16 using Lipofectamine 2000 (Invitrogen). .. The β -galactosidase plasmid was used as a transfection control.

    Reporter Gene Assay:

    Article Title: MicroRNA-137 Upregulation Increases Bladder Cancer Cell Proliferation and Invasion by Targeting PAQR3
    Article Snippet: .. For the reporter gene assay, the cells were cotransfected with 0.5 µg of pGL3-PAQR3-3′UTR or pGL3- PAQR3-3′UTR Mut plasmid, 0.05 ng of the phRL-SV40 control vector (Promega, USA), and 100 nM miR-137 or control RNA using Lipofectamine 2000 (Invitrogen, USA). .. The firefly and renilla luciferase activities were measured consecutively through a dual luciferase assay (Promega, USA) 24 h after transfection.

    Plasmid Preparation:

    Article Title: MicroRNA-137 Upregulation Increases Bladder Cancer Cell Proliferation and Invasion by Targeting PAQR3
    Article Snippet: .. For the reporter gene assay, the cells were cotransfected with 0.5 µg of pGL3-PAQR3-3′UTR or pGL3- PAQR3-3′UTR Mut plasmid, 0.05 ng of the phRL-SV40 control vector (Promega, USA), and 100 nM miR-137 or control RNA using Lipofectamine 2000 (Invitrogen, USA). .. The firefly and renilla luciferase activities were measured consecutively through a dual luciferase assay (Promega, USA) 24 h after transfection.

    Article Title: miR-16 and miR-26a target checkpoint kinases Wee1 and Chk1 in response to p53 activation by genotoxic stress
    Article Snippet: .. For luciferase reporter assays, cells were cultured in six-well plates, and each well was transfected with 0.5 μ g of firefly luciferase reporter plasmid, 0.5 μ g of β -galactosidase expression plasmid (Ambion), and equal amounts of scrambled negative control RNA, pre-miR-26a, pre-miR-16, or anti-miR-16 using Lipofectamine 2000 (Invitrogen). .. The β -galactosidase plasmid was used as a transfection control.

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    Thermo Fisher quantitative rt pcr analysis total rna
    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger <t>RNA</t> (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time <t>(qRT)‐PCR.</t> ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P
    Quantitative Rt Pcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time polymerase chain reaction qrt pcr total rna
    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by <t>qRT-PCR;</t> b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna
    MD001 stimulates fatty acid oxidation in vitro . HepG2 ( A ), differentiated 3T3-L1 adipocyte ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle or MD001 (10 μM) for 24 h and total <t>RNA</t> was isolated for <t>cDNA</t> synthesis. Relative gene expressions were analysed by qRT-PCR. HepG2 ( D ), differentiated 3T3-L1 adipocytes ( E ), and differentiated C2C12 myotubes ( F ) were treated with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h and the fatty acid oxidation rate was analysed as described in the Methods section. To confirm whether the fatty acid oxidation rate enhanced by MD001 is mediated by PPARα, HepG2 ( G ), differentiated 3T3-L1 ( H ), and differentiated C2C12 myotubes ( I ) were transfected with control or PPARα siRNA (20 nM) for 48 h, followed by treatment with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h. WY14643 was treated as positive control. *, **, vs. vehicle; † and ‡ , vs. vehicle/control siRNA; § , vs. WY/control siRNA; ¶ , vs. MD001/control siRNA. Data represent the mean ± SD of three independent experiments. * P
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 3653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher real time quantitative pcr qpcr total rna
    Pcf11 Expression Is Regulated by IPA (A) Schematic of mouse Pcf11 gene. Multiple PASs are indicated. EPA, internal exonic polyadenylation; IPA, intronic polyadenylation; TPA, 3′ terminal exon polyadenylation. 3′READS data of brain, heart, and testis are shown. (B) Features of intron 1 compared with other introns in the mouse genome. Each feature has ten bins, and the bin containing intron 1 of Pcf11 is highlighted in red. Distance to TSS is based on all IPA sites in PolyA_DB. (C) <t>RT-qPCR</t> analysis of IPA and TPA isoform expression in siPcf11 versus siCtrl cells. Error bars are SD on the basis of three replicates. (D) Top: schematic showing two sgRNAs used to remove a region flanking the IPA site. Bottom: validation of IPA site knockout (IPA Pcf11 -KO) by genomic DNA <t>PCR</t> using primers flanking the KO region. WT, wild-type 4T1 cells. (E) RT-qPCR analysis of Pcf11 isoform expression in WT and IPA Pcf11 -KO cells. Error bars are SD on the basis of three replicates. (F) Western blot analysis of PCF11 in WT and IPA Pcf11 -KO cells. (G) 3′UTR APA changes in IPA Pcf11 -KO cells. (H) IPA changes in IPA Pcf11 -KO cells. IPA isoforms (all combined) were compared with TPA isoforms (all combined) in analysis. (I) IPA distribution maps of activated IPA events in IPA Pcf11 -KO cells. (J) Left: schematic of PASs around the transcription start site (TSS); right: metagene analysis of PAS usage around the TSS in WT and IPA Pcf1 1 -KO cells. (K) Gene expression changes in IPA Pcf11 -KO cells for different groups of genes on the basis of IPA site frequency in PolyA_DB. ***p
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    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Migration, In Vitro, Quantitative RT-PCR, Western Blot

    Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Migration, In Vitro, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot

    MD001 stimulates fatty acid oxidation in vitro . HepG2 ( A ), differentiated 3T3-L1 adipocyte ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle or MD001 (10 μM) for 24 h and total RNA was isolated for cDNA synthesis. Relative gene expressions were analysed by qRT-PCR. HepG2 ( D ), differentiated 3T3-L1 adipocytes ( E ), and differentiated C2C12 myotubes ( F ) were treated with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h and the fatty acid oxidation rate was analysed as described in the Methods section. To confirm whether the fatty acid oxidation rate enhanced by MD001 is mediated by PPARα, HepG2 ( G ), differentiated 3T3-L1 ( H ), and differentiated C2C12 myotubes ( I ) were transfected with control or PPARα siRNA (20 nM) for 48 h, followed by treatment with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h. WY14643 was treated as positive control. *, **, vs. vehicle; † and ‡ , vs. vehicle/control siRNA; § , vs. WY/control siRNA; ¶ , vs. MD001/control siRNA. Data represent the mean ± SD of three independent experiments. * P

    Journal: Scientific Reports

    Article Title: MD001, a Novel Peroxisome Proliferator-activated Receptor α/γ Agonist, Improves Glucose and Lipid Metabolism

    doi: 10.1038/s41598-018-38281-0

    Figure Lengend Snippet: MD001 stimulates fatty acid oxidation in vitro . HepG2 ( A ), differentiated 3T3-L1 adipocyte ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle or MD001 (10 μM) for 24 h and total RNA was isolated for cDNA synthesis. Relative gene expressions were analysed by qRT-PCR. HepG2 ( D ), differentiated 3T3-L1 adipocytes ( E ), and differentiated C2C12 myotubes ( F ) were treated with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h and the fatty acid oxidation rate was analysed as described in the Methods section. To confirm whether the fatty acid oxidation rate enhanced by MD001 is mediated by PPARα, HepG2 ( G ), differentiated 3T3-L1 ( H ), and differentiated C2C12 myotubes ( I ) were transfected with control or PPARα siRNA (20 nM) for 48 h, followed by treatment with vehicle, MD001 (10 μM), or WY14643 (WY) (10 μM) for 24 h. WY14643 was treated as positive control. *, **, vs. vehicle; † and ‡ , vs. vehicle/control siRNA; § , vs. WY/control siRNA; ¶ , vs. MD001/control siRNA. Data represent the mean ± SD of three independent experiments. * P

    Article Snippet: Complementary DNA (cDNA) was synthesised by reverse transcription using 0.5 μg of total RNA, and quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (ABI Thermo Fisher Scientific, Foster City, CA, USA) on a StepOne 48-well real-time PCR system (ABI/Thermo Fisher Scientific).

