oligo dt primers Search Results


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  • 99
    Thermo Fisher oligo
    RT-PCR confirmation of antisense transcription. a) A graphic representation of um02794 transcription. The grey line represents the genomic sequence (middle), the blue arrow represents predicted gene structure, and the red (top) and green (bottom) arrows represent sense and anti-sense ESTs respectively. The range of the genome coordinates was included. b) Detecting antisense transcripts corresponding to um02794 via strand specific RT-PCR. In lanes 2 to 5 first strand synthesis was carried out on <t>RNA</t> of CM grown haploid cells. In lanes 6 to 9 first strand synthesis was carried out on RNA of MN grown haploid cells. First synthesis reactions of lanes 2 and 6 were prepared using sense strand specific primers; lanes 3 and 7 anti sense specific primers; lane 4 and 8 <t>oligo</t> dT and lanes 5 and 9 DEPC-treated water. Lane 10 used genomic DNA from U. maydis strain 521 and lane 11 used water a PCR template. Lane 1 and 12: Full Ranger DNA ladder.
    Oligo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 38632 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore oligo
    RT-PCR confirmation of antisense transcription. a) A graphic representation of um02794 transcription. The grey line represents the genomic sequence (middle), the blue arrow represents predicted gene structure, and the red (top) and green (bottom) arrows represent sense and anti-sense ESTs respectively. The range of the genome coordinates was included. b) Detecting antisense transcripts corresponding to um02794 via strand specific RT-PCR. In lanes 2 to 5 first strand synthesis was carried out on <t>RNA</t> of CM grown haploid cells. In lanes 6 to 9 first strand synthesis was carried out on RNA of MN grown haploid cells. First synthesis reactions of lanes 2 and 6 were prepared using sense strand specific primers; lanes 3 and 7 anti sense specific primers; lane 4 and 8 <t>oligo</t> dT and lanes 5 and 9 DEPC-treated water. Lane 10 used genomic DNA from U. maydis strain 521 and lane 11 used water a PCR template. Lane 1 and 12: Full Ranger DNA ladder.
    Oligo, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega oligo
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 9287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega oligo dt
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo Dt, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 5577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega oligo dt primers
    p120 308 is a null allele. (A) p120 308 deletes the entire p120 coding region, but does not affect other genes. Schematic as in Fig. 1 A. Genomic DNA from single wild-type or homozygous mutant flies was PCR amplified using primer pairs from the indicated regions between LD05623 and CG17486 . (B) The p120 mutants are mRNA nulls. <t>cDNA</t> generated from <t>oligo-dT-primed</t> total RNA from p120 mutants and wild-type was amplified with primers spanning the p120 third intron. An unrelated gene, CG2905 , is a control. A DNA control confirmed we were examining mRNA.
    Oligo Dt Primers, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 4572 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen oligo dt primers
    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with <t>Oligo</t> dT-primers, cDNA was analyzed by semi-quantitative <t>PCR</t> for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.
    Oligo Dt Primers, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1677 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher oligo dt 18 primer
    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with <t>Oligo</t> dT-primers, cDNA was analyzed by semi-quantitative <t>PCR</t> for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.
    Oligo Dt 18 Primer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1030 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher oligo dt 12 18 primer
    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with <t>Oligo</t> dT-primers, cDNA was analyzed by semi-quantitative <t>PCR</t> for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.
    Oligo Dt 12 18 Primer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 644 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad oligo dt primers
    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with <t>Oligo</t> dT-primers, cDNA was analyzed by semi-quantitative <t>PCR</t> for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.
    Oligo Dt Primers, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 855 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt primers/product/Bio-Rad
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    Image Search Results


    RT-PCR confirmation of antisense transcription. a) A graphic representation of um02794 transcription. The grey line represents the genomic sequence (middle), the blue arrow represents predicted gene structure, and the red (top) and green (bottom) arrows represent sense and anti-sense ESTs respectively. The range of the genome coordinates was included. b) Detecting antisense transcripts corresponding to um02794 via strand specific RT-PCR. In lanes 2 to 5 first strand synthesis was carried out on RNA of CM grown haploid cells. In lanes 6 to 9 first strand synthesis was carried out on RNA of MN grown haploid cells. First synthesis reactions of lanes 2 and 6 were prepared using sense strand specific primers; lanes 3 and 7 anti sense specific primers; lane 4 and 8 oligo dT and lanes 5 and 9 DEPC-treated water. Lane 10 used genomic DNA from U. maydis strain 521 and lane 11 used water a PCR template. Lane 1 and 12: Full Ranger DNA ladder.

