rt-pcr analysis Search Results


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  • 99
    Thermo Fisher rt pcr analysis
    Effects of DIM and Herceptin on <t>miR-200</t> family. ( A ) Comparative expression analysis of miR-200 in SKBR3 and MDA-MB-468 cells by real-time miRNA <t>RT-PCR.</t> ( B and C ) Relative expression analysis of miR-200 in SKBR3 and MDA-MB-468 cells treated with 15 µM DIM, 0.75 µg/ml Herceptin or their combinations for 24 h. ( D ) Effect of anti-miR-200s (anti-miR-200a+anti-miR-200b+anti-miR-200c) on cell proliferation of SKBR3 cells treated with 15 µM DIM and 0.75 µg/ml Herceptin for 72 hours, as measured by MTT assay. ( E ) Efficacy of anti-miR-200s transfections as well the effect of DIM plus Herceptin (15 µM DIM and 0.75 µg/ml Herceptin for 72 hours) on expression levels of anti-miR-200s (miR-200a/miR-200b/miR-200c) was evaluated by real time RT-PCR. ( F ) Effect of anti-miR-200s (anti-miR-200a+anti-miR-200b+anti-miR-200c) on expression of FoxM1 and pAkt in SKBR3 cells treated with 15 µM DIM and 0.75 µg/ml Herceptin for 72 hours, as determined by western blotting. The numbers represent percent of corresponding control normalized to β-actin levels. Her, Herceptin; anti-miR-200s, anti-miR-200a+anti-miR-200b+anti-miR-200c. *, P
    Rt Pcr Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8597 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rt pcr analysis
    <t>RT-PCR</t> analysis of MUC2 and <t>IL-1β</t> expression in Shigella dysenteriae infected HT 29 cells. HT 29 cells were infected with Shigella dysenteriae for different time intervals. After infection, mRNA was isolated for MUC2 and IL-1β gene expression analysis as described in methods . HT-29 control cells showed basal level expression of both MUC2 and IL-1β. Whereas, higher level (intensity) expression of both MUC2 (∼4–5 fold higher for 9 h) and IL-1β (25 fold higher for 9 h) were seen in a time dependent manner upon HT-29 cells infected with S. dysenteriae . HT 29 cells pre-incubated with Actinomycin D (1 µg/ml) for 30 min, prior to S. dysenteriae infection, showed lower level of expression of IL-1β. The band intensity was measured by using image J program. Amplification of β-actin was used as an internal control.
    Rt Pcr Analysis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad real time rt pcr analysis
    <t>RNA-seq</t> expression analysis of ESC and HC11 MEC undergoing lactogenic differentiation and its validation by real-time <t>PCR.</t> ( A ) Venn diagram showing expression of total, unique and overlapping genes ( > 1 FPKM value) in ESC, Normal (N), GC primed (P) and PRL treated HC11 cells. ( B ) Venn diagram showing unique and overlapping, upregulated genes between ESC vs N, N vs P and P vs PRL treated HC11 cells (Log2 ≥1). ( C ) Venn diagram showing unique and overlapping, downregulated genes between ESC vs N, N vs P and P vs PRL treated HC11 cells (Log2 ≤−1). ( D ) Schematic diagram showing a total number of genes which are differentially upregulated (Up arrow) and downregulated (Down arrow) between ESC vs Normal (N), N vs GC primed (P) and PRL treated HC11 cells. ( E ) Real-time PCR analysis of the top upregulated and ( F ) downregulated in Normal (N), GC primed (P) and PRL treated HC11 cells and their respective RNA-seq FPKM values of ( E’ ) upregulated genes and ( F’ ) downregulated genes.
    Real Time Rt Pcr Analysis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 1116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative rt pcr analysis
    QKI influences <t>pre-mRNA</t> splicing in naive PB monocytes and macrophages. ( a ) SpliceTrap assessment of the proximal ACUAA RNA motif enrichment in 50-bp windows upstream and downstream of alternatively spliced cassette exons (as compared with a background set of exons; grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment over the genomic locus are depicted. ( b ) Sashimi plots illustrate RNA-seq read coverage for selected alternative splicing events in Pat- QKI +/− versus Sib- QKI +/+ PB monocytes (orange) and macrophages (blue). Splicing events (se) are highlighted by inverted brackets. The location of ACUAA motifs and QKI PAR-CLIPs are provided below. Splicing events were defined based on the genomic organization of RefSeq transcripts (bottom tracks). Full event details are provided in Supplementary Data 3 . ( c ) <t>PCR</t> validation of alternatively spliced cassette exons in Sib- QKI +/+ and Pat- QKI +/ − PB-derived monocytes and macrophages. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). ( d ) Phase-contrast and fluorescence-microscopy photographs (scale bar, 50 μm) of primary human, PB macrophages of healthy controls that have been treated with FAM-labelled GapmeRs, to reduce QKI expression. ( e ) Quantitative RT–PCR (qRT–PCR) of QKI mRNA isoform expression in GapmeR-treated macrophages ( n =3). Data expressed as mean±s.e.m.; Student's t -test, with ** P
    Quantitative Rt Pcr Analysis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 510 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa cellamp rt pcr for direct prep kit real time protein analysis
    QKI influences <t>pre-mRNA</t> splicing in naive PB monocytes and macrophages. ( a ) SpliceTrap assessment of the proximal ACUAA RNA motif enrichment in 50-bp windows upstream and downstream of alternatively spliced cassette exons (as compared with a background set of exons; grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment over the genomic locus are depicted. ( b ) Sashimi plots illustrate RNA-seq read coverage for selected alternative splicing events in Pat- QKI +/− versus Sib- QKI +/+ PB monocytes (orange) and macrophages (blue). Splicing events (se) are highlighted by inverted brackets. The location of ACUAA motifs and QKI PAR-CLIPs are provided below. Splicing events were defined based on the genomic organization of RefSeq transcripts (bottom tracks). Full event details are provided in Supplementary Data 3 . ( c ) <t>PCR</t> validation of alternatively spliced cassette exons in Sib- QKI +/+ and Pat- QKI +/ − PB-derived monocytes and macrophages. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). ( d ) Phase-contrast and fluorescence-microscopy photographs (scale bar, 50 μm) of primary human, PB macrophages of healthy controls that have been treated with FAM-labelled GapmeRs, to reduce QKI expression. ( e ) Quantitative RT–PCR (qRT–PCR) of QKI mRNA isoform expression in GapmeR-treated macrophages ( n =3). Data expressed as mean±s.e.m.; Student's t -test, with ** P
    Cellamp Rt Pcr For Direct Prep Kit Real Time Protein Analysis, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative rt pcr analysis
    In vivo expression of <t>mEF</t> . (A) A schema of the wildtype and the mEF alleles before and after Cre recombination. The arrows indicate the three primers used in the genomic <t>PCR</t> analysis. Genomic DNAs from tamoxifen-treated EF;CreER+ (EF/Cre), EF Stop ; CreER
    Quantitative Rt Pcr Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher platinum quantitative rt pcr thermoscript one step system
    In vivo expression of <t>mEF</t> . (A) A schema of the wildtype and the mEF alleles before and after Cre recombination. The arrows indicate the three primers used in the genomic <t>PCR</t> analysis. Genomic DNAs from tamoxifen-treated EF;CreER+ (EF/Cre), EF Stop ; CreER
    Platinum Quantitative Rt Pcr Thermoscript One Step System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 269 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative rt pcr qrt pcr analysis
    <t>QRT-PCR</t> analysis of genes responsible for B cell differentiation. Purified MZ B cells and FO B cells from control WT mice or CD40LBTg mice were cultured in vitro with a combination of IL-4, anti-IgM [F(ab′) 2 ], and anti-CD40 Ab or with CpGODN for
    Quantitative Rt Pcr Qrt Pcr Analysis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taqpath 1 step rt qpcr master mix
    <t>QRT-PCR</t> analysis of genes responsible for B cell differentiation. Purified MZ B cells and FO B cells from control WT mice or CD40LBTg mice were cultured in vitro with a combination of IL-4, anti-IgM [F(ab′) 2 ], and anti-CD40 Ab or with CpGODN for
    Taqpath 1 Step Rt Qpcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co rt pcr analysis total rna
    Expression of the ldhA and pyk2 genes in C. glutamicum . a Identification of the co-transcription of ldhA and pyk2 in the ldhA-pyk2 cluster using <t>RT-PCR.</t> The C. glutamicum WT strain was cultured in minimal medium with glucose under aerobic conditions. The templates used for the PCR were as follows: lanes 1 and 4, total <t>RNA</t> reverse transcribed without reverse transcriptase; lanes 2 and 5, genomic DNA; and lanes 3 and 6, cDNA. The fragments in lanes 1, 2 and 3 were amplified using the primers WZ1181/WZ868 for rpoB. In addition, the primers WZ1171 and WZ1156 were used for the ldhA-pyk2 region in lanes 4, 5 and 6. b The relative transcription levels of the pyk1 , pyk2 and ldhA genes were analyzed by qRT-PCR. Total RNA was isolated from WT cells harvested at the exponential phase under aerobic conditions and at 3 h cultivation under oxygen-deprived conditions. The expression levels of pyk1 , pyk2 and ldhA under different conditions were compared against the expression of pyk1 under aerobic conditions (=1). The mean values from at least three independent cultures are shown with the standard deviations
    Rt Pcr Analysis Total Rna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 92/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SABiosciences real time rt pcr analysis
    Hormonal regulation of EGFR signaling in E- or E+P-treated tumors. a , b Real-time <t>RT-PCR</t> analysis of a EGFR and b EGFR ligand <t>mRNA</t> levels. Bars represent the mean ± SEM fold change compared to levels in E-treated tumors ( n = 4–5 tumors/group). c Representative merged images of immunolabeling with Areg- ( green ), PRA- ( blue ), and PRB- ( red ) specific antibodies. White arrows indicate cells co-expressing Areg and PRA. Yellow arrows indicate cells co-expressing Areg, PRB, and PRA. Scale bar , 75 μm. d Immunoblot analysis of Areg, phospho-Akt, phospho-JNK, and phospho-Erk for the same E- or E+P-treated tumors. Numbers above bands indicate relative band intensity normalized to β-actin
    Real Time Rt Pcr Analysis, supplied by SABiosciences, used in various techniques. Bioz Stars score: 92/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 7900ht fast real time pcr system plus minus assay guide
    Hormonal regulation of EGFR signaling in E- or E+P-treated tumors. a , b Real-time <t>RT-PCR</t> analysis of a EGFR and b EGFR ligand <t>mRNA</t> levels. Bars represent the mean ± SEM fold change compared to levels in E-treated tumors ( n = 4–5 tumors/group). c Representative merged images of immunolabeling with Areg- ( green ), PRA- ( blue ), and PRB- ( red ) specific antibodies. White arrows indicate cells co-expressing Areg and PRA. Yellow arrows indicate cells co-expressing Areg, PRB, and PRA. Scale bar , 75 μm. d Immunoblot analysis of Areg, phospho-Akt, phospho-JNK, and phospho-Erk for the same E- or E+P-treated tumors. Numbers above bands indicate relative band intensity normalized to β-actin
    7900ht Fast Real Time Pcr System Plus Minus Assay Guide, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies quantitative rt pcr analysis
    Hormonal regulation of EGFR signaling in E- or E+P-treated tumors. a , b Real-time <t>RT-PCR</t> analysis of a EGFR and b EGFR ligand <t>mRNA</t> levels. Bars represent the mean ± SEM fold change compared to levels in E-treated tumors ( n = 4–5 tumors/group). c Representative merged images of immunolabeling with Areg- ( green ), PRA- ( blue ), and PRB- ( red ) specific antibodies. White arrows indicate cells co-expressing Areg and PRA. Yellow arrows indicate cells co-expressing Areg, PRB, and PRA. Scale bar , 75 μm. d Immunoblot analysis of Areg, phospho-Akt, phospho-JNK, and phospho-Erk for the same E- or E+P-treated tumors. Numbers above bands indicate relative band intensity normalized to β-actin
    Quantitative Rt Pcr Analysis, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    fluidigm rt pcr analysis software
    Overview of the experimental strategy. CD34+ cells from healthy, mobilized apheresis donors were immunostained with a 10-fluorochrome panel and single cells were index-sorted into 96-well <t>PCR</t> plates for multiplex qRT-PCR analysis using the <t>Fluidigm</t> Biomark platform. MEP subpopulations were identified by principal component analysis (PCA) and correlated with the original index sorting data and mRNA levels of surface antigens. Identified cellular subsets were validated transcriptionally at the population level and functionally in single-cell differentiation assays. Finally, the cells were ordered in pseudotime to assess differentiation trajectories which were then further validated in functional assays. FACS, fluorescence-activated cell sorting; IF, immunofluorescence; qRT-PCR, quantitative real-time polymerase chain reaction
    Rt Pcr Analysis Software, supplied by fluidigm, used in various techniques. Bioz Stars score: 85/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Marker Gene Technologies rt pcr analysis
    <t>RT-PCR</t> analysis of MMP-2 ( A ), MMP-9 ( B ), TIMP-1 ( C ) and TIMP-2 ( D ) gene expression in <t>HepG2</t> cells after treatment with FXT and the attenuating effect of GNP. HepG2 cells were seeded onto a 6-well plate and incubated overnight, treated with FXT and GNP at dose as indicated and further incubated for 72 h. DMSO (1%) was used as vehicle control. Values are expressed as mean ± SD ( n = 3). Different letters indicate statistical significance ( p
    Rt Pcr Analysis, supplied by Marker Gene Technologies, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mirvana qrt pcr mirna detection kit
    <t>RT-PCR</t> analysis of MMP-2 ( A ), MMP-9 ( B ), TIMP-1 ( C ) and TIMP-2 ( D ) gene expression in <t>HepG2</t> cells after treatment with FXT and the attenuating effect of GNP. HepG2 cells were seeded onto a 6-well plate and incubated overnight, treated with FXT and GNP at dose as indicated and further incubated for 72 h. DMSO (1%) was used as vehicle control. Values are expressed as mean ± SD ( n = 3). Different letters indicate statistical significance ( p
    Mirvana Qrt Pcr Mirna Detection Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co quantitative rt pcr analysis total rna
    <t>RT-PCR</t> analysis of MMP-2 ( A ), MMP-9 ( B ), TIMP-1 ( C ) and TIMP-2 ( D ) gene expression in <t>HepG2</t> cells after treatment with FXT and the attenuating effect of GNP. HepG2 cells were seeded onto a 6-well plate and incubated overnight, treated with FXT and GNP at dose as indicated and further incubated for 72 h. DMSO (1%) was used as vehicle control. Values are expressed as mean ± SD ( n = 3). Different letters indicate statistical significance ( p
    Quantitative Rt Pcr Analysis Total Rna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 92/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc rt pcr analysis
    <t>RT-PCR</t> analysis reveals differentially expressed transcripts. RNA from the wild type control and the <t>heph</t> 2 mutant was analyzed by RT-PCR analysis. Gene-specific primer pairs were used for amplification of several randomly picked genes that showed significant differential expression. For Tequila , primer pairs were designed to test alternative 5’ ends (transcription start site usage).
    Rt Pcr Analysis, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of DIM and Herceptin on miR-200 family. ( A ) Comparative expression analysis of miR-200 in SKBR3 and MDA-MB-468 cells by real-time miRNA RT-PCR. ( B and C ) Relative expression analysis of miR-200 in SKBR3 and MDA-MB-468 cells treated with 15 µM DIM, 0.75 µg/ml Herceptin or their combinations for 24 h. ( D ) Effect of anti-miR-200s (anti-miR-200a+anti-miR-200b+anti-miR-200c) on cell proliferation of SKBR3 cells treated with 15 µM DIM and 0.75 µg/ml Herceptin for 72 hours, as measured by MTT assay. ( E ) Efficacy of anti-miR-200s transfections as well the effect of DIM plus Herceptin (15 µM DIM and 0.75 µg/ml Herceptin for 72 hours) on expression levels of anti-miR-200s (miR-200a/miR-200b/miR-200c) was evaluated by real time RT-PCR. ( F ) Effect of anti-miR-200s (anti-miR-200a+anti-miR-200b+anti-miR-200c) on expression of FoxM1 and pAkt in SKBR3 cells treated with 15 µM DIM and 0.75 µg/ml Herceptin for 72 hours, as determined by western blotting. The numbers represent percent of corresponding control normalized to β-actin levels. Her, Herceptin; anti-miR-200s, anti-miR-200a+anti-miR-200b+anti-miR-200c. *, P