    Techniques: In Vitro, Isolation, Quantitative RT-PCR, Transfection, Positive Control

    MD001 induces the expression of target genes through PPARα and PPARγ activation. HEK293 cells were transiently co-transfected with human HA-PPARα ( A ) and HA-PPARγ ( B ) expression vectors along with the reporter plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) with Renilla vector for 24 h. Cells were treated with MD001 (10 μM), rosiglitazone (rosi) (10 μM) or WY14643 (WY) (10 μM) for 24 h. Luciferase activity was normalised to Renilla luciferase activity as described in the Methods section. **, vs. vehicle. ( C ) HepG2 cells were treated with MD001 (10 μM) for 24 h, and total RNA was isolated and synthesised into cDNA. Relative expression was quantitated using qRT-PCR. **, vs. vehicle. ( D , E ) HepG2 cells were transfected with control siRNA, PPARα siRNA ( D ), or PPARγ siRNA ( E ) for 48 h and treated with vehicle, WY14643 (10 μM), rosiglitazone (10 μM) or MD001 (10 μM) for 24 h. Subsequently, cells were harvested for total RNA isolation. Relative expression of target genes was quantitated using qRT-PCR. # , vs. control siRNA; ‡ , vs. control siRNA/vehicle; § , vs. control siRNA/WY; * and **, vs. control siRNA/MD001. Data represent the mean ± SD of three independent experiments. * P

    Journal: Scientific Reports

    Article Title: MD001, a Novel Peroxisome Proliferator-activated Receptor α/γ Agonist, Improves Glucose and Lipid Metabolism

    doi: 10.1038/s41598-018-38281-0

    Figure Lengend Snippet: MD001 induces the expression of target genes through PPARα and PPARγ activation. HEK293 cells were transiently co-transfected with human HA-PPARα ( A ) and HA-PPARγ ( B ) expression vectors along with the reporter plasmid (PPRE-pk-Luc) or control reporter plasmid (pk-Luc) with Renilla vector for 24 h. Cells were treated with MD001 (10 μM), rosiglitazone (rosi) (10 μM) or WY14643 (WY) (10 μM) for 24 h. Luciferase activity was normalised to Renilla luciferase activity as described in the Methods section. **, vs. vehicle. ( C ) HepG2 cells were treated with MD001 (10 μM) for 24 h, and total RNA was isolated and synthesised into cDNA. Relative expression was quantitated using qRT-PCR. **, vs. vehicle. ( D , E ) HepG2 cells were transfected with control siRNA, PPARα siRNA ( D ), or PPARγ siRNA ( E ) for 48 h and treated with vehicle, WY14643 (10 μM), rosiglitazone (10 μM) or MD001 (10 μM) for 24 h. Subsequently, cells were harvested for total RNA isolation. Relative expression of target genes was quantitated using qRT-PCR. # , vs. control siRNA; ‡ , vs. control siRNA/vehicle; § , vs. control siRNA/WY; * and **, vs. control siRNA/MD001. Data represent the mean ± SD of three independent experiments. * P

    Article Snippet: Complementary DNA (cDNA) was synthesised by reverse transcription using 0.5 μg of total RNA, and quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (ABI Thermo Fisher Scientific, Foster City, CA, USA) on a StepOne 48-well real-time PCR system (ABI/Thermo Fisher Scientific).

    Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Isolation, Quantitative RT-PCR

    MD001 promotes glucose consumption by induction of GLUT expression. HepG2 ( A ), differentiated 3T3-L1 adipocytes ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle, MD001 (10, 50 μM), or rosiglitazone (rosi, 10 μM) for 24 h; glucose consumption was examined as described in the Methods section. Cells were harvested for total RNA isolation. Relative expression levels of GLUT2 ( D ) and GLUT4 ( E , F ) were quantitated using qRT-PCR. *, **, and † vs. vehicle. Data represent the mean ± SD of three independent experiments. * P

    Journal: Scientific Reports

    Article Title: MD001, a Novel Peroxisome Proliferator-activated Receptor α/γ Agonist, Improves Glucose and Lipid Metabolism

    doi: 10.1038/s41598-018-38281-0

    Figure Lengend Snippet: MD001 promotes glucose consumption by induction of GLUT expression. HepG2 ( A ), differentiated 3T3-L1 adipocytes ( B ), and differentiated C2C12 myotubes ( C ) were treated with vehicle, MD001 (10, 50 μM), or rosiglitazone (rosi, 10 μM) for 24 h; glucose consumption was examined as described in the Methods section. Cells were harvested for total RNA isolation. Relative expression levels of GLUT2 ( D ) and GLUT4 ( E , F ) were quantitated using qRT-PCR. *, **, and † vs. vehicle. Data represent the mean ± SD of three independent experiments. * P

    Article Snippet: Complementary DNA (cDNA) was synthesised by reverse transcription using 0.5 μg of total RNA, and quantitative RT-PCR was performed using Power SYBR Green PCR Master Mix (ABI Thermo Fisher Scientific, Foster City, CA, USA) on a StepOne 48-well real-time PCR system (ABI/Thermo Fisher Scientific).