    Journal: BMC Genomics

    Article Title: Gene discovery and transcript analyses in the corn smut pathogen Ustilago maydis: expressed sequence tag and genome sequence comparison

    doi: 10.1186/1471-2164-8-334

    Figure Lengend Snippet: RT-PCR confirmation of antisense transcription. a) A graphic representation of um02794 transcription. The grey line represents the genomic sequence (middle), the blue arrow represents predicted gene structure, and the red (top) and green (bottom) arrows represent sense and anti-sense ESTs respectively. The range of the genome coordinates was included. b) Detecting antisense transcripts corresponding to um02794 via strand specific RT-PCR. In lanes 2 to 5 first strand synthesis was carried out on RNA of CM grown haploid cells. In lanes 6 to 9 first strand synthesis was carried out on RNA of MN grown haploid cells. First synthesis reactions of lanes 2 and 6 were prepared using sense strand specific primers; lanes 3 and 7 anti sense specific primers; lane 4 and 8 oligo dT and lanes 5 and 9 DEPC-treated water. Lane 10 used genomic DNA from U. maydis strain 521 and lane 11 used water a PCR template. Lane 1 and 12: Full Ranger DNA ladder.

    Article Snippet: Reverse transcription was carried out on 5 μg of DNaseI treated RNA using Superscript III (Invitrogen) and oligo (dT)12–18 primers (Invitrogen).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction

    Measurements of influenza transcription. HIV-1-infected and -uninfected macrophages were infected with influenza A(H1N1)pdm09. After 24h, cells were lysed, RNA extracted and cDNA synthetized using oligo.dT and UNI-12 primers. Quantitative real time PCR was performed for the M1 gene. As a control, ribavirin-treated and -untreated influenza-infected macrophages were submitted to the same procedure described above (inset). * P

    Journal: PLoS ONE

    Article Title: HIV-1 and Its gp120 Inhibits the Influenza A(H1N1)pdm09 Life Cycle in an IFITM3-Dependent Fashion

    doi: 10.1371/journal.pone.0101056

    Figure Lengend Snippet: Measurements of influenza transcription. HIV-1-infected and -uninfected macrophages were infected with influenza A(H1N1)pdm09. After 24h, cells were lysed, RNA extracted and cDNA synthetized using oligo.dT and UNI-12 primers. Quantitative real time PCR was performed for the M1 gene. As a control, ribavirin-treated and -untreated influenza-infected macrophages were submitted to the same procedure described above (inset). * P

    Article Snippet: CDNA was synthetized with SuperScript III (Life Technologies) using oligo.dT (Life Technologies) or UNI-12 (5′-AGCRAAAGCAGG-3′) as the primers for first-strand synthesis, for 1 h at 45°C.

    Techniques: Infection, Real-time Polymerase Chain Reaction

    Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p

    Journal: Scientific Reports

    Article Title: The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

    doi: 10.1038/s41598-018-31095-0

    Figure Lengend Snippet: Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p

    Article Snippet: 0.6–1 μg of total RNA was used for reverse transcription to produce cDNA using oligo-dT primers (Invitrogen), 10 mM dNTP mix, RNasin plus (Promega) and M-MLV Reverse transcriptase enzyme (Invitrogen) according to manufacturer recommendations.

    Techniques: Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Bradford Assay, SDS Page, Western Blot, Positive Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p

    Journal: Scientific Reports

    Article Title: The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

    doi: 10.1038/s41598-018-31095-0

    Figure Lengend Snippet: IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p

    Article Snippet: 0.6–1 μg of total RNA was used for reverse transcription to produce cDNA using oligo-dT primers (Invitrogen), 10 mM dNTP mix, RNasin plus (Promega) and M-MLV Reverse transcriptase enzyme (Invitrogen) according to manufacturer recommendations.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    Effect of osajin on the expression of Fas/CD95, FasL/CD95L, GRP78/BiP, CHOP/GADD153 and Bcl-2 family proteins. TW04 cells were treated with various concentrations of osajin for 24 h. ( A ) RNA was isolated from cells treated with 5 µM or 7.5 µM osajin. Two µg of RNA was reversely transcribed into cDNA using oligo (dT) primers. RT-PCR analysis was performed using primers specific for Fas, FasL, GRP78, CHOP, Bax and Bcl-2 genes and also the internal control gene, β-actin. ( B ) Cell lysates were prepared for SDS-PAGE followed by Western blot for Fas, FasL, GRP78, CHOP, Bax and Bcl-2, with β-actin as a loading control. ( C ) to ( E ) Results are presented as the relative densities of protein bands normalized to β-actin. The data shown are the means ± SE of three individual experiments (* P

    Journal: PLoS ONE

    Article Title: Activation of Multiple Apoptotic Pathways in Human Nasopharyngeal Carcinoma Cells by the Prenylated Isoflavone, Osajin

    doi: 10.1371/journal.pone.0018308

    Figure Lengend Snippet: Effect of osajin on the expression of Fas/CD95, FasL/CD95L, GRP78/BiP, CHOP/GADD153 and Bcl-2 family proteins. TW04 cells were treated with various concentrations of osajin for 24 h. ( A ) RNA was isolated from cells treated with 5 µM or 7.5 µM osajin. Two µg of RNA was reversely transcribed into cDNA using oligo (dT) primers. RT-PCR analysis was performed using primers specific for Fas, FasL, GRP78, CHOP, Bax and Bcl-2 genes and also the internal control gene, β-actin. ( B ) Cell lysates were prepared for SDS-PAGE followed by Western blot for Fas, FasL, GRP78, CHOP, Bax and Bcl-2, with β-actin as a loading control. ( C ) to ( E ) Results are presented as the relative densities of protein bands normalized to β-actin. The data shown are the means ± SE of three individual experiments (* P

    Article Snippet: Two micrograms of RNA were reversely transcribed in 20 µl reaction volumes containing an oligo (dT) primer (Invitrogen) and the M-MLV reverse transcriptase (Promega, Madison, WI, USA).

    Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot

    BAX knockdown by siRNA mimics promotes tumor growth in vivo. siNC and siBAX oligos were injected into A431 cell xenografts every three days. ( A ) Loss of BAX promotes subcutaneous tumor growth in a mouse xenograft model. Tumor volumes (mm 3 ) were plotted according to days. Tumor volume statistical data represent the average of three independent experiments ± s.d., respectively; ( B ) the mice were sacrificed at the end of the experiment and images taken along with the dissected tumors from three representative mice are shown. White arrows indicate the siNC-treated xenografts, while black arrows indicate siBAX-treated xenografts; ( C ) the expression of BAX was measured in the dissected tumors by qRT-PCR. qRT-PCR statistical data represent the average of three independent experiments ± s.d. Expression folds are shown with respect to NC where normalized copy numbers were set to 1; ( D ) the protein expression of BAX was detected in xenografts after siBAX treatment by Western blot; ( E ) histopathology analysis (IHC staining) of BAX on tumor sections. The quantification was done by counting positively-stained cells from 20 randomly-chosen fields from a total of five sections per tumor. Magnification, 400×, Scale bars, 50 µm; ( F ) TUNEL assay of apoptosis on tumor sections after BAX depletion. The quantification was done by counting positively-stained signals from 20 randomly-chosen fields from a total of five sections per tumor. Magnification, 400×, Scale bars, 50 µm. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Loss of BAX by miR-365 Promotes Cutaneous Squamous Cell Carcinoma Progression by Suppressing Apoptosis

    doi: 10.3390/ijms18061157

    Figure Lengend Snippet: BAX knockdown by siRNA mimics promotes tumor growth in vivo. siNC and siBAX oligos were injected into A431 cell xenografts every three days. ( A ) Loss of BAX promotes subcutaneous tumor growth in a mouse xenograft model. Tumor volumes (mm 3 ) were plotted according to days. Tumor volume statistical data represent the average of three independent experiments ± s.d., respectively; ( B ) the mice were sacrificed at the end of the experiment and images taken along with the dissected tumors from three representative mice are shown. White arrows indicate the siNC-treated xenografts, while black arrows indicate siBAX-treated xenografts; ( C ) the expression of BAX was measured in the dissected tumors by qRT-PCR. qRT-PCR statistical data represent the average of three independent experiments ± s.d. Expression folds are shown with respect to NC where normalized copy numbers were set to 1; ( D ) the protein expression of BAX was detected in xenografts after siBAX treatment by Western blot; ( E ) histopathology analysis (IHC staining) of BAX on tumor sections. The quantification was done by counting positively-stained cells from 20 randomly-chosen fields from a total of five sections per tumor. Magnification, 400×, Scale bars, 50 µm; ( F ) TUNEL assay of apoptosis on tumor sections after BAX depletion. The quantification was done by counting positively-stained signals from 20 randomly-chosen fields from a total of five sections per tumor. Magnification, 400×, Scale bars, 50 µm. ** p

    Article Snippet: Oligos were prepared by pre-incubating 3 nM siRNA per mouse with Lipofectamine 2000 (Life Technologies) for 15 min; injections were made using a final volume of 50 µL in serum free DMEM (Life Technologies).

    Techniques: In Vivo, Injection, Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot, Histopathology, Immunohistochemistry, Staining, TUNEL Assay

    DDR1 affects IR expression (a) IR protein and mRNA expression in DDR1-depleted cells. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 siRNA oligos. After 48h, cells were lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. Blot is representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization against β-actin. In the same transfected cell lines, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. In parallel, DDR1 mRNA was evaluated by qRT-PCR to confirm DDR1 depletion (graphs on the right). (b) IR protein and mRNA expression after DDR1 overexpression. Breast cancer cells were transiently transfected with either constitutive empty (pCMV6-EV) or human DDR1 (pCMV6-DDR1) expressing vectors. After 48h, cells were lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative blot of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization over β-actin. In the same transfected cells, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. The overexpression of DDR1 was confirmed measuring DDR1 mRNA by qRT-PCR (graphs on the right). (a-b) *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 affects IR expression (a) IR protein and mRNA expression in DDR1-depleted cells. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 siRNA oligos. After 48h, cells were lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. Blot is representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization against β-actin. In the same transfected cell lines, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. In parallel, DDR1 mRNA was evaluated by qRT-PCR to confirm DDR1 depletion (graphs on the right). (b) IR protein and mRNA expression after DDR1 overexpression. Breast cancer cells were transiently transfected with either constitutive empty (pCMV6-EV) or human DDR1 (pCMV6-DDR1) expressing vectors. After 48h, cells were lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative blot of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization over β-actin. In the same transfected cells, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. The overexpression of DDR1 was confirmed measuring DDR1 mRNA by qRT-PCR (graphs on the right). (a-b) *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, SDS Page, Quantitative RT-PCR, Over Expression