    Journal: PLoS ONE

    Article Title: 3, 3?-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

    doi: 10.1371/journal.pone.0054657

    Figure Lengend Snippet: Effects of DIM and Herceptin on miR-200 family. ( A ) Comparative expression analysis of miR-200 in SKBR3 and MDA-MB-468 cells by real-time miRNA RT-PCR. ( B and C ) Relative expression analysis of miR-200 in SKBR3 and MDA-MB-468 cells treated with 15 µM DIM, 0.75 µg/ml Herceptin or their combinations for 24 h. ( D ) Effect of anti-miR-200s (anti-miR-200a+anti-miR-200b+anti-miR-200c) on cell proliferation of SKBR3 cells treated with 15 µM DIM and 0.75 µg/ml Herceptin for 72 hours, as measured by MTT assay. ( E ) Efficacy of anti-miR-200s transfections as well the effect of DIM plus Herceptin (15 µM DIM and 0.75 µg/ml Herceptin for 72 hours) on expression levels of anti-miR-200s (miR-200a/miR-200b/miR-200c) was evaluated by real time RT-PCR. ( F ) Effect of anti-miR-200s (anti-miR-200a+anti-miR-200b+anti-miR-200c) on expression of FoxM1 and pAkt in SKBR3 cells treated with 15 µM DIM and 0.75 µg/ml Herceptin for 72 hours, as determined by western blotting. The numbers represent percent of corresponding control normalized to β-actin levels. Her, Herceptin; anti-miR-200s, anti-miR-200a+anti-miR-200b+anti-miR-200c. *, P

    Article Snippet: miRNA Real-time Reverse Transcription-PCR To verify the alterations in the expression of miR-200 in DIM and Herceptin-treated breast cancer cells, we performed RT-PCR analysis using TaqMan MicroRNA Assay Kit (Applied Biosystems) following the manufacturer’s protocol.

    Techniques: Expressing, Multiple Displacement Amplification, miRNA RT, Polymerase Chain Reaction, MTT Assay, Transfection, Quantitative RT-PCR, Western Blot

    Knockdown of Nrf2 decreases DEM-inducible expression of HO-1 and NQO1 . (A) Immunoblot analysis of BRG1 and hBRM in SW480 and SW13 cells. Nuclear extracts of SW480 and SW13 cells were analyzed by anti-BRG1, anti-hBRM, or anti-lamin B antibodies. (B) SW480 cells were transfected with control siRNA (siCon) or Nrf2 siRNA (siNrf2). At 24 h posttransfection, cells were treated with 100 μM DEM for 4 h and then subjected to immunoblot analysis using anti-Nrf2 (upper panel) or anti-lamin B (lower panel) antibodies. (C) HO-1 and NQO1 expression in siCon and siNrf2 cells after DEM treatment. SW480 cells were transfected with siCon or siNrf2. Following treatment with 100 μM DEM for the indicated time periods, total RNA was isolated and HO-1 and NQO1 mRNA expression was determined by quantitative RT-PCR, with 18S rRNA used as an internal standard. The means of three independent experiments performed in duplicate are shown, and error bars represent SE. HO-1 and NQO1 expression in siCon cells without DEM treatment was set at 1.

    Journal: Molecular and Cellular Biology

    Article Title: BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress ▿

    doi: 10.1128/MCB.00700-06

    Figure Lengend Snippet: Knockdown of Nrf2 decreases DEM-inducible expression of HO-1 and NQO1 . (A) Immunoblot analysis of BRG1 and hBRM in SW480 and SW13 cells. Nuclear extracts of SW480 and SW13 cells were analyzed by anti-BRG1, anti-hBRM, or anti-lamin B antibodies. (B) SW480 cells were transfected with control siRNA (siCon) or Nrf2 siRNA (siNrf2). At 24 h posttransfection, cells were treated with 100 μM DEM for 4 h and then subjected to immunoblot analysis using anti-Nrf2 (upper panel) or anti-lamin B (lower panel) antibodies. (C) HO-1 and NQO1 expression in siCon and siNrf2 cells after DEM treatment. SW480 cells were transfected with siCon or siNrf2. Following treatment with 100 μM DEM for the indicated time periods, total RNA was isolated and HO-1 and NQO1 mRNA expression was determined by quantitative RT-PCR, with 18S rRNA used as an internal standard. The means of three independent experiments performed in duplicate are shown, and error bars represent SE. HO-1 and NQO1 expression in siCon cells without DEM treatment was set at 1.

    Article Snippet: Total RNA (1 μg) was reverse transcribed into cDNA and used for real-time (RT)-PCR analysis (Invitrogen).

    Techniques: Expressing, Transfection, Isolation, Quantitative RT-PCR

    Knockdown of BRG1 reduces the recruitment of RNA Pol II to the HO-1 promoter but not the NOQ1 promoter. SHCon and SH4 cells were treated with 100 μM DEM for 3.5 h for HO-1 or 12 h for NQO1 , and ChIP analyses were performed with antibodies against RNA Pol II and primers for the HO-1 and NQO1 promoter regions (pr). Normal rabbit IgG was used as a control. The 5′ upstream region of the CSF-1 ) was used as a control. In the input lane, 5% unprecipitated chromosomal DNA was amplified by PCR.

    Journal: Molecular and Cellular Biology

    Article Title: BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress ▿

    doi: 10.1128/MCB.00700-06

    Figure Lengend Snippet: Knockdown of BRG1 reduces the recruitment of RNA Pol II to the HO-1 promoter but not the NOQ1 promoter. SHCon and SH4 cells were treated with 100 μM DEM for 3.5 h for HO-1 or 12 h for NQO1 , and ChIP analyses were performed with antibodies against RNA Pol II and primers for the HO-1 and NQO1 promoter regions (pr). Normal rabbit IgG was used as a control. The 5′ upstream region of the CSF-1 ) was used as a control. In the input lane, 5% unprecipitated chromosomal DNA was amplified by PCR.

    Article Snippet: Total RNA (1 μg) was reverse transcribed into cDNA and used for real-time (RT)-PCR analysis (Invitrogen).

    Techniques: Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction

    BRG1 knockdown selectively downregulates DEM-inducible expression of HO-1 . (A) Immunoblot analysis of BRG1 knockdown cell lines. SHCon, SH4, and SH7 cells were treated with 100 μM DEM for 4 h, and the nuclear extracts were subjected to immunoblot analysis with antibodies against BRG1, hBRM, Nrf2, or lamin B. (B and C) RNA blot analysis of BRG1 knockdown cell lines. SHCon, SH4, and SH7 cells were treated with 100 μM DEM for the indicated time periods, and total RNA was isolated and analyzed by RNA blotting for HO-1 , NQO1 , and GAPDH (B). Band intensity was quantified by NIH Image and plotted after normalization with the GAPDH signal (C). The maximal induction level in SHCon cells was set at 100, and the mean relative expression levels from two independent experiments at each time point are presented. (D) Quantitative RT-PCR analysis of GCSL , GCSH , and AKR1C1 . The induction levels of the GCSL , GCSH , and AKR1C1 genes at the 12-h time point in SHCon cells were set at 100, and the means of relative expression from two independent experiments each carried out in duplicate at each time point are presented; 18S rRNA was used as an internal standard.

