amplification primers Search Results


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  • 96
    Integrated DNA Technologies antisense oligonucleotides
    Detection of DHPR α 1S gene expression in C2C12 cells and mouse skeletal muscle A 140 bp protected fragment obtained by specific <t>antisense</t> RNA probe hybridized to the same amount of total RNA (25 μg) from mouse skeletal muscle, C2C12 cell myoblasts in growth medium and C2C12 cell myotubes after 5 days in differentiation medium. The relative amount of total RNA loaded for each sample is indicated by the signal resulting from the hybridization of the 115 bp protected fragment for 28S rRNA in the same reaction.
    Antisense Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 96/100, based on 279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antisense oligonucleotides/product/Integrated DNA Technologies
    Average 96 stars, based on 279 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs nebnext multiplex oligos
    Detection of DHPR α 1S gene expression in C2C12 cells and mouse skeletal muscle A 140 bp protected fragment obtained by specific <t>antisense</t> RNA probe hybridized to the same amount of total RNA (25 μg) from mouse skeletal muscle, C2C12 cell myoblasts in growth medium and C2C12 cell myotubes after 5 days in differentiation medium. The relative amount of total RNA loaded for each sample is indicated by the signal resulting from the hybridization of the 115 bp protected fragment for 28S rRNA in the same reaction.
    Nebnext Multiplex Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4661 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher reagents rnai oligonucleotides oligo
    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool <t>oligonucleotides,</t> seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP <t>RNAi</t> oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P
    Reagents Rnai Oligonucleotides Oligo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Eurofins pcr amplification oligonucleotides
    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool <t>oligonucleotides,</t> seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP <t>RNAi</t> oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P
    Pcr Amplification Oligonucleotides, supplied by Eurofins, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr amplification oligonucleotides/product/Eurofins
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    86
    Thermo Fisher pcr amplifications oligonucleotides
    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool <t>oligonucleotides,</t> seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP <t>RNAi</t> oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P
    Pcr Amplifications Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 3 article reviews
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    91
    Illumina Inc oligonucleotides “oligos
    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool <t>oligonucleotides,</t> seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP <t>RNAi</t> oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P
    Oligonucleotides “Oligos, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche amplification oligos
    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool <t>oligonucleotides,</t> seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP <t>RNAi</t> oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P
    Amplification Oligos, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 3 oligonucleotide primers
    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool <t>oligonucleotides,</t> seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP <t>RNAi</t> oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P
    3 Oligonucleotide Primers, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    5 PRIME primer oligonucleotides
    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool <t>oligonucleotides,</t> seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP <t>RNAi</t> oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P
    Primer Oligonucleotides, supplied by 5 PRIME, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/primer oligonucleotides/product/5 PRIME
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    90
    Roche primer oligonucleotides
    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool <t>oligonucleotides,</t> seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP <t>RNAi</t> oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P
    Primer Oligonucleotides, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/primer oligonucleotides/product/Roche
    Average 90 stars, based on 39 article reviews
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    99
    Thermo Fisher sirna oligonucleotides
    Rottlerin and IL-1β-induced COX-2 expression is independent of PKC δ inhibition. (A) MDA-MB-231 cells were <t>transfected</t> with PKC δ <t>siRNA</t> or control siRNA. After transfection, cells were treated with or without the indicated concentrations
    Sirna Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna oligonucleotides/product/Thermo Fisher
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    99
    Integrated DNA Technologies hairpin amplifiers dna oligonucleotides
    Combined detection of miRNAs and mRNAs by in situ <t>HCR</t> for cell identification. ( a–e ) miR-182 expression overlaps with Rhodopsin (Rho) expression in mouse rods. ( f – j ) miR-182 expression also overlaps with Pcp2, a marker of rod and ON cone bipolar cells. ( k – o ) miR-124 is absent in Müller glial cells expressing Rlbp1. ( e , j , o ) show enlargements of the indicated regions in b, g, l. Magenta: Cy3, green: Cy5, blue: <t>DNA.</t> Scale bars: 25 µm ( a – d , g – i , k–m ); 10 µm ( e , j , k ).
    Hairpin Amplifiers Dna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Detection of DHPR α 1S gene expression in C2C12 cells and mouse skeletal muscle A 140 bp protected fragment obtained by specific antisense RNA probe hybridized to the same amount of total RNA (25 μg) from mouse skeletal muscle, C2C12 cell myoblasts in growth medium and C2C12 cell myotubes after 5 days in differentiation medium. The relative amount of total RNA loaded for each sample is indicated by the signal resulting from the hybridization of the 115 bp protected fragment for 28S rRNA in the same reaction.