    DDR1 affects IR – dependent transcription factors (a) Protein levels of IR-dependent transcription factors after DDR1 depletion. MCF-7 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 specific siRNA oligos. After 48h, protein expression of IR, and of p53, HMGA, and Sp1 transcription factors was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (b) Protein levels of IR-related transcription factors after DDR1 overexpression. MCF-7 cells were transiently transfected with either either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 48h, protein expression of IR, p53, HMGA, and Sp1 was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (c) mRNA expression of IR-related transcription factors after DDR1 depletion. MCF-7 cells transfected as in (a) were evaluated by qRT-PCR analysis for IR, p53, HMGA, and Sp1 mRNA levels. Values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as control for DDR1 depletion efficiency. Values represent the mean±SEM of three independent experiments. (d) mRNA expression of IR-related transcription factors after DDR1 overexpression. In cells transfected as in (b) , IR, p53, HMGA, and Sp1 mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as silencing control. Values represent the mean±SEM of three independent experiments. (a-d) NS, p > 0.05; *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 affects IR – dependent transcription factors (a) Protein levels of IR-dependent transcription factors after DDR1 depletion. MCF-7 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 specific siRNA oligos. After 48h, protein expression of IR, and of p53, HMGA, and Sp1 transcription factors was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (b) Protein levels of IR-related transcription factors after DDR1 overexpression. MCF-7 cells were transiently transfected with either either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 48h, protein expression of IR, p53, HMGA, and Sp1 was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (c) mRNA expression of IR-related transcription factors after DDR1 depletion. MCF-7 cells transfected as in (a) were evaluated by qRT-PCR analysis for IR, p53, HMGA, and Sp1 mRNA levels. Values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as control for DDR1 depletion efficiency. Values represent the mean±SEM of three independent experiments. (d) mRNA expression of IR-related transcription factors after DDR1 overexpression. In cells transfected as in (b) , IR, p53, HMGA, and Sp1 mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as silencing control. Values represent the mean±SEM of three independent experiments. (a-d) NS, p > 0.05; *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Transfection, Expressing, Western Blot, Over Expression, Quantitative RT-PCR

    DDR1 stabilizes IR both at the protein and mRNA level in MCF-7 cells (a) DDR1 depletion affects IR proteasomal degradation. MCF-7 cells were transiently transfected with either a pool of four scramble or four DDR1-specific siRNA oligos. After 48h, cells were treated with 1μM of the proteasome inhibitor MG132 for 6h. (b) DDR1 depletion does not affect IR lysosomal degradation. MCF-7 cells were transiently transfected as in (a) . After 48 h, cells were treated with 10μM of the cloroquine (CHL) for 2, 6, and 24h. (c) Cycloheximide treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected with either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 24 h, cells were treated with 100 μM of cycloheximide (CHX) for 8, 16, and 24h. (d) Actinomycin D treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected as in (c) . After 24h, cells were treated with 2.5μg/mL of actinomycin D (ACT D) for 2, 4, 6, and 16h. IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 overexpression was confirmed by western blot analysis as shown on the right of the histogram. (a-c) Samples were analyzed by western blotting for DDR1 and IR expression. β-actin antibody was used as control. A representative blot of three independent experiments is shown. The histogram represents the mean±SEM of densitometric analysis after normalization against β-actin. (a-d) NS, p > 0.05; *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 stabilizes IR both at the protein and mRNA level in MCF-7 cells (a) DDR1 depletion affects IR proteasomal degradation. MCF-7 cells were transiently transfected with either a pool of four scramble or four DDR1-specific siRNA oligos. After 48h, cells were treated with 1μM of the proteasome inhibitor MG132 for 6h. (b) DDR1 depletion does not affect IR lysosomal degradation. MCF-7 cells were transiently transfected as in (a) . After 48 h, cells were treated with 10μM of the cloroquine (CHL) for 2, 6, and 24h. (c) Cycloheximide treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected with either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 24 h, cells were treated with 100 μM of cycloheximide (CHX) for 8, 16, and 24h. (d) Actinomycin D treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected as in (c) . After 24h, cells were treated with 2.5μg/mL of actinomycin D (ACT D) for 2, 4, 6, and 16h. IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 overexpression was confirmed by western blot analysis as shown on the right of the histogram. (a-c) Samples were analyzed by western blotting for DDR1 and IR expression. β-actin antibody was used as control. A representative blot of three independent experiments is shown. The histogram represents the mean±SEM of densitometric analysis after normalization against β-actin. (a-d) NS, p > 0.05; *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Transfection, Activated Clotting Time Assay, Quantitative RT-PCR, Over Expression, Western Blot, Expressing