    Journal: Molecular and Cellular Biology

    Article Title: BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress ▿

    doi: 10.1128/MCB.00700-06

    Figure Lengend Snippet: BRG1 knockdown selectively downregulates DEM-inducible expression of HO-1 . (A) Immunoblot analysis of BRG1 knockdown cell lines. SHCon, SH4, and SH7 cells were treated with 100 μM DEM for 4 h, and the nuclear extracts were subjected to immunoblot analysis with antibodies against BRG1, hBRM, Nrf2, or lamin B. (B and C) RNA blot analysis of BRG1 knockdown cell lines. SHCon, SH4, and SH7 cells were treated with 100 μM DEM for the indicated time periods, and total RNA was isolated and analyzed by RNA blotting for HO-1 , NQO1 , and GAPDH (B). Band intensity was quantified by NIH Image and plotted after normalization with the GAPDH signal (C). The maximal induction level in SHCon cells was set at 100, and the mean relative expression levels from two independent experiments at each time point are presented. (D) Quantitative RT-PCR analysis of GCSL , GCSH , and AKR1C1 . The induction levels of the GCSL , GCSH , and AKR1C1 genes at the 12-h time point in SHCon cells were set at 100, and the means of relative expression from two independent experiments each carried out in duplicate at each time point are presented; 18S rRNA was used as an internal standard.

    Article Snippet: Total RNA (1 μg) was reverse transcribed into cDNA and used for real-time (RT)-PCR analysis (Invitrogen).

    Techniques: Expressing, Northern blot, Isolation, Quantitative RT-PCR

    Expression of BRG1, but not the BRG1K785A mutant or hBRM, enhances inducible expression of the HO-1 gene by DEM in SW13 cells. (A and B) Reconstitution by BRG1 activity in SW13 cells. SW13 cells were cotransfected with either control vector or BRG1 expression plasmid along with pSUPER-puro control vector. Cells were selected with puromycin for 2 days, followed by treatment with 100 μM DEM for the indicated periods. Total RNA was subjected to RNA blot analysis for HO-1 , NQO1 , BRG1 , and GAPDH (A). Band intensities (A) were quantified by NIH Image and plotted after normalization to the GAPDH signal (B). (C) hBRM does not activate HO-1 gene expression. SW13 cells were cotransfected with either control vector, Flag-BRG1, or Flag-hBRM expression plasmids, along with pSUPER-puro control vector. HO-1 mRNA levels were determined by quantitative RT-PCR using 18S rRNA as an internal standard. HO-1 expression in BRG1-overexpressing cells at 24 h after 100 μM DEM treatment was arbitrarily set at 100, and the means of two independent experiments performed in triplicate are presented. (D and E) ATPase activity of BRG1 is indispensable for transactivation. SW13 cells were cotransfected with either control vector or BRG1K785A mutant expression plasmid along with pSUPER-puro control vector. The cells were analyzed as described for panel A. Band intensities in panel D were quantified and plotted after normalization by GAPDH signals (E). (F) Expression of BRG1, BRG1K785A, and hBRM proteins in SW13 cells. SW13 cells were cotransfected with either control vector or BRG1, BRG1K785A mutant, or hBRM expression plasmid along with pSUPER-puro control vector. The nuclear extracts were subjected to immunoblot analysis with anti-Flag (upper panel) or anti-lamin B (bottom panel) antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress ▿

    doi: 10.1128/MCB.00700-06

    Figure Lengend Snippet: Expression of BRG1, but not the BRG1K785A mutant or hBRM, enhances inducible expression of the HO-1 gene by DEM in SW13 cells. (A and B) Reconstitution by BRG1 activity in SW13 cells. SW13 cells were cotransfected with either control vector or BRG1 expression plasmid along with pSUPER-puro control vector. Cells were selected with puromycin for 2 days, followed by treatment with 100 μM DEM for the indicated periods. Total RNA was subjected to RNA blot analysis for HO-1 , NQO1 , BRG1 , and GAPDH (A). Band intensities (A) were quantified by NIH Image and plotted after normalization to the GAPDH signal (B). (C) hBRM does not activate HO-1 gene expression. SW13 cells were cotransfected with either control vector, Flag-BRG1, or Flag-hBRM expression plasmids, along with pSUPER-puro control vector. HO-1 mRNA levels were determined by quantitative RT-PCR using 18S rRNA as an internal standard. HO-1 expression in BRG1-overexpressing cells at 24 h after 100 μM DEM treatment was arbitrarily set at 100, and the means of two independent experiments performed in triplicate are presented. (D and E) ATPase activity of BRG1 is indispensable for transactivation. SW13 cells were cotransfected with either control vector or BRG1K785A mutant expression plasmid along with pSUPER-puro control vector. The cells were analyzed as described for panel A. Band intensities in panel D were quantified and plotted after normalization by GAPDH signals (E). (F) Expression of BRG1, BRG1K785A, and hBRM proteins in SW13 cells. SW13 cells were cotransfected with either control vector or BRG1, BRG1K785A mutant, or hBRM expression plasmid along with pSUPER-puro control vector. The nuclear extracts were subjected to immunoblot analysis with anti-Flag (upper panel) or anti-lamin B (bottom panel) antibodies.

    Article Snippet: Total RNA (1 μg) was reverse transcribed into cDNA and used for real-time (RT)-PCR analysis (Invitrogen).

    Techniques: Expressing, Mutagenesis, Activity Assay, Plasmid Preparation, Northern blot, Quantitative RT-PCR

    RT-PCR analysis of MUC2 and IL-1β expression in Shigella dysenteriae infected HT 29 cells. HT 29 cells were infected with Shigella dysenteriae for different time intervals. After infection, mRNA was isolated for MUC2 and IL-1β gene expression analysis as described in methods . HT-29 control cells showed basal level expression of both MUC2 and IL-1β. Whereas, higher level (intensity) expression of both MUC2 (∼4–5 fold higher for 9 h) and IL-1β (25 fold higher for 9 h) were seen in a time dependent manner upon HT-29 cells infected with S. dysenteriae . HT 29 cells pre-incubated with Actinomycin D (1 µg/ml) for 30 min, prior to S. dysenteriae infection, showed lower level of expression of IL-1β. The band intensity was measured by using image J program. Amplification of β-actin was used as an internal control.

    Journal: PLoS ONE

    Article Title: Up-Regulation of MUC2 and IL-1? Expression in Human Colonic Epithelial Cells by Shigella and Its Interaction with Mucins

    doi: 10.1371/journal.pone.0027046

    Figure Lengend Snippet: RT-PCR analysis of MUC2 and IL-1β expression in Shigella dysenteriae infected HT 29 cells. HT 29 cells were infected with Shigella dysenteriae for different time intervals. After infection, mRNA was isolated for MUC2 and IL-1β gene expression analysis as described in methods . HT-29 control cells showed basal level expression of both MUC2 and IL-1β. Whereas, higher level (intensity) expression of both MUC2 (∼4–5 fold higher for 9 h) and IL-1β (25 fold higher for 9 h) were seen in a time dependent manner upon HT-29 cells infected with S. dysenteriae . HT 29 cells pre-incubated with Actinomycin D (1 µg/ml) for 30 min, prior to S. dysenteriae infection, showed lower level of expression of IL-1β. The band intensity was measured by using image J program. Amplification of β-actin was used as an internal control.

    Article Snippet: RT-PCR analysis of Mucin and IL-1β gene expression Total RNA was extracted from control cells, cells preincubated with actinomycin D (1 µg/ml) for 30 min and Shigella dysenteriae infected HT 29 cells, using Trizol reagent (Sigma- Aldrich, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Isolation, Incubation, Amplification

    PDGF up-regulated miR-150 expression and secretion in SMC in IRE1α / XBP1 dependent manner. ( A ) PDGF increased miR-150 expression and secretion in SMC in an XBP1 splicing dependent manner. HSMCs were infected with non-target ( NTsh ), IRE1α ( Ish ) or XBP1 ( Xsh ) shRNA lentivirus at 100 IU for 48 hr, and then treated with DMEM supplemented with 0.5% FBS for 24 hr, followed by 20 ng/ml PDGF-BB treatment for 4 hr. The cellular and EV miR-150 levels were assessed by quantitative RT-PCR of three independent experiments. (mean ± SEM, * P

    Journal: Scientific Reports

    Article Title: XBP1 splicing triggers miR-150 transfer from smooth muscle cells to endothelial cells via extracellular vesicles

    doi: 10.1038/srep28627

    Figure Lengend Snippet: PDGF up-regulated miR-150 expression and secretion in SMC in IRE1α / XBP1 dependent manner. ( A ) PDGF increased miR-150 expression and secretion in SMC in an XBP1 splicing dependent manner. HSMCs were infected with non-target ( NTsh ), IRE1α ( Ish ) or XBP1 ( Xsh ) shRNA lentivirus at 100 IU for 48 hr, and then treated with DMEM supplemented with 0.5% FBS for 24 hr, followed by 20 ng/ml PDGF-BB treatment for 4 hr. The cellular and EV miR-150 levels were assessed by quantitative RT-PCR of three independent experiments. (mean ± SEM, * P

    Article Snippet: The EVs pellets were subjected to miRNAs isolation and quantitative RT-PCR analysis for miR-150 or reconstituted into the original volume with M199 medium containing 0.5% FBS ( , , except ) or M199 medium supplemented with 5 μg/ml insulin (Sigma) and 5 μg/ml transferrin (Sigma) ( , and ) for cellular function assays.

    Techniques: Expressing, Infection, In Situ Hybridization, shRNA, Quantitative RT-PCR

    VEGF-PI3K/Akt pathway was responsible for Ad- XBP1s /HSMC-derived EVs-mediated EC migration. ( A,B ) Ad- XBP1s /HSMC-derived EVs increased VEGF-A mRNA and Akt phosphorylation in HUVECs. EVs were isolated from Ad- XBP1s /HSMC condition medium (CM), reconstituted in M199 medium supplemented with 5 μg/ml insulin and 5 μg/ml transferrin and applied to HUVECs for time indicated, followed by quantitative RT-PCR analysis of VEGF-A mRNA (A) or western blot analysis of ErK and Akt (S473) phosphorylation ( B ). ( C,D ) Anti-miR-150 abolished XBP1s /HSMC-derived EVs-mediated VEGF-A expression and Akt. EVs were isolated from control (ctl) or anti-miR-150 transfected with Ad- XBP1s -infected HSMCs CM, reconstituted and applied to HUVECs for 2 h, followed by VEGF-A mRNA ( C ) and Akt (S473) phosphorylation assays ( D ). ( E ) SU5416 ablated Ad- XBP1s /HSMC-derived EVs-induced Akt phosphorylation. HUVECs were treated with EVs isolated from Ad- XBP1s in the presence of 10 μM SU5416 for 2 h, followed by Akt phosphorylation assay. ( F ) SU5416 and LY294002 attenuated Ad- XBP1s /HSMC-derived EVs-induced HUVEC migration. Transwell migration assays were performed with EVs isolated from Ad- XBP1s -infected HSMC CM in M199 medium supplemented with insulin and transferrin in the presence of SU5416 (SU) or 10 10 μM LY294002 (LY) for 8 h. Left panel shows the representative images of migrated cells while right panel shows average migrated cells per 10x view. Ad-null and DMSO (DM) were included as control. Data presented are representative images or mean of three independent experiments. (mean ± SEM, * P