    Journal: The Journal of Physiology

    Article Title: Charge movement and transcription regulation of L-type calcium channel ?1S in skeletal muscle cells

    doi: 10.1113/jphysiol.2001.013464

    Figure Lengend Snippet: Detection of DHPR α 1S gene expression in C2C12 cells and mouse skeletal muscle A 140 bp protected fragment obtained by specific antisense RNA probe hybridized to the same amount of total RNA (25 μg) from mouse skeletal muscle, C2C12 cell myoblasts in growth medium and C2C12 cell myotubes after 5 days in differentiation medium. The relative amount of total RNA loaded for each sample is indicated by the signal resulting from the hybridization of the 115 bp protected fragment for 28S rRNA in the same reaction.

    Article Snippet: Sense and antisense oligonucleotides were synthesized and phosphorothioated in three nucleotides at both ends (IDT, Integrated DNA Technologies, Coralville, IA, USA).

    Techniques: Expressing, Hybridization

    Determination of the mouse DHPR α 1S gene transcription start site by RPA A protected fragment was obtained using antisense probe to hybridize to total RNA from skeletal muscle (lane RPA Mu), yeast tRNA (lane RPA Yt) as negative RNA control, and without RNA (lane RPA Pb). Lanes G, A, T and C in the DNA sequence show the control M13mp18 vector sequenced with control primer (Sequenase Sequence Kit, Amersham).

    Journal: The Journal of Physiology

    Article Title: Charge movement and transcription regulation of L-type calcium channel ?1S in skeletal muscle cells

    doi: 10.1113/jphysiol.2001.013464

    Figure Lengend Snippet: Determination of the mouse DHPR α 1S gene transcription start site by RPA A protected fragment was obtained using antisense probe to hybridize to total RNA from skeletal muscle (lane RPA Mu), yeast tRNA (lane RPA Yt) as negative RNA control, and without RNA (lane RPA Pb). Lanes G, A, T and C in the DNA sequence show the control M13mp18 vector sequenced with control primer (Sequenase Sequence Kit, Amersham).

    Article Snippet: Sense and antisense oligonucleotides were synthesized and phosphorothioated in three nucleotides at both ends (IDT, Integrated DNA Technologies, Coralville, IA, USA).

    Techniques: Recombinase Polymerase Amplification, Sequencing, Plasmid Preparation

    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool oligonucleotides, seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP RNAi oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: RCP-driven ?5?1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1-IQGAP1 complex

    doi: 10.1083/jcb.201302041

    Figure Lengend Snippet: RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool oligonucleotides, seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP RNAi oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P

    Article Snippet: Plasmids and reagents RNAi oligonucleotides (oligo) were purchased from Thermo Fisher Scientific as follows: ON-TARGETplus nontargeting siRNA (single oligo or pool as appropriate); IQGAP1 #1 (5′-GAACGUGGCUUAUGAGUAC-3′); IQGAP1 #2 (J-004694-08); RacGAP1 (SMARTpool, oligo 6, 5′-GCGAAGUGCUCUGGAUGUU-3′; and oligo 8, 5′-GAAGUCACAUCUGCCUGUU-3′); Rac1 (SMARTpool or Rac1 #1, 5′-CGGCACCACUGUCCCAACA-3′); RhoA (SMARTpool or RhoA #1, 5′-AUGGAAAGCAGGUAGAGUU-3′); and RCP (J-015968-10). shRNA vectors for PKB/Akt isoforms were prepared using mU6Pro and the following sequences: Akt1 #1, 5′-GCTACTTCCTCCTCAAGAA-3′; Akt1 #2, 5′-CGAGTTTGAGTACCTGAAG-3′; Akt2 #1, 5′-CGTGGTGAATACATCAAGA-3′; and Akt2# 2, 5′-TCTGTCATCAAAGAAGGCT-3′.

    Techniques: Transfection, Stable Transfection, Expressing, Multiple Displacement Amplification, Mutagenesis, Plasmid Preparation

    Integrin trafficking suppresses Rac activity and activates RhoA through the RacGAP1–IQGAP1 complex. (A) A2780 cells were subjected to control or RacGAP1 oligo #6 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C ; n ≥ 15/condition). (B) A2780 cells were subjected to control or IQGAP1 oligo #1 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto the CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C ; n ≥ 8/condition). (C) A2780 cells stably expressing RacGAP1 WT , RacGAP1 249A , or RacGAP1 249D were transfected with Raichu-Rac and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C) . Representative images are shown ( n ≥ 8/condition). (D) A2780 cells stably expressing RacGAP1 WT , RacGAP1 249A , or RacGAP1 249D were transfected with Raichu-RhoA and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C) . Representative images are shown ( n ≥ 4/condition). Zoomed insets correspond to areas indicated by dotted ROIs. Yellow lines represent the baseline activity as determined by an inactive mutant of the probe. Data represent means ± SEM from at least three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: RCP-driven ?5?1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1-IQGAP1 complex