    DDR1 depletion affects insulin and IGF-2 mediated biological effects in human cancer cells (a) Western blot before and after DDR1 depletion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four DDR1 siRNA oligos or scramble siRNA oligos. After 24h, cells were grown in medium containing 2.5% of CS-FCS for 24h. DDR1 depletion was confirmed for each cells line by western blot analysis as shown in the panel. (b) Cell proliferation. Cell viability was evaluated by MTT assay. Values are expressed as percentages of untreated scramble-transfected cells (basal) and represent the mean±SEM of three independent experiments in triplicate. (c) Cell cycle progression. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24h. Cells were then incubated with or without insulin or IGF-2 at a dose of 10nM for additional 48h and analyzed for cell-cycle profiles. Cell populations positive for propidium iodine staining were evaluated by FACS analysis, and G0/G1 and G2/M phases were scored. The graph shows the percentage of cells in S and G2/M phases. Values are expressed as percent of basal (untreated scramble transfected cells) and are the mean±SEM of three independent experiments. (d) Cell invasion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24 h. Cells were then removed from plates with 0.01% trypsin and seeded on polycarbonate filters coated with 25μg/mL fibronectin. Cells were allowed to migrate for 6h (MCF-7 and MDA-MB-157) or 8h (BT-474 cells) in response to 10nM of insulin or IGF-2 added to the lower chamber. Values are mean±SEM of three independent experiments done in duplicate and are expressed as percent increase over untreated scramble cells (basal). (e) Colony formation. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) , and seeded in soft-agar, as described in Materials and Methods. Cells were plated in triplicate and cultured in serum free medium containing 2.5% CS-FCS for 3 weeks. Colonies developed only from plated MCF-7 and MDA-MB-157 cells but not from BT-474. Colonies were stained with MTT and then photographed. The histogram represents the mean number of colonies shown in (e) . Error bars indicate SEM (n = 3 wells). (b-e) *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 depletion affects insulin and IGF-2 mediated biological effects in human cancer cells (a) Western blot before and after DDR1 depletion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four DDR1 siRNA oligos or scramble siRNA oligos. After 24h, cells were grown in medium containing 2.5% of CS-FCS for 24h. DDR1 depletion was confirmed for each cells line by western blot analysis as shown in the panel. (b) Cell proliferation. Cell viability was evaluated by MTT assay. Values are expressed as percentages of untreated scramble-transfected cells (basal) and represent the mean±SEM of three independent experiments in triplicate. (c) Cell cycle progression. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24h. Cells were then incubated with or without insulin or IGF-2 at a dose of 10nM for additional 48h and analyzed for cell-cycle profiles. Cell populations positive for propidium iodine staining were evaluated by FACS analysis, and G0/G1 and G2/M phases were scored. The graph shows the percentage of cells in S and G2/M phases. Values are expressed as percent of basal (untreated scramble transfected cells) and are the mean±SEM of three independent experiments. (d) Cell invasion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24 h. Cells were then removed from plates with 0.01% trypsin and seeded on polycarbonate filters coated with 25μg/mL fibronectin. Cells were allowed to migrate for 6h (MCF-7 and MDA-MB-157) or 8h (BT-474 cells) in response to 10nM of insulin or IGF-2 added to the lower chamber. Values are mean±SEM of three independent experiments done in duplicate and are expressed as percent increase over untreated scramble cells (basal). (e) Colony formation. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) , and seeded in soft-agar, as described in Materials and Methods. Cells were plated in triplicate and cultured in serum free medium containing 2.5% CS-FCS for 3 weeks. Colonies developed only from plated MCF-7 and MDA-MB-157 cells but not from BT-474. Colonies were stained with MTT and then photographed. The histogram represents the mean number of colonies shown in (e) . Error bars indicate SEM (n = 3 wells). (b-e) *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Western Blot, Multiple Displacement Amplification, Transfection, MTT Assay, Incubation, Staining, FACS, Cell Culture

    DDR1 depletion or overexpression affects insulin and IGF-2 downstream signaling in human breast cancer cells (a) Insulin and IGF-2 signaling after DDR1 depletion. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with a pool of four scramble or of four siRNA oligos against DDR1. After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. Cells were then lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative of three experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoprotein against β-actin. (b) Insulin and IGF-2 signaling after DDR1 overexpression. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with plasmids encoding either the human DDR1 cDNA (pCMV6-DDR1) or the corresponding empty vector (pCMV6-EV). After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. The activation of downstream signaling was assessed as in (a) . Blots are representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoproteins against β-actin. (a-b) NS, p > 0.05; *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 depletion or overexpression affects insulin and IGF-2 downstream signaling in human breast cancer cells (a) Insulin and IGF-2 signaling after DDR1 depletion. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with a pool of four scramble or of four siRNA oligos against DDR1. After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. Cells were then lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative of three experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoprotein against β-actin. (b) Insulin and IGF-2 signaling after DDR1 overexpression. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with plasmids encoding either the human DDR1 cDNA (pCMV6-DDR1) or the corresponding empty vector (pCMV6-EV). After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. The activation of downstream signaling was assessed as in (a) . Blots are representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoproteins against β-actin. (a-b) NS, p > 0.05; *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Over Expression, Multiple Displacement Amplification, Transfection, SDS Page, Plasmid Preparation, Activation Assay