    Journal: Scientific Reports

    Article Title: XBP1 splicing triggers miR-150 transfer from smooth muscle cells to endothelial cells via extracellular vesicles

    doi: 10.1038/srep28627

    Figure Lengend Snippet: VEGF-PI3K/Akt pathway was responsible for Ad- XBP1s /HSMC-derived EVs-mediated EC migration. ( A,B ) Ad- XBP1s /HSMC-derived EVs increased VEGF-A mRNA and Akt phosphorylation in HUVECs. EVs were isolated from Ad- XBP1s /HSMC condition medium (CM), reconstituted in M199 medium supplemented with 5 μg/ml insulin and 5 μg/ml transferrin and applied to HUVECs for time indicated, followed by quantitative RT-PCR analysis of VEGF-A mRNA (A) or western blot analysis of ErK and Akt (S473) phosphorylation ( B ). ( C,D ) Anti-miR-150 abolished XBP1s /HSMC-derived EVs-mediated VEGF-A expression and Akt. EVs were isolated from control (ctl) or anti-miR-150 transfected with Ad- XBP1s -infected HSMCs CM, reconstituted and applied to HUVECs for 2 h, followed by VEGF-A mRNA ( C ) and Akt (S473) phosphorylation assays ( D ). ( E ) SU5416 ablated Ad- XBP1s /HSMC-derived EVs-induced Akt phosphorylation. HUVECs were treated with EVs isolated from Ad- XBP1s in the presence of 10 μM SU5416 for 2 h, followed by Akt phosphorylation assay. ( F ) SU5416 and LY294002 attenuated Ad- XBP1s /HSMC-derived EVs-induced HUVEC migration. Transwell migration assays were performed with EVs isolated from Ad- XBP1s -infected HSMC CM in M199 medium supplemented with insulin and transferrin in the presence of SU5416 (SU) or 10 10 μM LY294002 (LY) for 8 h. Left panel shows the representative images of migrated cells while right panel shows average migrated cells per 10x view. Ad-null and DMSO (DM) were included as control. Data presented are representative images or mean of three independent experiments. (mean ± SEM, * P

    Article Snippet: The EVs pellets were subjected to miRNAs isolation and quantitative RT-PCR analysis for miR-150 or reconstituted into the original volume with M199 medium containing 0.5% FBS ( , , except ) or M199 medium supplemented with 5 μg/ml insulin (Sigma) and 5 μg/ml transferrin (Sigma) ( , and ) for cellular function assays.

    Techniques: Derivative Assay, Migration, Isolation, Quantitative RT-PCR, Western Blot, Expressing, CTL Assay, Transfection, Infection, Phosphorylation Assay

    Over-expression of premiR-150 increased VEGF-A expression and VEGF receptor dependent Akt phosphorylation in HUVECs. ( A,B ) Over-expression of premiR-150 increased VEGF-A expression and secretion. HUVECs were transfected with premiR-150 or anti-miR-150 in serum free M199 medium for 5 h, then treated with M199 medium supplemented with 5 μg/ml insulin and 5 μg/ml transferrin for 3 h, followed by quantitative RT-PCR analysis of VEGF-A mRNA ( A ) or ELISA assay to detect VEGF-A in cell culture medium ( B ) premiR and anti-miR control RNA fragments were included as controls. ( C ) SU5416 ablated premiR-150 -induced Akt and ErK phosphorylation. HUVECs were transfected with premiR-150 or anti-miR-150 in serum free M199 medium for 5 h, then treated with M199 medium supplemented with 5 μg/ml insulin and 5 μg/ml transferrin in the presence of 10 μM SU5416 for 3 h, followed by Western blot analysis of Akt and ErK phosphorylation and VEGF expression. premiR RNA fragments DMSO were included as controls. Data presented are representative images or mean of three independent experiments. (mean ± SEM, * P

    Journal: Scientific Reports

    Article Title: XBP1 splicing triggers miR-150 transfer from smooth muscle cells to endothelial cells via extracellular vesicles

    doi: 10.1038/srep28627

    Figure Lengend Snippet: Over-expression of premiR-150 increased VEGF-A expression and VEGF receptor dependent Akt phosphorylation in HUVECs. ( A,B ) Over-expression of premiR-150 increased VEGF-A expression and secretion. HUVECs were transfected with premiR-150 or anti-miR-150 in serum free M199 medium for 5 h, then treated with M199 medium supplemented with 5 μg/ml insulin and 5 μg/ml transferrin for 3 h, followed by quantitative RT-PCR analysis of VEGF-A mRNA ( A ) or ELISA assay to detect VEGF-A in cell culture medium ( B ) premiR and anti-miR control RNA fragments were included as controls. ( C ) SU5416 ablated premiR-150 -induced Akt and ErK phosphorylation. HUVECs were transfected with premiR-150 or anti-miR-150 in serum free M199 medium for 5 h, then treated with M199 medium supplemented with 5 μg/ml insulin and 5 μg/ml transferrin in the presence of 10 μM SU5416 for 3 h, followed by Western blot analysis of Akt and ErK phosphorylation and VEGF expression. premiR RNA fragments DMSO were included as controls. Data presented are representative images or mean of three independent experiments. (mean ± SEM, * P

    Article Snippet: The EVs pellets were subjected to miRNAs isolation and quantitative RT-PCR analysis for miR-150 or reconstituted into the original volume with M199 medium containing 0.5% FBS ( , , except ) or M199 medium supplemented with 5 μg/ml insulin (Sigma) and 5 μg/ml transferrin (Sigma) ( , and ) for cellular function assays.

    Techniques: Over Expression, Expressing, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot

    RNA-seq expression analysis of ESC and HC11 MEC undergoing lactogenic differentiation and its validation by real-time PCR. ( A ) Venn diagram showing expression of total, unique and overlapping genes ( > 1 FPKM value) in ESC, Normal (N), GC primed (P) and PRL treated HC11 cells. ( B ) Venn diagram showing unique and overlapping, upregulated genes between ESC vs N, N vs P and P vs PRL treated HC11 cells (Log2 ≥1). ( C ) Venn diagram showing unique and overlapping, downregulated genes between ESC vs N, N vs P and P vs PRL treated HC11 cells (Log2 ≤−1). ( D ) Schematic diagram showing a total number of genes which are differentially upregulated (Up arrow) and downregulated (Down arrow) between ESC vs Normal (N), N vs GC primed (P) and PRL treated HC11 cells. ( E ) Real-time PCR analysis of the top upregulated and ( F ) downregulated in Normal (N), GC primed (P) and PRL treated HC11 cells and their respective RNA-seq FPKM values of ( E’ ) upregulated genes and ( F’ ) downregulated genes.

    Journal: Scientific Reports

    Article Title: Comprehensive profiling of transcriptional networks specific for lactogenic differentiation of HC11 mammary epithelial stem-like cells

    doi: 10.1038/s41598-018-30122-4

    Figure Lengend Snippet: RNA-seq expression analysis of ESC and HC11 MEC undergoing lactogenic differentiation and its validation by real-time PCR. ( A ) Venn diagram showing expression of total, unique and overlapping genes ( > 1 FPKM value) in ESC, Normal (N), GC primed (P) and PRL treated HC11 cells. ( B ) Venn diagram showing unique and overlapping, upregulated genes between ESC vs N, N vs P and P vs PRL treated HC11 cells (Log2 ≥1). ( C ) Venn diagram showing unique and overlapping, downregulated genes between ESC vs N, N vs P and P vs PRL treated HC11 cells (Log2 ≤−1). ( D ) Schematic diagram showing a total number of genes which are differentially upregulated (Up arrow) and downregulated (Down arrow) between ESC vs Normal (N), N vs GC primed (P) and PRL treated HC11 cells. ( E ) Real-time PCR analysis of the top upregulated and ( F ) downregulated in Normal (N), GC primed (P) and PRL treated HC11 cells and their respective RNA-seq FPKM values of ( E’ ) upregulated genes and ( F’ ) downregulated genes.

    Article Snippet: Real-time RT-PCR analysis Differentially expressed genes in RNA-seq analysis were validated by quantitative real-time PCR (CFX96#Bio-Rad).

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

    Characterization of HC11 MEC undergoing lactogenic differentiation. ( A ) Bright field microscopic images of actively growing ESC, undifferentiated HC11 cells in presence of EGF and INS (N) and HC11 cells primed with GC (P) alone and HC11 cells treated with GC and PRL. Note the formation of clear dome-shaped mammospheres under PRL condition. Scale bar represents 100 μM. ( B ) Immunoblot analysis of cell cycle regulators in actively growing (N*), confluent stage undifferentiated normal (N’) HC11 cells along with HC primed (P) and PRL treated cells showing a gradual reduction in their levels in comparison with Actin-B. Full-length blot ECL images are provided in Supplementary Fig. S2 . ( B’ ) Quantitative analysis of cell cycle regulators normalized against β-Actin showing a gradual reduction in their levels during lactogenic differentiation. ( C ) Table showing the percentage of ESC, N, P and PRL treated HC11 cells at G0/G1, S and G2/M phase of cell cycle showing Predominantly in S phase for ESCs and G0/G1 phage for rest of HC11 cell types. ( D ) Real-time PCR analysis of cell-type specific gene expression analysis representing ESC, N, P, and PRL treated HC11 cells. ( D’ ) RNA-seq data presentative FPKM values for the respective cell-type-specific genes.

    Journal: Scientific Reports

    Article Title: Comprehensive profiling of transcriptional networks specific for lactogenic differentiation of HC11 mammary epithelial stem-like cells

    doi: 10.1038/s41598-018-30122-4

    Figure Lengend Snippet: Characterization of HC11 MEC undergoing lactogenic differentiation. ( A ) Bright field microscopic images of actively growing ESC, undifferentiated HC11 cells in presence of EGF and INS (N) and HC11 cells primed with GC (P) alone and HC11 cells treated with GC and PRL. Note the formation of clear dome-shaped mammospheres under PRL condition. Scale bar represents 100 μM. ( B ) Immunoblot analysis of cell cycle regulators in actively growing (N*), confluent stage undifferentiated normal (N’) HC11 cells along with HC primed (P) and PRL treated cells showing a gradual reduction in their levels in comparison with Actin-B. Full-length blot ECL images are provided in Supplementary Fig. S2 . ( B’ ) Quantitative analysis of cell cycle regulators normalized against β-Actin showing a gradual reduction in their levels during lactogenic differentiation. ( C ) Table showing the percentage of ESC, N, P and PRL treated HC11 cells at G0/G1, S and G2/M phase of cell cycle showing Predominantly in S phase for ESCs and G0/G1 phage for rest of HC11 cell types. ( D ) Real-time PCR analysis of cell-type specific gene expression analysis representing ESC, N, P, and PRL treated HC11 cells. ( D’ ) RNA-seq data presentative FPKM values for the respective cell-type-specific genes.

    Article Snippet: Real-time RT-PCR analysis Differentially expressed genes in RNA-seq analysis were validated by quantitative real-time PCR (CFX96#Bio-Rad).

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

    Real-time PCR validation of top up or downregulated TFs and ERs in ESC and HC11 MEC undergoing lactogenic differentiation. ( A ) Bar chart showing real-time PCR measured relative expression of upregulated and ( B ) downregulated TFs along with its respective RNA-seq FPKM values ( A’ and B’ ) in ESC, N, P and PRL treated HC11 cells. ( C ) Bar chart showing real-time PCR measured relative expression of up-regulated and ( D ) down-regulated ERs along with its respective RNA-seq FPKM values ( C’ and D’ ) in ESC, N, P and PRL treated HC11 cells.

    Journal: Scientific Reports

    Article Title: Comprehensive profiling of transcriptional networks specific for lactogenic differentiation of HC11 mammary epithelial stem-like cells

    doi: 10.1038/s41598-018-30122-4

    Figure Lengend Snippet: Real-time PCR validation of top up or downregulated TFs and ERs in ESC and HC11 MEC undergoing lactogenic differentiation. ( A ) Bar chart showing real-time PCR measured relative expression of upregulated and ( B ) downregulated TFs along with its respective RNA-seq FPKM values ( A’ and B’ ) in ESC, N, P and PRL treated HC11 cells. ( C ) Bar chart showing real-time PCR measured relative expression of up-regulated and ( D ) down-regulated ERs along with its respective RNA-seq FPKM values ( C’ and D’ ) in ESC, N, P and PRL treated HC11 cells.