    doi: 10.1083/jcb.201302041

    Figure Lengend Snippet: Integrin trafficking suppresses Rac activity and activates RhoA through the RacGAP1–IQGAP1 complex. (A) A2780 cells were subjected to control or RacGAP1 oligo #6 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C ; n ≥ 15/condition). (B) A2780 cells were subjected to control or IQGAP1 oligo #1 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto the CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C ; n ≥ 8/condition). (C) A2780 cells stably expressing RacGAP1 WT , RacGAP1 249A , or RacGAP1 249D were transfected with Raichu-Rac and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C) . Representative images are shown ( n ≥ 8/condition). (D) A2780 cells stably expressing RacGAP1 WT , RacGAP1 249A , or RacGAP1 249D were transfected with Raichu-RhoA and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C) . Representative images are shown ( n ≥ 4/condition). Zoomed insets correspond to areas indicated by dotted ROIs. Yellow lines represent the baseline activity as determined by an inactive mutant of the probe. Data represent means ± SEM from at least three independent experiments. *, P

    Article Snippet: Plasmids and reagents RNAi oligonucleotides (oligo) were purchased from Thermo Fisher Scientific as follows: ON-TARGETplus nontargeting siRNA (single oligo or pool as appropriate); IQGAP1 #1 (5′-GAACGUGGCUUAUGAGUAC-3′); IQGAP1 #2 (J-004694-08); RacGAP1 (SMARTpool, oligo 6, 5′-GCGAAGUGCUCUGGAUGUU-3′; and oligo 8, 5′-GAAGUCACAUCUGCCUGUU-3′); Rac1 (SMARTpool or Rac1 #1, 5′-CGGCACCACUGUCCCAACA-3′); RhoA (SMARTpool or RhoA #1, 5′-AUGGAAAGCAGGUAGAGUU-3′); and RCP (J-015968-10). shRNA vectors for PKB/Akt isoforms were prepared using mU6Pro and the following sequences: Akt1 #1, 5′-GCTACTTCCTCCTCAAGAA-3′; Akt1 #2, 5′-CGAGTTTGAGTACCTGAAG-3′; Akt2 #1, 5′-CGTGGTGAATACATCAAGA-3′; and Akt2# 2, 5′-TCTGTCATCAAAGAAGGCT-3′.

    Techniques: Activity Assay, Transfection, Stable Transfection, Expressing, Mutagenesis

    Effects of zinc and hypoxia on nuclear localization and DNA binding of MTF-1. (A) C 2 C 12 cells were untreated (control) with 100 μmol/L ZnCl 2 , or exposed to hypoxia (pO 2 = 1%) for 16 h, as indicated. Extracts were analyzed by electrophoretic mobility shift assays (EMSA) using labeled MRE-s oligos. Only the region of the gel containing the specific MTF-1 complexes is shown. (B) Immunoblotting detection of MTF-1 in whole-cell (WCE), nuclear, and cytosolic extracts. Extracts were prepared from L cells that were untreated (0), treated with 100 μmol/L ZnCl 2 (Zn), or exposed to hypoxia (Hx) (pO 2 = 1%), as indicated, and analyzed by immunoblotting with antibodies against MTF-1 and tubulin. Because tubulin is not an appropriate loading control for nuclear extracts, a particular band detected on the blot by staining with amido black is shown. The results shown in panels A and B are representative of 3 independent experiments. (C) ChIP assays were performed using chromatin isolated from L cells that were untreated or treatedwith 100 μmol/L ZnCl 2 for 3 h or exposed to hypoxia for 16 h, prior to formaldehyde crosslinking. Immunoprecipitation of crosslinked chromatin was done with MTF-1 (anti-MTF-1) or HIF-1α (anti-HIF1α) antibodies or a pre-immune normal rabbit serum (PI), as indicated. DNA from both the IP input (prior to immunoprecipitation) and the IP-bound fractions was amplified by PCR with primer pairs for the mouse Mt-1 or Glut-1 promoter. Input, amplification of DNA prior to immunoprecipitation. The input sample contained 0.4% of the supernatant used for immunoprecipitation of crosslinked MTF-1. The PCR products were analyzed by agarose gel electrophoresis. These ChIP assays were performed 3 times using 3 different chromatin preparations with similar results.