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    Pcf11 Expression Is Regulated by IPA (A) Schematic of mouse Pcf11 gene. Multiple PASs are indicated. EPA, internal exonic polyadenylation; IPA, intronic polyadenylation; TPA, 3′ terminal exon polyadenylation. 3′READS data of brain, heart, and testis are shown. (B) Features of intron 1 compared with other introns in the mouse genome. Each feature has ten bins, and the bin containing intron 1 of Pcf11 is highlighted in red. Distance to TSS is based on all IPA sites in PolyA_DB. (C) RT-qPCR analysis of IPA and TPA isoform expression in siPcf11 versus siCtrl cells. Error bars are SD on the basis of three replicates. (D) Top: schematic showing two sgRNAs used to remove a region flanking the IPA site. Bottom: validation of IPA site knockout (IPA Pcf11 -KO) by genomic DNA PCR using primers flanking the KO region. WT, wild-type 4T1 cells. (E) RT-qPCR analysis of Pcf11 isoform expression in WT and IPA Pcf11 -KO cells. Error bars are SD on the basis of three replicates. (F) Western blot analysis of PCF11 in WT and IPA Pcf11 -KO cells. (G) 3′UTR APA changes in IPA Pcf11 -KO cells. (H) IPA changes in IPA Pcf11 -KO cells. IPA isoforms (all combined) were compared with TPA isoforms (all combined) in analysis. (I) IPA distribution maps of activated IPA events in IPA Pcf11 -KO cells. (J) Left: schematic of PASs around the transcription start site (TSS); right: metagene analysis of PAS usage around the TSS in WT and IPA Pcf1 1 -KO cells. (K) Gene expression changes in IPA Pcf11 -KO cells for different groups of genes on the basis of IPA site frequency in PolyA_DB. ***p

    Journal: Cell reports

    Article Title: Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes

    doi: 10.1016/j.celrep.2019.02.049

    Figure Lengend Snippet: Pcf11 Expression Is Regulated by IPA (A) Schematic of mouse Pcf11 gene. Multiple PASs are indicated. EPA, internal exonic polyadenylation; IPA, intronic polyadenylation; TPA, 3′ terminal exon polyadenylation. 3′READS data of brain, heart, and testis are shown. (B) Features of intron 1 compared with other introns in the mouse genome. Each feature has ten bins, and the bin containing intron 1 of Pcf11 is highlighted in red. Distance to TSS is based on all IPA sites in PolyA_DB. (C) RT-qPCR analysis of IPA and TPA isoform expression in siPcf11 versus siCtrl cells. Error bars are SD on the basis of three replicates. (D) Top: schematic showing two sgRNAs used to remove a region flanking the IPA site. Bottom: validation of IPA site knockout (IPA Pcf11 -KO) by genomic DNA PCR using primers flanking the KO region. WT, wild-type 4T1 cells. (E) RT-qPCR analysis of Pcf11 isoform expression in WT and IPA Pcf11 -KO cells. Error bars are SD on the basis of three replicates. (F) Western blot analysis of PCF11 in WT and IPA Pcf11 -KO cells. (G) 3′UTR APA changes in IPA Pcf11 -KO cells. (H) IPA changes in IPA Pcf11 -KO cells. IPA isoforms (all combined) were compared with TPA isoforms (all combined) in analysis. (I) IPA distribution maps of activated IPA events in IPA Pcf11 -KO cells. (J) Left: schematic of PASs around the transcription start site (TSS); right: metagene analysis of PAS usage around the TSS in WT and IPA Pcf1 1 -KO cells. (K) Gene expression changes in IPA Pcf11 -KO cells for different groups of genes on the basis of IPA site frequency in PolyA_DB. ***p

    Article Snippet: Real-time quantitative PCR (qPCR) Total RNA was extracted using TRIzol (Thermo Fisher) and treated with Turbo DNase (Thermo Fisher). cDNA was synthesized using oligo(dT) and M-MLV reverse transcriptase with 1.5–2 μg of RNA. cDNA was then mixed with real-time PCR primers and Luna qPCR master mix (NEB).

    Techniques: Expressing, Indirect Immunoperoxidase Assay, Quantitative RT-PCR, Knock-Out, Polymerase Chain Reaction, Western Blot