    Article Snippet: Real-time RT-PCR analysis Differentially expressed genes in RNA-seq analysis were validated by quantitative real-time PCR (CFX96#Bio-Rad).

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

    Depletion of ChREBPβ attenuates glucose-stimulated β-cell proliferation in isolated rat islet cells. A and B : Isolated rat islet cells were treated with lipid-conjugated (Accell) siRNAs and cultured in 5.5 or 15 mmol/L glucose for 96 h. Total RNA was collected and subjected to RT-PCR using primers specific for ChREBPβ or ChREBPα mRNAs. C : Protein extracts were subjected to immunoblotting using an antibody against ChREBPα and β-actin. D : To determine the effects of the siRNAs on ChREBP translocation, cells were treated with control or Accell siRNA against ChREBPβ cultured in 5.5 or 15 mmol/L glucose for 32 h and were fixed and stained for insulin, ChREBP, and DNA (DAPI). E : Isolated rat islet cells were treated with Accell siRNAs and cultured in 5.5 or 15 mmol/L glucose for 96 h and fixed and stained with Ki67 and insulin. F : Quantification of the results in E , wherein at least 1,000 insulin-positive cells were counted. Results are from four different rat islet isolations. siChREBPβ-01 and -02, siRNAs directed against ChREBPβ; siCon, scramble control siRNA. Error bars are the SEM. * P

    Journal: Diabetes

    Article Title: Induction of the ChREBPβ Isoform Is Essential for Glucose-Stimulated β-Cell Proliferation

    doi: 10.2337/db15-0239

    Figure Lengend Snippet: Depletion of ChREBPβ attenuates glucose-stimulated β-cell proliferation in isolated rat islet cells. A and B : Isolated rat islet cells were treated with lipid-conjugated (Accell) siRNAs and cultured in 5.5 or 15 mmol/L glucose for 96 h. Total RNA was collected and subjected to RT-PCR using primers specific for ChREBPβ or ChREBPα mRNAs. C : Protein extracts were subjected to immunoblotting using an antibody against ChREBPα and β-actin. D : To determine the effects of the siRNAs on ChREBP translocation, cells were treated with control or Accell siRNA against ChREBPβ cultured in 5.5 or 15 mmol/L glucose for 32 h and were fixed and stained for insulin, ChREBP, and DNA (DAPI). E : Isolated rat islet cells were treated with Accell siRNAs and cultured in 5.5 or 15 mmol/L glucose for 96 h and fixed and stained with Ki67 and insulin. F : Quantification of the results in E , wherein at least 1,000 insulin-positive cells were counted. Results are from four different rat islet isolations. siChREBPβ-01 and -02, siRNAs directed against ChREBPβ; siCon, scramble control siRNA. Error bars are the SEM. * P

    Article Snippet: RNA Isolation and Quantitative RT-PCR Analysis RNA (0.5–1 μg) was used for reverse transcription, and RT-PCR analysis was conducted using The iTaq Universal SYBR Green Supermix (Bio-Rad, Cat. #172-5124) on the 7500 Applied Biosystems Real-Time System.

    Techniques: Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Translocation Assay, Staining

    Glucose induces ChREBPβ in primary rat and human islet cells. A – G : Dispersed rat islet cells were incubated in media containing 5.5 or 15 mmol/L glucose for 1 to 4 days, and the expression of the indicated genes were determined and shown as fold change relative to each day after normalizing to β-actin using the ΔΔCT method. H : Isolated human islet cells were cultured in media containing 5.5 or 15 mmol/L glucose for 1, 2, or 4 days. Total RNA was isolated and subjected to RT-PCR using primers specific for the indicated genes. The data are expressed as a fold change from 5.5 mmol/L glucose. I : Rat islets were isolated, dispersed, and incubated with lipid-conjugated Accell siRNA for 4 days. Total RNA was isolated and subjected to RT-PCR. Data are presented as relative to the scramble control (SiCon) 15 mmol/L treatment, after normalization to β-actin using the ΔΔCT method. Error bars are the SEM ( n = 3–4 for rat islets, n = 5–9 for human islets). ChREBPComm, ChREBP-common; siChREBPβ01 and -02, siRNAs directed against ChREBPβ. * P

    Journal: Diabetes

    Article Title: Induction of the ChREBPβ Isoform Is Essential for Glucose-Stimulated β-Cell Proliferation

    doi: 10.2337/db15-0239

    Figure Lengend Snippet: Glucose induces ChREBPβ in primary rat and human islet cells. A – G : Dispersed rat islet cells were incubated in media containing 5.5 or 15 mmol/L glucose for 1 to 4 days, and the expression of the indicated genes were determined and shown as fold change relative to each day after normalizing to β-actin using the ΔΔCT method. H : Isolated human islet cells were cultured in media containing 5.5 or 15 mmol/L glucose for 1, 2, or 4 days. Total RNA was isolated and subjected to RT-PCR using primers specific for the indicated genes. The data are expressed as a fold change from 5.5 mmol/L glucose. I : Rat islets were isolated, dispersed, and incubated with lipid-conjugated Accell siRNA for 4 days. Total RNA was isolated and subjected to RT-PCR. Data are presented as relative to the scramble control (SiCon) 15 mmol/L treatment, after normalization to β-actin using the ΔΔCT method. Error bars are the SEM ( n = 3–4 for rat islets, n = 5–9 for human islets). ChREBPComm, ChREBP-common; siChREBPβ01 and -02, siRNAs directed against ChREBPβ. * P

    Article Snippet: RNA Isolation and Quantitative RT-PCR Analysis RNA (0.5–1 μg) was used for reverse transcription, and RT-PCR analysis was conducted using The iTaq Universal SYBR Green Supermix (Bio-Rad, Cat. #172-5124) on the 7500 Applied Biosystems Real-Time System.

    Techniques: Incubation, Expressing, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    TIP60 inhibits Sp1 function. (A, B) TIP60 and Sp1 were depleted in HeLa cells using siRNA transfection. Cells were harvested 72 h post transfection, RNA and protein was isolated to use for real time PCR and western blotting analysis. Real-time PCR analysis was used to study TERT expression upon TIP60 and Sp1 knockdown. All mRNA expression data was normalized to GAPDH and plotted as fold change. Endogenous TIP60 and Sp1 were detected using anti-TIP60 and anti-Sp1 antibodies respectively. All the bands observed when probed with anti-TIP60 correspond to TIP60 protein since they are specifically reduced upon depletion of TIP60. α-Actinin serves as the loading control for the western blot. ( C ) Quantification of TIP60 bands using Image J software, from three independent experiments has also been depicted. (D) Relative telomerase activity was measured by qTRAP in the same set of samples. Error bars reflect the standard error of mean (SEM) of 3 independent experiments and significance is represented as ***, P

    Journal: PLoS Pathogens

    Article Title: TIP60 represses telomerase expression by inhibiting Sp1 binding to the TERT promoter

    doi: 10.1371/journal.ppat.1006681

    Figure Lengend Snippet: TIP60 inhibits Sp1 function. (A, B) TIP60 and Sp1 were depleted in HeLa cells using siRNA transfection. Cells were harvested 72 h post transfection, RNA and protein was isolated to use for real time PCR and western blotting analysis. Real-time PCR analysis was used to study TERT expression upon TIP60 and Sp1 knockdown. All mRNA expression data was normalized to GAPDH and plotted as fold change. Endogenous TIP60 and Sp1 were detected using anti-TIP60 and anti-Sp1 antibodies respectively. All the bands observed when probed with anti-TIP60 correspond to TIP60 protein since they are specifically reduced upon depletion of TIP60. α-Actinin serves as the loading control for the western blot. ( C ) Quantification of TIP60 bands using Image J software, from three independent experiments has also been depicted. (D) Relative telomerase activity was measured by qTRAP in the same set of samples. Error bars reflect the standard error of mean (SEM) of 3 independent experiments and significance is represented as ***, P

    Article Snippet: Real-time PCR (RT-PCR) analysis The total RNA was converted into complementary DNA (cDNA) using iSCRIPT cDNA synthesis kit (Bio-Rad Cat. No. 170–8891).

    Techniques: Transfection, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Software, Activity Assay

    TIP60 regulates TERT expression and activity. (A) Telomerase activity was measured by qTRAP, using the whole cell lysates from HeLa-LPCX and HeLa-TIP60 cells. Heat inactivated samples served as negative control. Stable overexpression of Flag-tagged TIP60 in HeLa cells causes a decrease in the telomerase activity. (B) The stable cell lines generated were validated by western blotting using anti-Flag antibody. α-Actinin serves as a loading control to indicate equal protein amount used for qTRAP assay. (C) RNA was isolated from HeLa-LPCX and HeLa-TIP60 cells and real time PCR was performed to check for TERT expression. (D) TIP60 was transiently depleted in HeLa-TIP60 cells using siRNA and cells were harvested 72 h post transfection. Telomerase activity was measured. (E) Efficiency of TIP60 depletion was confirmed by western blotting. TIP60 was detected using an endogenous TIP60 antibody and α-Actinin serves as loading control. (F) Cellular RNA was isolated and analyzed by real time PCR after knockdown to determine TERT expression. mRNA expression was normalized to GAPDH and plotted as fold change. ( G ) Relative telomerase activity was measured for HK (human primary keratinocytes) and HK 18 (human keratinocytes expressing integrated HPV 18 genome) cells. ( H ) Detection of TIP60, 18 E6 and α-Actinin using antibodies for endogenous proteins in HK and HK18 cells. ( I ) RNA was isolated from HK and HK 18 and TERT expression was detected by qPCR. ( J ) Transient depletion of EDD1 in HeLa, the ubiquitin ligase known to destabilize TIP60 and measurement of relative telomerase activity. ( K ) Western blotting of the same samples harvested 72 h after siRNA transfection, with the indicated antibodies to detect endogenous levels of TIP60, EDD1 and E6. ( L ) qPCR to detect changes in TERT mRNA expression upon EDD1 depletion. Error bars reflect the standard error of mean (SEM) of 3 independent experiments and significance is represented as *, P

    Journal: PLoS Pathogens

    Article Title: TIP60 represses telomerase expression by inhibiting Sp1 binding to the TERT promoter

    doi: 10.1371/journal.ppat.1006681

    Figure Lengend Snippet: TIP60 regulates TERT expression and activity. (A) Telomerase activity was measured by qTRAP, using the whole cell lysates from HeLa-LPCX and HeLa-TIP60 cells. Heat inactivated samples served as negative control. Stable overexpression of Flag-tagged TIP60 in HeLa cells causes a decrease in the telomerase activity. (B) The stable cell lines generated were validated by western blotting using anti-Flag antibody. α-Actinin serves as a loading control to indicate equal protein amount used for qTRAP assay. (C) RNA was isolated from HeLa-LPCX and HeLa-TIP60 cells and real time PCR was performed to check for TERT expression. (D) TIP60 was transiently depleted in HeLa-TIP60 cells using siRNA and cells were harvested 72 h post transfection. Telomerase activity was measured. (E) Efficiency of TIP60 depletion was confirmed by western blotting. TIP60 was detected using an endogenous TIP60 antibody and α-Actinin serves as loading control. (F) Cellular RNA was isolated and analyzed by real time PCR after knockdown to determine TERT expression. mRNA expression was normalized to GAPDH and plotted as fold change. ( G ) Relative telomerase activity was measured for HK (human primary keratinocytes) and HK 18 (human keratinocytes expressing integrated HPV 18 genome) cells. ( H ) Detection of TIP60, 18 E6 and α-Actinin using antibodies for endogenous proteins in HK and HK18 cells. ( I ) RNA was isolated from HK and HK 18 and TERT expression was detected by qPCR. ( J ) Transient depletion of EDD1 in HeLa, the ubiquitin ligase known to destabilize TIP60 and measurement of relative telomerase activity. ( K ) Western blotting of the same samples harvested 72 h after siRNA transfection, with the indicated antibodies to detect endogenous levels of TIP60, EDD1 and E6. ( L ) qPCR to detect changes in TERT mRNA expression upon EDD1 depletion. Error bars reflect the standard error of mean (SEM) of 3 independent experiments and significance is represented as *, P

    Article Snippet: Real-time PCR (RT-PCR) analysis The total RNA was converted into complementary DNA (cDNA) using iSCRIPT cDNA synthesis kit (Bio-Rad Cat. No. 170–8891).