    RNA Pol II kinetics with a slower transcription elongation rate affects pA site selection. ( A ) Pol II ChIP across polo and polo-snap intergenic region. In the top panel, a schematic diagram is shown where polo and snap genes are depicted to scale (adapted from genome.ucsc.edu). ChIP was performed using α-Rpb3 antibody on wild-type ( w 1118 ) and slow Pol II ( RpII215 mutant, C4 mutation) adult flies chromatin. Numbers below each graph bar represent the position of real-time PCR primers. ( B ) RpII215 mutant generates similar levels of polo mRNA as compared with w 1118 . Graph represents RT–qPCR quantification of polo mRNAs in adult flies, relative to rp49 mRNA. ( C ) RpII215 mutant presents similar levels of Polo protein as w 1118 . Western blot from total protein extracts of w 1118 and RpII215 third instar larvae brains. MA294 Polo and DM1A α-tubulin antibodies were used. Quantification was made by densitometry (see Materials and methods) and Polo/α-tubulin ratio was set at 1 for w 1118 . ( D ) Representative gel of fractionated 3′RACE products of polo mRNA from w 1118 and RpII215 adult flies is shown. Arrows indicate the bands corresponding to polo pA1 and pA2; −RT is a control reaction without reverse transcriptase. In both panels, phased-anchored oligodT was used for reverse transcription, as depicted in the diagram. ( E ) RpII215 shows an increase in the ratio of total/pA2 mRNAs. Diagram shows primer positions for qPCR analysis. Levels of total polo mRNAs and pA2 mRNAs were measured by real-time PCR and their ratio (total/pA2) in adult flies is shown. ( F ) RpII215 shows an increase in proximal site usage in several genes. RT–qPCR quantification was performed as in ( E ). cDNA synthesis was performed with random primers (see Materials and methods). The ratio for w 1118 was set at 1. For all the panels, error bars show s.e.m. from at least three independent experiments.

    Journal: The EMBO Journal

    Article Title: RNA polymerase II kinetics in polo polyadenylation signal selection

    doi: 10.1038/emboj.2011.156

    Figure Lengend Snippet: RNA Pol II kinetics with a slower transcription elongation rate affects pA site selection. ( A ) Pol II ChIP across polo and polo-snap intergenic region. In the top panel, a schematic diagram is shown where polo and snap genes are depicted to scale (adapted from genome.ucsc.edu). ChIP was performed using α-Rpb3 antibody on wild-type ( w 1118 ) and slow Pol II ( RpII215 mutant, C4 mutation) adult flies chromatin. Numbers below each graph bar represent the position of real-time PCR primers. ( B ) RpII215 mutant generates similar levels of polo mRNA as compared with w 1118 . Graph represents RT–qPCR quantification of polo mRNAs in adult flies, relative to rp49 mRNA. ( C ) RpII215 mutant presents similar levels of Polo protein as w 1118 . Western blot from total protein extracts of w 1118 and RpII215 third instar larvae brains. MA294 Polo and DM1A α-tubulin antibodies were used. Quantification was made by densitometry (see Materials and methods) and Polo/α-tubulin ratio was set at 1 for w 1118 . ( D ) Representative gel of fractionated 3′RACE products of polo mRNA from w 1118 and RpII215 adult flies is shown. Arrows indicate the bands corresponding to polo pA1 and pA2; −RT is a control reaction without reverse transcriptase. In both panels, phased-anchored oligodT was used for reverse transcription, as depicted in the diagram. ( E ) RpII215 shows an increase in the ratio of total/pA2 mRNAs. Diagram shows primer positions for qPCR analysis. Levels of total polo mRNAs and pA2 mRNAs were measured by real-time PCR and their ratio (total/pA2) in adult flies is shown. ( F ) RpII215 shows an increase in proximal site usage in several genes. RT–qPCR quantification was performed as in ( E ). cDNA synthesis was performed with random primers (see Materials and methods). The ratio for w 1118 was set at 1. For all the panels, error bars show s.e.m. from at least three independent experiments.

    Article Snippet: cDNA synthesis was performed using either oligodT or random hexamers with Superscript III following the manufacturer's instructions (Invitrogen). cDNA was then quantified in an iQ5 Bio-Rad real time PCR machine with iQ™ Sybr® Green Supermix. rp49 was used for normalization in all assays.