    Journal: Biochemistry and cell biology = Biochimie et biologie cellulaire

    Article Title: Hypoxia acts through multiple signaling pathways to induce metallothionein transactivation by the metal-responsive transcription factor-1 (MTF-1)

    doi: 10.1139/o11-063

    Figure Lengend Snippet: Effects of zinc and hypoxia on nuclear localization and DNA binding of MTF-1. (A) C 2 C 12 cells were untreated (control) with 100 μmol/L ZnCl 2 , or exposed to hypoxia (pO 2 = 1%) for 16 h, as indicated. Extracts were analyzed by electrophoretic mobility shift assays (EMSA) using labeled MRE-s oligos. Only the region of the gel containing the specific MTF-1 complexes is shown. (B) Immunoblotting detection of MTF-1 in whole-cell (WCE), nuclear, and cytosolic extracts. Extracts were prepared from L cells that were untreated (0), treated with 100 μmol/L ZnCl 2 (Zn), or exposed to hypoxia (Hx) (pO 2 = 1%), as indicated, and analyzed by immunoblotting with antibodies against MTF-1 and tubulin. Because tubulin is not an appropriate loading control for nuclear extracts, a particular band detected on the blot by staining with amido black is shown. The results shown in panels A and B are representative of 3 independent experiments. (C) ChIP assays were performed using chromatin isolated from L cells that were untreated or treatedwith 100 μmol/L ZnCl 2 for 3 h or exposed to hypoxia for 16 h, prior to formaldehyde crosslinking. Immunoprecipitation of crosslinked chromatin was done with MTF-1 (anti-MTF-1) or HIF-1α (anti-HIF1α) antibodies or a pre-immune normal rabbit serum (PI), as indicated. DNA from both the IP input (prior to immunoprecipitation) and the IP-bound fractions was amplified by PCR with primer pairs for the mouse Mt-1 or Glut-1 promoter. Input, amplification of DNA prior to immunoprecipitation. The input sample contained 0.4% of the supernatant used for immunoprecipitation of crosslinked MTF-1. The PCR products were analyzed by agarose gel electrophoresis. These ChIP assays were performed 3 times using 3 different chromatin preparations with similar results.

    Article Snippet: The transfection reagent ExGen500 was purchased from MBI Fermentas (Burlington, Ont.), DNA modifying enzymes were obtained from New England Biolabs (Pickering, Ont.), and synthetic oligonucleotides (oligos) were from Invitrogen (Carlsbad, Calif.) or Sigma–Aldrich (St-Louis, Mo.).

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Staining, Chromatin Immunoprecipitation, Isolation, Cross-linking Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    iFISH implementation. a Scheme of iFISH4U. Pre-designed genome-wide databases of oligos (left) are used as input by the iFISH4U web interface (center) to select oligos within one or more user-specified genomic regions, based on the indicated features. Features 1–3 are used while designing single probes, whereas all the four features are used to design multiple probes on the same chromosome. The black dashed boxes indicate examples of probes within the same region of interest, with the same number of oligos (vertical bars), but suboptimal size (1), homogeneity (2), or centrality (3), whereas the orange box represents the probe of choice having optimal size, homogeneity and centrality. b Cumulative distribution of the distances between consecutive oligos in the human 40-mers database. c Median standard deviation (s.d.) of the distance between consecutive oligos, inside non-overlapping genomic windows of the indicated size, in the 40-mers database and OligoMiner (OM) hg19 databases. OMB, OM ‘Balance’. OMC, OM ‘Coverage’. OMS, OM ‘Stringent’. d Percentage of non-overlapping genomic windows of the indicated size, containing at least 96 oligos, in the 40-mers database and OM hg19 databases. e Scheme of oligos in iFISH probes. Each probe consists of n oligos differing in the T sequence. f Location of the 330 iFISH probes targeting all the human autosomes and chrX. Red dots, individually tested probes (see Fig 2a, b ). g Scheme of the pipeline used to produce iFISH probes. (1) Up to 12,000 oligos, corresponding to a maximum of 125 probes each containing 96 oligos, are synthesized on an array and then pooled together. (2) The oligo-pool is dispensed into n 96-well plates, depending on the total number of probes ( p ) and colors per probe ( c ). (3) In each well, the oligos corresponding to the same probe are selectively amplified using a probe-specific PCR primer pair that incorporates the T7 promoter sequence (T7) and color adapter sequence (C), and (4) successfully amplified probes are purified and linearly amplified by in vitro transcription (IVT). (5) Purified IVT products are reverse transcribed (RT), (6) RNA is hydrolyzed, and finally (7) single-stranded DNA (ssDNA) is purified to obtain ready-to-use probes