    Techniques: Expressing, Activity Assay, Negative Control, Over Expression, Stable Transfection, Generated, Western Blot, Qtrap Assay, Isolation, Real-time Polymerase Chain Reaction, Transfection

    QKI influences pre-mRNA splicing in naive PB monocytes and macrophages. ( a ) SpliceTrap assessment of the proximal ACUAA RNA motif enrichment in 50-bp windows upstream and downstream of alternatively spliced cassette exons (as compared with a background set of exons; grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment over the genomic locus are depicted. ( b ) Sashimi plots illustrate RNA-seq read coverage for selected alternative splicing events in Pat- QKI +/− versus Sib- QKI +/+ PB monocytes (orange) and macrophages (blue). Splicing events (se) are highlighted by inverted brackets. The location of ACUAA motifs and QKI PAR-CLIPs are provided below. Splicing events were defined based on the genomic organization of RefSeq transcripts (bottom tracks). Full event details are provided in Supplementary Data 3 . ( c ) PCR validation of alternatively spliced cassette exons in Sib- QKI +/+ and Pat- QKI +/ − PB-derived monocytes and macrophages. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). ( d ) Phase-contrast and fluorescence-microscopy photographs (scale bar, 50 μm) of primary human, PB macrophages of healthy controls that have been treated with FAM-labelled GapmeRs, to reduce QKI expression. ( e ) Quantitative RT–PCR (qRT–PCR) of QKI mRNA isoform expression in GapmeR-treated macrophages ( n =3). Data expressed as mean±s.e.m.; Student's t -test, with ** P

    Journal: Nature Communications

    Article Title: Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression

    doi: 10.1038/ncomms10846

    Figure Lengend Snippet: QKI influences pre-mRNA splicing in naive PB monocytes and macrophages. ( a ) SpliceTrap assessment of the proximal ACUAA RNA motif enrichment in 50-bp windows upstream and downstream of alternatively spliced cassette exons (as compared with a background set of exons; grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment over the genomic locus are depicted. ( b ) Sashimi plots illustrate RNA-seq read coverage for selected alternative splicing events in Pat- QKI +/− versus Sib- QKI +/+ PB monocytes (orange) and macrophages (blue). Splicing events (se) are highlighted by inverted brackets. The location of ACUAA motifs and QKI PAR-CLIPs are provided below. Splicing events were defined based on the genomic organization of RefSeq transcripts (bottom tracks). Full event details are provided in Supplementary Data 3 . ( c ) PCR validation of alternatively spliced cassette exons in Sib- QKI +/+ and Pat- QKI +/ − PB-derived monocytes and macrophages. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). ( d ) Phase-contrast and fluorescence-microscopy photographs (scale bar, 50 μm) of primary human, PB macrophages of healthy controls that have been treated with FAM-labelled GapmeRs, to reduce QKI expression. ( e ) Quantitative RT–PCR (qRT–PCR) of QKI mRNA isoform expression in GapmeR-treated macrophages ( n =3). Data expressed as mean±s.e.m.; Student's t -test, with ** P

    Article Snippet: Quantitative RT–PCR analysis for designated mRNA products was performed using SYBR Green master mix (Bio-Rad, Veenendaal, The Netherlands).

    Techniques: RNA Sequencing Assay, Polymerase Chain Reaction, Derivative Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR

    QKI expression levels influence pre-mRNA splicing during THP-1-based monocyte-like to macrophage-like cell differentiation. ( a ) Schematic depicting detectable alternative splicing events with the splicing-sensitive microarray platform and number of inclusion (incl.; top lines) or exclusion (excl.; bottom lines) events observed in unstimulated THP-1 ‘monocytes' (left) and 3-day PMA-stimulated THP-1 ‘macrophages' ( n =3, q ≤0.05). ( b ) Scatterplots of skip ( y axis) and include ( x axis) probe set intensity for selected alternative splicing events in sh-Cont (blue boxes) versus sh-QKI (orange circles) in unstimulated and 3 days PMA-stimulated THP-1 ‘monocytes' and ‘macrophages', respectively. Regression coefficients (constrained to pass the origin) are depicted as solid lines. The log 2 difference in the slopes (termed separation score; ss) are provided to the right of the plots for each event, with for example, an ss of −1.72, indicating a 3.3-fold more inclusion of ADD3 exon 13 in sh-QKI versus sh-Cont THP-1 ‘monocytes'. Full event details are provided in Supplementary Data 6 . CE, cassette exon; Alt 5′ or 3′, alternative 5′ or 3′ splice site; RI, retained intron. ( c ) SpliceTrap assessment of average proximal ACUAA RNA motif enrichment in 50 bp windows upstream and downstream of alternatively spliced cassette exons as compared with a background set of exons (grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment are depicted. ( d ) PCR validation of alternatively spliced cassette exons in sh-Cont and sh-QKI THP-1 ‘monocytes' and ‘macrophages'. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). All experiments depict biological n =3. ( e ) PCR validation of three splicing events in wt and qk v mouse-derived primary monocytes and 7 days M-CSF-stimulated macrophages. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). Depicted is a representative PCR for at least a biological n ≥3.

    Journal: Nature Communications

    Article Title: Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression

    doi: 10.1038/ncomms10846

    Figure Lengend Snippet: QKI expression levels influence pre-mRNA splicing during THP-1-based monocyte-like to macrophage-like cell differentiation. ( a ) Schematic depicting detectable alternative splicing events with the splicing-sensitive microarray platform and number of inclusion (incl.; top lines) or exclusion (excl.; bottom lines) events observed in unstimulated THP-1 ‘monocytes' (left) and 3-day PMA-stimulated THP-1 ‘macrophages' ( n =3, q ≤0.05). ( b ) Scatterplots of skip ( y axis) and include ( x axis) probe set intensity for selected alternative splicing events in sh-Cont (blue boxes) versus sh-QKI (orange circles) in unstimulated and 3 days PMA-stimulated THP-1 ‘monocytes' and ‘macrophages', respectively. Regression coefficients (constrained to pass the origin) are depicted as solid lines. The log 2 difference in the slopes (termed separation score; ss) are provided to the right of the plots for each event, with for example, an ss of −1.72, indicating a 3.3-fold more inclusion of ADD3 exon 13 in sh-QKI versus sh-Cont THP-1 ‘monocytes'. Full event details are provided in Supplementary Data 6 . CE, cassette exon; Alt 5′ or 3′, alternative 5′ or 3′ splice site; RI, retained intron. ( c ) SpliceTrap assessment of average proximal ACUAA RNA motif enrichment in 50 bp windows upstream and downstream of alternatively spliced cassette exons as compared with a background set of exons (grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment are depicted. ( d ) PCR validation of alternatively spliced cassette exons in sh-Cont and sh-QKI THP-1 ‘monocytes' and ‘macrophages'. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). All experiments depict biological n =3. ( e ) PCR validation of three splicing events in wt and qk v mouse-derived primary monocytes and 7 days M-CSF-stimulated macrophages. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). Depicted is a representative PCR for at least a biological n ≥3.

    Article Snippet: Quantitative RT–PCR analysis for designated mRNA products was performed using SYBR Green master mix (Bio-Rad, Veenendaal, The Netherlands).

    Techniques: Expressing, Cell Differentiation, Microarray, Polymerase Chain Reaction, Derivative Assay

    Effect of IS on inflammatory cytokine expression on C2C12 myoblast cells. Effect of IS on inflammatory cytokines expression was determined by real time RT-PCR. C2C12 myoblast cells were starved with serum free medium for 2 hr, and then treated with IS for 24, 48 or 72 hr. After the incubation, total RNA was collected and the mRNA expression of ( A ) TNF-α, ( C ) IL-6 or ( E ) TGF-β1 in C2C12 myoblast cells were determined. To determine the effect of inhibitors, cells were co-incubated in the presence or absence of ascorbic acid (AsA, a ROS scavenger), probenecid (Prob, Oat inhibitor) and CH-223191 (AHR inhibitor) for 72 hr, and the mRNA expression of ( B ) TNF-α ( D ) IL-6 or ( F ) TGF-β1 in C2C12 myoblast cells were determined. Data are expressed the means ± SEM (n = 3~5). * p

    Journal: Scientific Reports

    Article Title: Indoxyl sulfate potentiates skeletal muscle atrophy by inducing the oxidative stress-mediated expression of myostatin and atrogin-1

    doi: 10.1038/srep32084

    Figure Lengend Snippet: Effect of IS on inflammatory cytokine expression on C2C12 myoblast cells. Effect of IS on inflammatory cytokines expression was determined by real time RT-PCR. C2C12 myoblast cells were starved with serum free medium for 2 hr, and then treated with IS for 24, 48 or 72 hr. After the incubation, total RNA was collected and the mRNA expression of ( A ) TNF-α, ( C ) IL-6 or ( E ) TGF-β1 in C2C12 myoblast cells were determined. To determine the effect of inhibitors, cells were co-incubated in the presence or absence of ascorbic acid (AsA, a ROS scavenger), probenecid (Prob, Oat inhibitor) and CH-223191 (AHR inhibitor) for 72 hr, and the mRNA expression of ( B ) TNF-α ( D ) IL-6 or ( F ) TGF-β1 in C2C12 myoblast cells were determined. Data are expressed the means ± SEM (n = 3~5). * p

    Article Snippet: Quantitative real time RT-PCR analysis of IL-6, TNF-α, TGF-β1, myostatin, atrogin-1, MyoD, myogenin and GAPDH was performed in an iCycler thermal cycler (Bio-Rad) with an iQ5 qRT-PCR detection system attached (Bio-Rad) using SYBR® Premix Ex TaqII (Perfect Real Time) (TaKaRa Bio Inc.).