    Techniques: Selection, Chromatin Immunoprecipitation, Mutagenesis, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    Sequence comparison of zebrafish foxo3b with other FOXOs and developmental expressing patterns of zebrafish foxo3b . ( A ) Sequence alignment of zebrafish foxo3b and human FOXO3a protein. ( B ) Neighbor-Joining Analysis of vertebrate FOXO protein sequences. Phylogenetic analysis was conducted using MEGA version 5 (Tamura, Peterson, Stecher, Nei, and Kumar 2011). h Foxo3a ( Homo sapiens , Accession number NM_001455), h Foxo6 (XM_002342102), h Foxo4 (NM_005938), h Foxo1 (NM_002015), m Foxo3 ( Mus musculus , NM_019740), m Foxo6 (NM_194060), m Foxo4 (NM_018789), mFoxo1 (NM_019739), ra Foxo3 ( Rattus norvegicus , NM_001106395), ra Foxo6 (XM_001057233), ra Foxo4 (NM_001106943), ra Foxo1 (NM_001191846), z Foxo3b ( Danio rerio , NM_131085), z Foxo3a (NM_001009988), z Foxo1a (NM_001077257), z Foxo1b (NM_001082857), xe Foxo3 ( Xenopus laevis , NM_001092949), xe Foxo6 (NM_001159282), xe Foxo4 (FJ811896), xe Foxo1a (NM_001092948), xi foxo5 ( Xiphophorus maculates , AY040320). NJ bootstrap values were shown on the branches. ( C ) The expression pattern of zebrafish foxo3b during embryogenesis. (C1–C3) The expression of foxo3b was detected at 2-cell stage embryos, and became weaker at oblong stage. (C4, C5) Foxo3b was ubiquitously expressed at 40% epiboly stage, a stronger expression was observed at shield stage. (C6, C7) Foxo3b became weaker at bud stage; at 6-somite stage, its expression level almost recovered to that of shield stage embryos. (C8, C13) Foxo3b was observed in the developing eye, hindbrain and posterior mesoderm by 24 hpf. (C9, C10) By 55 hpf, foxo3b expression was confined to the anterior central nervous system (CNS), with weak expression in the heart. (C11, C12, C14) By 72 hpf, foxo3b expression became weaker, but continued in the CNS and heart. C1–C4, lateral view; C5, lateral view with dorsal to the right; C6, C7, lateral view with anterior on top; C8, C9, C11, C13, C14, lateral views with anterior to the left; C10, C12, dorsal views with anterior on top; r, retina; p, pectoral fin bud; hb, hindbrain; pm, posterior mesoderm; pd, pronephric duct; h, heart; t, telencephalon; ov, otic vesicle; m, mesencephalon; c, cell; s, somite; h, hours post-fertilization (hpf). ( D ) Relative RNA expression levels as determined by semi-quantitative RT-PCR. For each stage, oligo dT-primed cDNA was used as template for three separate PCR amplifications using primers for foxo3b and 18s (internal control), foxo3b reached a high expression level at shield stage. For quantitative purpose, mRNA expression levels were normalized to 6 hpf (1.00).

    Journal: PLoS ONE

    Article Title: Zebrafish foxo3b Negatively Regulates Canonical Wnt Signaling to Affect Early Embryogenesis

    doi: 10.1371/journal.pone.0024469

    Figure Lengend Snippet: Sequence comparison of zebrafish foxo3b with other FOXOs and developmental expressing patterns of zebrafish foxo3b . ( A ) Sequence alignment of zebrafish foxo3b and human FOXO3a protein. ( B ) Neighbor-Joining Analysis of vertebrate FOXO protein sequences. Phylogenetic analysis was conducted using MEGA version 5 (Tamura, Peterson, Stecher, Nei, and Kumar 2011). h Foxo3a ( Homo sapiens , Accession number NM_001455), h Foxo6 (XM_002342102), h Foxo4 (NM_005938), h Foxo1 (NM_002015), m Foxo3 ( Mus musculus , NM_019740), m Foxo6 (NM_194060), m Foxo4 (NM_018789), mFoxo1 (NM_019739), ra Foxo3 ( Rattus norvegicus , NM_001106395), ra Foxo6 (XM_001057233), ra Foxo4 (NM_001106943), ra Foxo1 (NM_001191846), z Foxo3b ( Danio rerio , NM_131085), z Foxo3a (NM_001009988), z Foxo1a (NM_001077257), z Foxo1b (NM_001082857), xe Foxo3 ( Xenopus laevis , NM_001092949), xe Foxo6 (NM_001159282), xe Foxo4 (FJ811896), xe Foxo1a (NM_001092948), xi foxo5 ( Xiphophorus maculates , AY040320). NJ bootstrap values were shown on the branches. ( C ) The expression pattern of zebrafish foxo3b during embryogenesis. (C1–C3) The expression of foxo3b was detected at 2-cell stage embryos, and became weaker at oblong stage. (C4, C5) Foxo3b was ubiquitously expressed at 40% epiboly stage, a stronger expression was observed at shield stage. (C6, C7) Foxo3b became weaker at bud stage; at 6-somite stage, its expression level almost recovered to that of shield stage embryos. (C8, C13) Foxo3b was observed in the developing eye, hindbrain and posterior mesoderm by 24 hpf. (C9, C10) By 55 hpf, foxo3b expression was confined to the anterior central nervous system (CNS), with weak expression in the heart. (C11, C12, C14) By 72 hpf, foxo3b expression became weaker, but continued in the CNS and heart. C1–C4, lateral view; C5, lateral view with dorsal to the right; C6, C7, lateral view with anterior on top; C8, C9, C11, C13, C14, lateral views with anterior to the left; C10, C12, dorsal views with anterior on top; r, retina; p, pectoral fin bud; hb, hindbrain; pm, posterior mesoderm; pd, pronephric duct; h, heart; t, telencephalon; ov, otic vesicle; m, mesencephalon; c, cell; s, somite; h, hours post-fertilization (hpf). ( D ) Relative RNA expression levels as determined by semi-quantitative RT-PCR. For each stage, oligo dT-primed cDNA was used as template for three separate PCR amplifications using primers for foxo3b and 18s (internal control), foxo3b reached a high expression level at shield stage. For quantitative purpose, mRNA expression levels were normalized to 6 hpf (1.00).