    Journal: Nature Communications

    Article Title: iFISH is a publically available resource enabling versatile DNA FISH to study genome architecture

    doi: 10.1038/s41467-019-09616-w

    Figure Lengend Snippet: iFISH implementation. a Scheme of iFISH4U. Pre-designed genome-wide databases of oligos (left) are used as input by the iFISH4U web interface (center) to select oligos within one or more user-specified genomic regions, based on the indicated features. Features 1–3 are used while designing single probes, whereas all the four features are used to design multiple probes on the same chromosome. The black dashed boxes indicate examples of probes within the same region of interest, with the same number of oligos (vertical bars), but suboptimal size (1), homogeneity (2), or centrality (3), whereas the orange box represents the probe of choice having optimal size, homogeneity and centrality. b Cumulative distribution of the distances between consecutive oligos in the human 40-mers database. c Median standard deviation (s.d.) of the distance between consecutive oligos, inside non-overlapping genomic windows of the indicated size, in the 40-mers database and OligoMiner (OM) hg19 databases. OMB, OM ‘Balance’. OMC, OM ‘Coverage’. OMS, OM ‘Stringent’. d Percentage of non-overlapping genomic windows of the indicated size, containing at least 96 oligos, in the 40-mers database and OM hg19 databases. e Scheme of oligos in iFISH probes. Each probe consists of n oligos differing in the T sequence. f Location of the 330 iFISH probes targeting all the human autosomes and chrX. Red dots, individually tested probes (see Fig 2a, b ). g Scheme of the pipeline used to produce iFISH probes. (1) Up to 12,000 oligos, corresponding to a maximum of 125 probes each containing 96 oligos, are synthesized on an array and then pooled together. (2) The oligo-pool is dispensed into n 96-well plates, depending on the total number of probes ( p ) and colors per probe ( c ). (3) In each well, the oligos corresponding to the same probe are selectively amplified using a probe-specific PCR primer pair that incorporates the T7 promoter sequence (T7) and color adapter sequence (C), and (4) successfully amplified probes are purified and linearly amplified by in vitro transcription (IVT). (5) Purified IVT products are reverse transcribed (RT), (6) RNA is hydrolyzed, and finally (7) single-stranded DNA (ssDNA) is purified to obtain ready-to-use probes

    Article Snippet: We then amplified the oligos in each well by real-time PCR using the SYBR Select Master Mix (Thermo Fisher Scientific, cat. no. 4472913).

    Techniques: Genome Wide, Standard Deviation, Sequencing, Synthesized, Amplification, Polymerase Chain Reaction, Purification, In Vitro

    Scheme of in vitro ligation selection and sequencing library preparation. For each ligase selected library, an equal amount of 4 random RNA oligos containing a constant region (solid line), a randomized region (wavy line) and a known 3′-nt were combined to make a random oligo pool and used as substrates in a ligation reaction with pre-adenylated SR1 DNA adapter using a specific T4 RNA ligase. The ligated products were reverse transcribed and amplified to introduce the required primer regions for Ion Torrent sequencing. To determine the sequence content of the random RNA oligo pool, each of the four RNA oligos was sequenced independently. First, the oligos were poly A tailed for the random RNA oligo U, C and G or poly C tailed for the random RNA oligo A using poly(A) polymerase. The tailed RNA oligos were then reverse transcribed using primers complementary to the polymer tails ( Supplementary Table S1 ). The cDNA libraries were amplified and processed in the same manner as the ligase selected libraries described above.

    Journal: Nucleic Acids Research

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation

    doi: 10.1093/nar/gkr1263

    Figure Lengend Snippet: Scheme of in vitro ligation selection and sequencing library preparation. For each ligase selected library, an equal amount of 4 random RNA oligos containing a constant region (solid line), a randomized region (wavy line) and a known 3′-nt were combined to make a random oligo pool and used as substrates in a ligation reaction with pre-adenylated SR1 DNA adapter using a specific T4 RNA ligase. The ligated products were reverse transcribed and amplified to introduce the required primer regions for Ion Torrent sequencing. To determine the sequence content of the random RNA oligo pool, each of the four RNA oligos was sequenced independently. First, the oligos were poly A tailed for the random RNA oligo U, C and G or poly C tailed for the random RNA oligo A using poly(A) polymerase. The tailed RNA oligos were then reverse transcribed using primers complementary to the polymer tails ( Supplementary Table S1 ). The cDNA libraries were amplified and processed in the same manner as the ligase selected libraries described above.

    Article Snippet: To assess the frequency of nucleotides at each randomized position in the oligo pool, the random RNA oligos were subjected to Ion Torrent sequencing without undergoing adapter ligation ( ).