    Techniques: Expressing, Quantitative RT-PCR, Incubation

    Effect of IS administration on myostatin expression and muscle atrophy- or myogenic-related genes expression or Akt phosphorylation in the skeletal muscle of 1/2 Nx mice. After IS administration for 12 weeks, ( A ) mRNA and ( B ) protein expressions of myostatin in gastrocnemius were determined by real time RT-PCR and Western blots. mRNA expression of ( C ) atrogin-1 in gastrocnemius were determined by real time RT-PCR. ( D ) Cryosections of tibialis anterior muscles were immunostained with anti-laminin to assess myofiber size. ( E ) Muscle degradation marker, 14 kDa actin fragment was determined by Western blot. ( F ) MyoD and myogenin expression in gastrocnemius were determined by Western blots. ( G ) Akt phosphorylation in gastrocnemius was determined by Western blots. Relative intensity of pAkt/Akt was quantified using the ImageJ software. Data are expressed the means ± SEM (n = 6~8). * p

    Journal: Scientific Reports

    Article Title: Indoxyl sulfate potentiates skeletal muscle atrophy by inducing the oxidative stress-mediated expression of myostatin and atrogin-1

    doi: 10.1038/srep32084

    Figure Lengend Snippet: Effect of IS administration on myostatin expression and muscle atrophy- or myogenic-related genes expression or Akt phosphorylation in the skeletal muscle of 1/2 Nx mice. After IS administration for 12 weeks, ( A ) mRNA and ( B ) protein expressions of myostatin in gastrocnemius were determined by real time RT-PCR and Western blots. mRNA expression of ( C ) atrogin-1 in gastrocnemius were determined by real time RT-PCR. ( D ) Cryosections of tibialis anterior muscles were immunostained with anti-laminin to assess myofiber size. ( E ) Muscle degradation marker, 14 kDa actin fragment was determined by Western blot. ( F ) MyoD and myogenin expression in gastrocnemius were determined by Western blots. ( G ) Akt phosphorylation in gastrocnemius was determined by Western blots. Relative intensity of pAkt/Akt was quantified using the ImageJ software. Data are expressed the means ± SEM (n = 6~8). * p

    Article Snippet: Quantitative real time RT-PCR analysis of IL-6, TNF-α, TGF-β1, myostatin, atrogin-1, MyoD, myogenin and GAPDH was performed in an iCycler thermal cycler (Bio-Rad) with an iQ5 qRT-PCR detection system attached (Bio-Rad) using SYBR® Premix Ex TaqII (Perfect Real Time) (TaKaRa Bio Inc.).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot, Marker, Software

    Effect of IS on the expression of myostatin, skeletal muscle atrophy- or myogenic-related genes and Akt phosphorylation in C2C12 myoblast cells. Effect of IS on ( A,B ) mRNA and ( C ) protein expression of myostatin, mRNA expression of ( D,E ) atrogin-1 were determined by real time RT-PCR. ( F ) Effect of AHR RNAi on IS-induced myostatin or atrogin-1 expression was determined by real time RT-PCR. ( G ) Protein expression of MyoD and myogenin in C2C12 myoblast cells were determined by Western blots. ( H ) Phosphorylation of Akt was detected by Western blots. C2C12 myoblast cells were starved with serum free medium for 2 hr, and then treated with IS for 24, 48 or 72 hr. After the incubation, total RNA or a whole cell lysate was collected and mRNA or protein expressions in the C2C12 myoblast cells were determined. To determine the effect of inhibitors, the cells were co-incubated in the presence or absence of ascorbic acid (AsA, a ROS scavenger), probenecid (Prob, Oat inhibitor) and CH-223191 (AHR inhibitor) for 72 hr, and the mRNA expression of ( B ) myostatin or ( E ) atrogin-1 in C2C12 myoblast cells were determined. Data are expressed the means ± SEM (n = 3~4). * p

    Journal: Scientific Reports

    Article Title: Indoxyl sulfate potentiates skeletal muscle atrophy by inducing the oxidative stress-mediated expression of myostatin and atrogin-1

    doi: 10.1038/srep32084

    Figure Lengend Snippet: Effect of IS on the expression of myostatin, skeletal muscle atrophy- or myogenic-related genes and Akt phosphorylation in C2C12 myoblast cells. Effect of IS on ( A,B ) mRNA and ( C ) protein expression of myostatin, mRNA expression of ( D,E ) atrogin-1 were determined by real time RT-PCR. ( F ) Effect of AHR RNAi on IS-induced myostatin or atrogin-1 expression was determined by real time RT-PCR. ( G ) Protein expression of MyoD and myogenin in C2C12 myoblast cells were determined by Western blots. ( H ) Phosphorylation of Akt was detected by Western blots. C2C12 myoblast cells were starved with serum free medium for 2 hr, and then treated with IS for 24, 48 or 72 hr. After the incubation, total RNA or a whole cell lysate was collected and mRNA or protein expressions in the C2C12 myoblast cells were determined. To determine the effect of inhibitors, the cells were co-incubated in the presence or absence of ascorbic acid (AsA, a ROS scavenger), probenecid (Prob, Oat inhibitor) and CH-223191 (AHR inhibitor) for 72 hr, and the mRNA expression of ( B ) myostatin or ( E ) atrogin-1 in C2C12 myoblast cells were determined. Data are expressed the means ± SEM (n = 3~4). * p

    Article Snippet: Quantitative real time RT-PCR analysis of IL-6, TNF-α, TGF-β1, myostatin, atrogin-1, MyoD, myogenin and GAPDH was performed in an iCycler thermal cycler (Bio-Rad) with an iQ5 qRT-PCR detection system attached (Bio-Rad) using SYBR® Premix Ex TaqII (Perfect Real Time) (TaKaRa Bio Inc.).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Incubation

    Effect of IS administration on inflammatory cytokines expression in the skeletal muscle of 1/2 Nx mice. After IS administration for 12 weeks, the mRNA expression of ( A ) TNF-α, ( B ) IL-6 and ( C ) TGF-β1 in gastrocnemius were determined by real time RT-PCR. Data are expressed the means ± SEM (n = 6~8). * p

    Journal: Scientific Reports

    Article Title: Indoxyl sulfate potentiates skeletal muscle atrophy by inducing the oxidative stress-mediated expression of myostatin and atrogin-1

    doi: 10.1038/srep32084

    Figure Lengend Snippet: Effect of IS administration on inflammatory cytokines expression in the skeletal muscle of 1/2 Nx mice. After IS administration for 12 weeks, the mRNA expression of ( A ) TNF-α, ( B ) IL-6 and ( C ) TGF-β1 in gastrocnemius were determined by real time RT-PCR. Data are expressed the means ± SEM (n = 6~8). * p

    Article Snippet: Quantitative real time RT-PCR analysis of IL-6, TNF-α, TGF-β1, myostatin, atrogin-1, MyoD, myogenin and GAPDH was performed in an iCycler thermal cycler (Bio-Rad) with an iQ5 qRT-PCR detection system attached (Bio-Rad) using SYBR® Premix Ex TaqII (Perfect Real Time) (TaKaRa Bio Inc.).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR

    LPS-induced iNOS expression and nitrite formation by monocytes in vitro are unaffected by the addition of UA, inosine, and inosinic acid. RAW 264.7 cells were activated by LPS as described in Experimental Procedures in the presence and absence of 200 μM UA, inosine, or inosinic acid. iNOS expression was determined by real-time quantitative RT-PCR ( A ), and nitrite production in culture medium was measured by the Griess reaction ( B ) as detailed in Experimental Procedures . Results are expressed as mean ± SD of a minimum of two independent samples assayed in duplicate.

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

    Article Title: Therapeutic intervention in experimental allergic encephalomyelitis by administration of uric acid precursors

    doi: 10.1073/pnas.212645999

    Figure Lengend Snippet: LPS-induced iNOS expression and nitrite formation by monocytes in vitro are unaffected by the addition of UA, inosine, and inosinic acid. RAW 264.7 cells were activated by LPS as described in Experimental Procedures in the presence and absence of 200 μM UA, inosine, or inosinic acid. iNOS expression was determined by real-time quantitative RT-PCR ( A ), and nitrite production in culture medium was measured by the Griess reaction ( B ) as detailed in Experimental Procedures . Results are expressed as mean ± SD of a minimum of two independent samples assayed in duplicate.

    Article Snippet: The activation of iNOS genes was assessed by real-time quantitative RT-PCR analysis of the expression of the specific mRNAs by using a Bio-Rad (Hercules, CA) iCycler iQ real-time detection system as detailed ( ).

    Techniques: Expressing, In Vitro, Quantitative RT-PCR

    In vivo expression of mEF . (A) A schema of the wildtype and the mEF alleles before and after Cre recombination. The arrows indicate the three primers used in the genomic PCR analysis. Genomic DNAs from tamoxifen-treated EF;CreER+ (EF/Cre), EF Stop ; CreER

    Journal: Cancer research

    Article Title: EWS/FLI1 Oncogene Activates Caspase 3 Transcription and Triggers Apoptosis In Vivo

    doi: 10.1158/0008-5472.CAN-09-1993

    Figure Lengend Snippet: In vivo expression of mEF . (A) A schema of the wildtype and the mEF alleles before and after Cre recombination. The arrows indicate the three primers used in the genomic PCR analysis. Genomic DNAs from tamoxifen-treated EF;CreER+ (EF/Cre), EF Stop ; CreER

    Article Snippet: We confirmed the induction of Casp3 following mEF expression in EF;CreER+ MEFs using a quantitative RT-PCR analysis (TaqMan, Applied Biosystems) (data not shown), as well as by immunoblotting ( ).

    Techniques: In Vivo, Expressing, Polymerase Chain Reaction

    QRT-PCR analysis of genes responsible for B cell differentiation. Purified MZ B cells and FO B cells from control WT mice or CD40LBTg mice were cultured in vitro with a combination of IL-4, anti-IgM [F(ab′) 2 ], and anti-CD40 Ab or with CpGODN for

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Constitutive CD40L Expression on B Cells Prematurely Terminates Germinal Center Response and Leads to Augmented Plasma Cell Production in T Cell Areas

    doi: 10.4049/jimmunol.0901689

    Figure Lengend Snippet: QRT-PCR analysis of genes responsible for B cell differentiation. Purified MZ B cells and FO B cells from control WT mice or CD40LBTg mice were cultured in vitro with a combination of IL-4, anti-IgM [F(ab′) 2 ], and anti-CD40 Ab or with CpGODN for

    Article Snippet: Quantitative RT-PCR (QRT-PCR) analysis was performed using primers from SABioscience with an iQ5 cycler (Bio-Rad, Hercules, CA).

    Techniques: Quantitative RT-PCR, Cell Differentiation, Purification, Mouse Assay, Cell Culture, In Vitro

    Expression of the ldhA and pyk2 genes in C. glutamicum . a Identification of the co-transcription of ldhA and pyk2 in the ldhA-pyk2 cluster using RT-PCR. The C. glutamicum WT strain was cultured in minimal medium with glucose under aerobic conditions. The templates used for the PCR were as follows: lanes 1 and 4, total RNA reverse transcribed without reverse transcriptase; lanes 2 and 5, genomic DNA; and lanes 3 and 6, cDNA. The fragments in lanes 1, 2 and 3 were amplified using the primers WZ1181/WZ868 for rpoB. In addition, the primers WZ1171 and WZ1156 were used for the ldhA-pyk2 region in lanes 4, 5 and 6. b The relative transcription levels of the pyk1 , pyk2 and ldhA genes were analyzed by qRT-PCR. Total RNA was isolated from WT cells harvested at the exponential phase under aerobic conditions and at 3 h cultivation under oxygen-deprived conditions. The expression levels of pyk1 , pyk2 and ldhA under different conditions were compared against the expression of pyk1 under aerobic conditions (=1). The mean values from at least three independent cultures are shown with the standard deviations

    Journal: BMC Biotechnology

    Article Title: A novel pyruvate kinase and its application in lactic acid production under oxygen deprivation in Corynebacterium glutamicum

    doi: 10.1186/s12896-016-0313-6

    Figure Lengend Snippet: Expression of the ldhA and pyk2 genes in C. glutamicum . a Identification of the co-transcription of ldhA and pyk2 in the ldhA-pyk2 cluster using RT-PCR. The C. glutamicum WT strain was cultured in minimal medium with glucose under aerobic conditions. The templates used for the PCR were as follows: lanes 1 and 4, total RNA reverse transcribed without reverse transcriptase; lanes 2 and 5, genomic DNA; and lanes 3 and 6, cDNA. The fragments in lanes 1, 2 and 3 were amplified using the primers WZ1181/WZ868 for rpoB. In addition, the primers WZ1171 and WZ1156 were used for the ldhA-pyk2 region in lanes 4, 5 and 6. b The relative transcription levels of the pyk1 , pyk2 and ldhA genes were analyzed by qRT-PCR. Total RNA was isolated from WT cells harvested at the exponential phase under aerobic conditions and at 3 h cultivation under oxygen-deprived conditions. The expression levels of pyk1 , pyk2 and ldhA under different conditions were compared against the expression of pyk1 under aerobic conditions (=1). The mean values from at least three independent cultures are shown with the standard deviations