    Article Snippet: Oligo-dT-primed cDNA was synthesized by using RevertAid™ first strand cDNA synthesis Kit (Fermentas) and random primers were used to reverse transcribe 2 µg RNA.

    Techniques: Sequencing, Expressing, RNA Expression, Quantitative RT-PCR, Polymerase Chain Reaction

    Nodal knockdown induces a decrease of cell proliferation and an increase of apoptosis in C18-4 cells. ( A ): Transfection efficiency was monitored by the uptake of BLOCK-iT ™ fluorescent oligo (green fluorescence, left panel) at 24 hours following

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Nodal Signaling via an Autocrine Pathway Promotes Proliferation of Mouse Spermatogonial Stem/Progenitor Cells through Smad2/3 and Oct-4 Activation

    doi: 10.1002/stem.198

    Figure Lengend Snippet: Nodal knockdown induces a decrease of cell proliferation and an increase of apoptosis in C18-4 cells. ( A ): Transfection efficiency was monitored by the uptake of BLOCK-iT ™ fluorescent oligo (green fluorescence, left panel) at 24 hours following

    Article Snippet: We found that Nodal siRNAs could be efficiently transfected into C18-4 cells using Lipofectamine™ 2000 as shown by a high uptake of the BLOCK-iT™ fluorescent oligo, whose fluorescent signal correlates with the delivery of active siRNAs (Invitrogen).

    Techniques: Transfection, Blocking Assay, Fluorescence

    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to RNA levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control oligo (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P

    Journal: Journal of Cell Science

    Article Title: The centrosomal deubiquitylase USP21 regulates Gli1 transcriptional activity and stability

    doi: 10.1242/jcs.188516

    Figure Lengend Snippet: USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to RNA levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control oligo (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P

    Article Snippet: Quantitative reverse-transcription PCR Cells were lysed, and mRNA was extracted using the RNAeasy mini kit (Qiagen). cDNA synthesis was performed using 1 µg RNA with RevertAid H-minus M-MuLV reverse transcriptase (Fermentas) using an oligo-dT primer (Promega).

    Techniques: Activity Assay, Incubation, Polymerase Chain Reaction, Transfection, Lysis, Luciferase, Expressing, Construct, Western Blot

    p120 308 is a null allele. (A) p120 308 deletes the entire p120 coding region, but does not affect other genes. Schematic as in Fig. 1 A. Genomic DNA from single wild-type or homozygous mutant flies was PCR amplified using primer pairs from the indicated regions between LD05623 and CG17486 . (B) The p120 mutants are mRNA nulls. cDNA generated from oligo-dT-primed total RNA from p120 mutants and wild-type was amplified with primers spanning the p120 third intron. An unrelated gene, CG2905 , is a control. A DNA control confirmed we were examining mRNA.

    Journal: The Journal of Cell Biology

    Article Title: Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component

    doi: 10.1083/jcb.200211083

    Figure Lengend Snippet: p120 308 is a null allele. (A) p120 308 deletes the entire p120 coding region, but does not affect other genes. Schematic as in Fig. 1 A. Genomic DNA from single wild-type or homozygous mutant flies was PCR amplified using primer pairs from the indicated regions between LD05623 and CG17486 . (B) The p120 mutants are mRNA nulls. cDNA generated from oligo-dT-primed total RNA from p120 mutants and wild-type was amplified with primers spanning the p120 third intron. An unrelated gene, CG2905 , is a control. A DNA control confirmed we were examining mRNA.

    Article Snippet: For RT-PCR, RNA was isolated from 20 larvae using TRIzol reagent and the manufacturer's protocol (Invitrogen Life Technologies), cDNA was generated using oligo-dT primers (Promega Reverse Transcription System), and 5 of 100 μl used for PCR.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Generated

    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with Oligo dT-primers, cDNA was analyzed by semi-quantitative PCR for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.

    Journal: BMC Genomics

    Article Title: Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

    doi: 10.1186/1471-2164-12-196

    Figure Lengend Snippet: KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with Oligo dT-primers, cDNA was analyzed by semi-quantitative PCR for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.

    Article Snippet: RT-PCR and qRT-PCR Reverse transcription was carried out using oligo dT primers and the Omniscript reverse transcriptase kit (Qiagen) following the instructions provided.

    Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction, Expressing, Marker, Polymerase Chain Reaction

    Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Journal: Nucleic Acids Research

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

    doi: 10.1093/nar/gkq131

    Figure Lengend Snippet: Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Article Snippet: RNA (500 ng) was reverse transcribed using an oligo-dT primer. cDNA was amplified with the QuantiTectTM SYBR® Green PCR Kit (Qiagen, Hilden, Germany).

    Techniques: Sequencing, Migration, Modification, Hybridization