    Techniques: In Vitro, Ligation, Selection, Sequencing, Amplification, Introduce

    miR-29 suppresses YY1 through binding to its 3’UTR (A) Predicted target site of miR-29c (green) in the 3’UTR of mouse YY1 (red) with the seed region underlined. (B) Predicted folding structure from mFOLD between miR-29c (green) and YY1 3’UTR (red). The minimal free energy (mfe) is indicated as well as the seed region shown by a line and arrow. (C) A wild type (WT) luciferase reporter was generated by fusing a ~ 500bp fragment of the YY1 3’UTR encompassing the miR-29 binding site downstream of the luciferase (Luc) reporter gene. The mutant plasmid was generated by deleting the miR-29 binding site. WT or Mutant reporter constructs were then transfected into MB with indicated precursor miRNA oligos. Luciferase was determined at 48h post-transfection and normalized to β-Galactosidase. Data represent the average of three independent experiments ± S.D. (D) MB were transfected with either precursor (Pre-NC) or Anti-miR control (Anti-NC) or precursor miR-29c (Pre-29) or anti-miR-29c (Anti-29) oligos. YY1 protein was then probed in extracts from cells differentiated for 48h. Blots were stripped and reprobed for α-tubulin. (E) P5 neonatal mice were injected with anti-miR control (Anti-NC) or anti-miR-29c (Anti-29) oligos into lower limb muscles. 48h post-injection, lysates were probed for YY1. (F) MB were transfected with siRNA control or siRNA to YY1. Cells where then differentiated for 24h, at which time they were photographed under phase contrast (scale bar = 200µm) or immunostained for MyHC (scale bar = 100µm). Numbers indicate averages of MyHC positive cells counted from a minimum of 10 randomly chosen fields.

    Journal: Cancer cell

    Article Title: NF-?B-YY1-miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

    doi: 10.1016/j.ccr.2008.10.006

    Figure Lengend Snippet: miR-29 suppresses YY1 through binding to its 3’UTR (A) Predicted target site of miR-29c (green) in the 3’UTR of mouse YY1 (red) with the seed region underlined. (B) Predicted folding structure from mFOLD between miR-29c (green) and YY1 3’UTR (red). The minimal free energy (mfe) is indicated as well as the seed region shown by a line and arrow. (C) A wild type (WT) luciferase reporter was generated by fusing a ~ 500bp fragment of the YY1 3’UTR encompassing the miR-29 binding site downstream of the luciferase (Luc) reporter gene. The mutant plasmid was generated by deleting the miR-29 binding site. WT or Mutant reporter constructs were then transfected into MB with indicated precursor miRNA oligos. Luciferase was determined at 48h post-transfection and normalized to β-Galactosidase. Data represent the average of three independent experiments ± S.D. (D) MB were transfected with either precursor (Pre-NC) or Anti-miR control (Anti-NC) or precursor miR-29c (Pre-29) or anti-miR-29c (Anti-29) oligos. YY1 protein was then probed in extracts from cells differentiated for 48h. Blots were stripped and reprobed for α-tubulin. (E) P5 neonatal mice were injected with anti-miR control (Anti-NC) or anti-miR-29c (Anti-29) oligos into lower limb muscles. 48h post-injection, lysates were probed for YY1. (F) MB were transfected with siRNA control or siRNA to YY1. Cells where then differentiated for 24h, at which time they were photographed under phase contrast (scale bar = 200µm) or immunostained for MyHC (scale bar = 100µm). Numbers indicate averages of MyHC positive cells counted from a minimum of 10 randomly chosen fields.

    Article Snippet: 5 µM of pre-miR oligos or siRNA oligos were pre-incubated with Lipofectamine (Invitrogen) for 15 minutes prior to injection into tumors in a final volume of 60µl of OPTI-EM (Invitrogen).

    Techniques: Binding Assay, Luciferase, Generated, Mutagenesis, Plasmid Preparation, Construct, Transfection, Mouse Assay, Injection

    NF-κBY–Y1–miR-29 circuitry is dysregulated in RMS (A) Extracts were prepared from normal human muscle and RMS cell lines and immunoblots were performed probing for YY1, Ezh2, and p65. (B) Lysates were prepared five RMS patient tumors and adjacent normal muscle tissue and probed for YY1, Ezh2, and p65 proteins. (C) RH30 cells were transfected with siRNA control oligos or siRNA-YY1. Cells were differentiated and semi-quantitative RT-PCR was performed probing for differentiation markers, or GAPDH used as a control. (D) RH30 tumors were established in nude mice and then injected with siRNA oligos every 3 days for 1 week. RNA and protein lysates were prepared from tumors and subsequently probed for MyHC, α-actin and troponin by RT-PCR and YY1 by Western. (E) ChIPs with either an YY1 antibody or control IgG were performed on chromatins isolated from human skeletal muscle cells (control) or RH30 cells. Precipitated DNA fragments were amplified with oligonucleotides spanning regions A-D of the human miR-29b/c regulatory region. Total inputs are indicated. (F) RH30 cells were infected with adenoviruses expressing vector control or IκBα-SR. YY1 and miR-29b levels were measured at 48h post-infection by qRT-PCR. (G) RH30 cells were infected with control or IκBá-SR adenovirus and differentiation markers were probed by qRT-PCR. (H) RH30 cells were transduced with vector or IκBα-SR expressing retroviruses to generate stable cell lines. Cells were then treated with differentiation medium for 2 days and immunostained for troponin or quantified for myofibrillar expression. (I) The model depicts the role of the NF-κB-YY1-miR-29 regulatory circuit in both normal myogenic differentiation and RMS. In myogenesis this circuit involves constitutive activity of NF-κB in myoblasts regulating YY1, which subsequently epigentically suppresses miR-29 and maintains cells in an undifferentiated state. As differentiation ensues, downregulation of the NF-κB–YY1 pathway leads to upregulation of miR-29 that in turns further decreases YY1 levels to ensure proper differentiation into myotubes. In RMS, this circuit becomes dysregulated due to an increase in the NF-κB–YY1 pathway that constitutively represses miR-29. In the absence of miR-29 tumor suppressor activity, YY1 is left uncontrolled thereby impairing differentiation leading to Rhabdomyosarcomagenesis.