    Article Snippet: RNA preparation and RT-PCR analysis Total RNA was isolated from the cells with the RNAprep Pure Cell/Bacteria Kit (Tiangen, China).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Isolation

    Hormonal regulation of EGFR signaling in E- or E+P-treated tumors. a , b Real-time RT-PCR analysis of a EGFR and b EGFR ligand mRNA levels. Bars represent the mean ± SEM fold change compared to levels in E-treated tumors ( n = 4–5 tumors/group). c Representative merged images of immunolabeling with Areg- ( green ), PRA- ( blue ), and PRB- ( red ) specific antibodies. White arrows indicate cells co-expressing Areg and PRA. Yellow arrows indicate cells co-expressing Areg, PRB, and PRA. Scale bar , 75 μm. d Immunoblot analysis of Areg, phospho-Akt, phospho-JNK, and phospho-Erk for the same E- or E+P-treated tumors. Numbers above bands indicate relative band intensity normalized to β-actin

    Journal: Hormones & Cancer

    Article Title: Amphiregulin Mediates Estrogen, Progesterone, and EGFR Signaling in the Normal Rat Mammary Gland and in Hormone-Dependent Rat Mammary Cancers

    doi: 10.1007/s12672-010-0048-0

    Figure Lengend Snippet: Hormonal regulation of EGFR signaling in E- or E+P-treated tumors. a , b Real-time RT-PCR analysis of a EGFR and b EGFR ligand mRNA levels. Bars represent the mean ± SEM fold change compared to levels in E-treated tumors ( n = 4–5 tumors/group). c Representative merged images of immunolabeling with Areg- ( green ), PRA- ( blue ), and PRB- ( red ) specific antibodies. White arrows indicate cells co-expressing Areg and PRA. Yellow arrows indicate cells co-expressing Areg, PRB, and PRA. Scale bar , 75 μm. d Immunoblot analysis of Areg, phospho-Akt, phospho-JNK, and phospho-Erk for the same E- or E+P-treated tumors. Numbers above bands indicate relative band intensity normalized to β-actin

    Article Snippet: Real-time RT-PCR analysis of Areg, RANKL, and Wnt4 mRNA expression was performed utilizing primers with equal amplification efficiencies (SA Biosciences).

    Techniques: Quantitative RT-PCR, Immunolabeling, Expressing

    Hormonal regulation of EGFR signaling. Mammary glands were obtained from OVX animals treated with vehicle (C), with E alone, P alone, or E+P. a Real-time PCR analysis of EGFR mRNA and immunoblot analysis of EGFR protein expression. Bars represent the mean ± SEM fold increase. * P

    Journal: Hormones & Cancer

    Article Title: Amphiregulin Mediates Estrogen, Progesterone, and EGFR Signaling in the Normal Rat Mammary Gland and in Hormone-Dependent Rat Mammary Cancers

    doi: 10.1007/s12672-010-0048-0

    Figure Lengend Snippet: Hormonal regulation of EGFR signaling. Mammary glands were obtained from OVX animals treated with vehicle (C), with E alone, P alone, or E+P. a Real-time PCR analysis of EGFR mRNA and immunoblot analysis of EGFR protein expression. Bars represent the mean ± SEM fold increase. * P

    Article Snippet: Real-time RT-PCR analysis of Areg, RANKL, and Wnt4 mRNA expression was performed utilizing primers with equal amplification efficiencies (SA Biosciences).

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Hormonal regulation of Areg expression and colocalization with PR isoforms. a Real-time RT-PCR analysis of EGFR ligand mRNA expression. Bars represent the mean ± SEM fold increase compared to the level of EGF mRNA in OVX control. * P

    Journal: Hormones & Cancer

    Article Title: Amphiregulin Mediates Estrogen, Progesterone, and EGFR Signaling in the Normal Rat Mammary Gland and in Hormone-Dependent Rat Mammary Cancers

    doi: 10.1007/s12672-010-0048-0

    Figure Lengend Snippet: Hormonal regulation of Areg expression and colocalization with PR isoforms. a Real-time RT-PCR analysis of EGFR ligand mRNA expression. Bars represent the mean ± SEM fold increase compared to the level of EGF mRNA in OVX control. * P

    Article Snippet: Real-time RT-PCR analysis of Areg, RANKL, and Wnt4 mRNA expression was performed utilizing primers with equal amplification efficiencies (SA Biosciences).

    Techniques: Expressing, Quantitative RT-PCR

    Overview of the experimental strategy. CD34+ cells from healthy, mobilized apheresis donors were immunostained with a 10-fluorochrome panel and single cells were index-sorted into 96-well PCR plates for multiplex qRT-PCR analysis using the Fluidigm Biomark platform. MEP subpopulations were identified by principal component analysis (PCA) and correlated with the original index sorting data and mRNA levels of surface antigens. Identified cellular subsets were validated transcriptionally at the population level and functionally in single-cell differentiation assays. Finally, the cells were ordered in pseudotime to assess differentiation trajectories which were then further validated in functional assays. FACS, fluorescence-activated cell sorting; IF, immunofluorescence; qRT-PCR, quantitative real-time polymerase chain reaction

    Journal: Genome Biology

    Article Title: Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways

    doi: 10.1186/s13059-016-0939-7

    Figure Lengend Snippet: Overview of the experimental strategy. CD34+ cells from healthy, mobilized apheresis donors were immunostained with a 10-fluorochrome panel and single cells were index-sorted into 96-well PCR plates for multiplex qRT-PCR analysis using the Fluidigm Biomark platform. MEP subpopulations were identified by principal component analysis (PCA) and correlated with the original index sorting data and mRNA levels of surface antigens. Identified cellular subsets were validated transcriptionally at the population level and functionally in single-cell differentiation assays. Finally, the cells were ordered in pseudotime to assess differentiation trajectories which were then further validated in functional assays. FACS, fluorescence-activated cell sorting; IF, immunofluorescence; qRT-PCR, quantitative real-time polymerase chain reaction

    Article Snippet: Data were analyzed using Fluidigm RT-PCR Analysis Software to standardize thresholds for each gene assay across plates.

    Techniques: Polymerase Chain Reaction, Multiplex Assay, Quantitative RT-PCR, Cell Differentiation, Functional Assay, FACS, Fluorescence, Immunofluorescence, Real-time Polymerase Chain Reaction

    RT-PCR analysis of MMP-2 ( A ), MMP-9 ( B ), TIMP-1 ( C ) and TIMP-2 ( D ) gene expression in HepG2 cells after treatment with FXT and the attenuating effect of GNP. HepG2 cells were seeded onto a 6-well plate and incubated overnight, treated with FXT and GNP at dose as indicated and further incubated for 72 h. DMSO (1%) was used as vehicle control. Values are expressed as mean ± SD ( n = 3). Different letters indicate statistical significance ( p

    Journal: Molecules

    Article Title: Evaluation of the Inhibitory Effects of Genipin on the Fluoxetine-Induced Invasive and Metastatic Model in Human HepG2 Cells

    doi: 10.3390/molecules23123327

    Figure Lengend Snippet: RT-PCR analysis of MMP-2 ( A ), MMP-9 ( B ), TIMP-1 ( C ) and TIMP-2 ( D ) gene expression in HepG2 cells after treatment with FXT and the attenuating effect of GNP. HepG2 cells were seeded onto a 6-well plate and incubated overnight, treated with FXT and GNP at dose as indicated and further incubated for 72 h. DMSO (1%) was used as vehicle control. Values are expressed as mean ± SD ( n = 3). Different letters indicate statistical significance ( p

    Article Snippet: RT-PCR Analysis of Marker Gene Expression in HepG2 Cells After FXT-GNP Co-Treatment The results of the RT-PCR analysis on the MMP-2 and MMP-9 gene expressions in HepG2 cells after treatment with FXT were shown in A,B, respectively.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation

    RT-PCR analysis reveals differentially expressed transcripts. RNA from the wild type control and the heph 2 mutant was analyzed by RT-PCR analysis. Gene-specific primer pairs were used for amplification of several randomly picked genes that showed significant differential expression. For Tequila , primer pairs were designed to test alternative 5’ ends (transcription start site usage).

    Journal: PLoS ONE

    Article Title: High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis

    doi: 10.1371/journal.pone.0150768

    Figure Lengend Snippet: RT-PCR analysis reveals differentially expressed transcripts. RNA from the wild type control and the heph 2 mutant was analyzed by RT-PCR analysis. Gene-specific primer pairs were used for amplification of several randomly picked genes that showed significant differential expression. For Tequila , primer pairs were designed to test alternative 5’ ends (transcription start site usage).

    Article Snippet: RT-PCR analysis confirms expression level differences We randomly selected several candidates from specific gene ontology categories for analysis by RT-PCR for their expression level differences between the wild-type control and the heph 2 mutant. shows that RT-PCR analysis recapitulated the up and down regulation observed from the Illumina sequencing reads.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Amplification, Expressing

    Use of alternative transcription start sites in the heph 2 mutant. ( A) RNA Seq read pileup across a Paramyosin alternative transcription start site, where alternative transcription start sites highlighted by the box. ( B) RNA Seq read pileup across a Tequila alternative transcription start site, where the 5’ end showing the most difference is highlighted by the box. For RT-PCR validation of Tequila alternative transcription start site usage, see Fig 3 .

    Journal: PLoS ONE

    Article Title: High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis

    doi: 10.1371/journal.pone.0150768

    Figure Lengend Snippet: Use of alternative transcription start sites in the heph 2 mutant. ( A) RNA Seq read pileup across a Paramyosin alternative transcription start site, where alternative transcription start sites highlighted by the box. ( B) RNA Seq read pileup across a Tequila alternative transcription start site, where the 5’ end showing the most difference is highlighted by the box. For RT-PCR validation of Tequila alternative transcription start site usage, see Fig 3 .

    Article Snippet: RT-PCR analysis confirms expression level differences We randomly selected several candidates from specific gene ontology categories for analysis by RT-PCR for their expression level differences between the wild-type control and the heph 2 mutant. shows that RT-PCR analysis recapitulated the up and down regulation observed from the Illumina sequencing reads.

    Techniques: Mutagenesis, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction

    Alternative splicing of the Mlc1 gene in the heph 2 mutant. RNA Seq pileups were generated using UCSC Genome Browser, y-axis is auto-scaled to show differences in isoform fractions, alternative exon is highlighted by the box. (A) RNA-Seq read pileup across the Mlc1 skipped exon in control (top) and heph 2 mutant (bottom) flies, showing significantly increased exon skipped in the heph 2 mutant. ( B) RT-PCR analysis of Mlc1 for exon skipping, using primers in the flanking exons shown by the arrows.

    Journal: PLoS ONE

    Article Title: High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis

    doi: 10.1371/journal.pone.0150768

    Figure Lengend Snippet: Alternative splicing of the Mlc1 gene in the heph 2 mutant. RNA Seq pileups were generated using UCSC Genome Browser, y-axis is auto-scaled to show differences in isoform fractions, alternative exon is highlighted by the box. (A) RNA-Seq read pileup across the Mlc1 skipped exon in control (top) and heph 2 mutant (bottom) flies, showing significantly increased exon skipped in the heph 2 mutant. ( B) RT-PCR analysis of Mlc1 for exon skipping, using primers in the flanking exons shown by the arrows.

    Article Snippet: RT-PCR analysis confirms expression level differences We randomly selected several candidates from specific gene ontology categories for analysis by RT-PCR for their expression level differences between the wild-type control and the heph 2 mutant. shows that RT-PCR analysis recapitulated the up and down regulation observed from the Illumina sequencing reads.

    Techniques: Mutagenesis, RNA Sequencing Assay, Generated, Reverse Transcription Polymerase Chain Reaction