    Journal: Cancer cell

    Article Title: NF-?B-YY1-miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

    doi: 10.1016/j.ccr.2008.10.006

    Figure Lengend Snippet: NF-κBY–Y1–miR-29 circuitry is dysregulated in RMS (A) Extracts were prepared from normal human muscle and RMS cell lines and immunoblots were performed probing for YY1, Ezh2, and p65. (B) Lysates were prepared five RMS patient tumors and adjacent normal muscle tissue and probed for YY1, Ezh2, and p65 proteins. (C) RH30 cells were transfected with siRNA control oligos or siRNA-YY1. Cells were differentiated and semi-quantitative RT-PCR was performed probing for differentiation markers, or GAPDH used as a control. (D) RH30 tumors were established in nude mice and then injected with siRNA oligos every 3 days for 1 week. RNA and protein lysates were prepared from tumors and subsequently probed for MyHC, α-actin and troponin by RT-PCR and YY1 by Western. (E) ChIPs with either an YY1 antibody or control IgG were performed on chromatins isolated from human skeletal muscle cells (control) or RH30 cells. Precipitated DNA fragments were amplified with oligonucleotides spanning regions A-D of the human miR-29b/c regulatory region. Total inputs are indicated. (F) RH30 cells were infected with adenoviruses expressing vector control or IκBα-SR. YY1 and miR-29b levels were measured at 48h post-infection by qRT-PCR. (G) RH30 cells were infected with control or IκBá-SR adenovirus and differentiation markers were probed by qRT-PCR. (H) RH30 cells were transduced with vector or IκBα-SR expressing retroviruses to generate stable cell lines. Cells were then treated with differentiation medium for 2 days and immunostained for troponin or quantified for myofibrillar expression. (I) The model depicts the role of the NF-κB-YY1-miR-29 regulatory circuit in both normal myogenic differentiation and RMS. In myogenesis this circuit involves constitutive activity of NF-κB in myoblasts regulating YY1, which subsequently epigentically suppresses miR-29 and maintains cells in an undifferentiated state. As differentiation ensues, downregulation of the NF-κB–YY1 pathway leads to upregulation of miR-29 that in turns further decreases YY1 levels to ensure proper differentiation into myotubes. In RMS, this circuit becomes dysregulated due to an increase in the NF-κB–YY1 pathway that constitutively represses miR-29. In the absence of miR-29 tumor suppressor activity, YY1 is left uncontrolled thereby impairing differentiation leading to Rhabdomyosarcomagenesis.

    Article Snippet: 5 µM of pre-miR oligos or siRNA oligos were pre-incubated with Lipofectamine (Invitrogen) for 15 minutes prior to injection into tumors in a final volume of 60µl of OPTI-EM (Invitrogen).

    Techniques: Western Blot, Transfection, Quantitative RT-PCR, Mouse Assay, Injection, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Infection, Expressing, Plasmid Preparation, Transduction, Stable Transfection, Activity Assay

    NF-κB negatively regulates miR-29b/c (A) C2C12 cells were treated with TNFα and miR-29 was measured by qRT-PCR normalized to U6. Fold changes are shown with respect to vector cells where miR-29 levels were set to a value of 1. (B) MB were transfected with vector or a p65 plasmid and miR-29b/c levels were measured 48h post-transfection. (C) MB were transfected with either vector or p65 siRNA oligos and miR-29 expression was then measured as in (B). (D) MiR-29b/c were measured in MB stably expressing vector or IκBα-SR. Fold changes are shown with respect to vector cells, which were set to a value of 1. (E) MyoD was stably expressed in p65 +/+ or p65 −/− ) and qRT-PCR was performed for miR-29b and miR-29c. (F) ChIPs with YY1 or control IgG were performed on chromatins derived from either vector control (V) or Iκ Bα-SR (SR) expressing MB. Primers specific to site D were used for the PCR amplification. Total inputs are indicated.

    Journal: Cancer cell

    Article Title: NF-?B-YY1-miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

    doi: 10.1016/j.ccr.2008.10.006

    Figure Lengend Snippet: NF-κB negatively regulates miR-29b/c (A) C2C12 cells were treated with TNFα and miR-29 was measured by qRT-PCR normalized to U6. Fold changes are shown with respect to vector cells where miR-29 levels were set to a value of 1. (B) MB were transfected with vector or a p65 plasmid and miR-29b/c levels were measured 48h post-transfection. (C) MB were transfected with either vector or p65 siRNA oligos and miR-29 expression was then measured as in (B). (D) MiR-29b/c were measured in MB stably expressing vector or IκBα-SR. Fold changes are shown with respect to vector cells, which were set to a value of 1. (E) MyoD was stably expressed in p65 +/+ or p65 −/− ) and qRT-PCR was performed for miR-29b and miR-29c. (F) ChIPs with YY1 or control IgG were performed on chromatins derived from either vector control (V) or Iκ Bα-SR (SR) expressing MB. Primers specific to site D were used for the PCR amplification. Total inputs are indicated.

    Article Snippet: 5 µM of pre-miR oligos or siRNA oligos were pre-incubated with Lipofectamine (Invitrogen) for 15 minutes prior to injection into tumors in a final volume of 60µl of OPTI-EM (Invitrogen).

    Techniques: Quantitative RT-PCR, Plasmid Preparation, Transfection, Expressing, Stable Transfection, Derivative Assay, Polymerase Chain Reaction, Amplification

    miR-29 accelerates muscle differentiation (A) C2C12 cells were transfected with 0.2µg of MyHC-Luc or Troponin-Luc reporters along with pCMV-LacZ and 50µM of precursor control oligos (Pre-NC) or miR-29c (Pre-29c) oligos. Cells were differentiated for 48h and luciferase was determined and normalized to β-Galactosidase. Relative activity is shown with respect to control cells where normalized luciferase values were set to 1. The data represents the average of three independent experiments ± S.D. (B) C2C12 MB were transfected with Pre-NC or Pre-29c oligos. Cells were then maintained as MB or differentiated into MT. Lysates were prepared and probed for MyHC and troponin T. (C) MB were transfected with Pre-NC or Pre-29a, b, or c members and differentiated (DM) for 1 or 2 days (d), at which time cells were immunostained for MyHC. Scale bar = 100µm. Cell morphology was visualized by phase-contrast microscopy; scale bar = 200µm. (D) C2C12 cells transfected with a MyHC-Luc along with anti-miR control (Anti-NC) or anti-miR-29c (Anti-29c). Cells were then maintained as MB or differentiated into MT for 48hrs at which time luciferase activity was determined. (E) MB were administered Anti-NC or Anti-29c and then MyHC and troponin were probed in cells maintained as MB or differentiated into MT. (F) MB were transfected with Anti-NC or Anti-29c oligos and cells were subsequently differentiated for 3 days at which time cultures were stained for MyHC. Positively stained cells were counted from a minimum of 10 randomly chosen fields from 3 individual plates.

    Journal: Cancer cell

    Article Title: NF-?B-YY1-miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

    doi: 10.1016/j.ccr.2008.10.006

    Figure Lengend Snippet: miR-29 accelerates muscle differentiation (A) C2C12 cells were transfected with 0.2µg of MyHC-Luc or Troponin-Luc reporters along with pCMV-LacZ and 50µM of precursor control oligos (Pre-NC) or miR-29c (Pre-29c) oligos. Cells were differentiated for 48h and luciferase was determined and normalized to β-Galactosidase. Relative activity is shown with respect to control cells where normalized luciferase values were set to 1. The data represents the average of three independent experiments ± S.D. (B) C2C12 MB were transfected with Pre-NC or Pre-29c oligos. Cells were then maintained as MB or differentiated into MT. Lysates were prepared and probed for MyHC and troponin T. (C) MB were transfected with Pre-NC or Pre-29a, b, or c members and differentiated (DM) for 1 or 2 days (d), at which time cells were immunostained for MyHC. Scale bar = 100µm. Cell morphology was visualized by phase-contrast microscopy; scale bar = 200µm. (D) C2C12 cells transfected with a MyHC-Luc along with anti-miR control (Anti-NC) or anti-miR-29c (Anti-29c). Cells were then maintained as MB or differentiated into MT for 48hrs at which time luciferase activity was determined. (E) MB were administered Anti-NC or Anti-29c and then MyHC and troponin were probed in cells maintained as MB or differentiated into MT. (F) MB were transfected with Anti-NC or Anti-29c oligos and cells were subsequently differentiated for 3 days at which time cultures were stained for MyHC. Positively stained cells were counted from a minimum of 10 randomly chosen fields from 3 individual plates.

    Article Snippet: 5 µM of pre-miR oligos or siRNA oligos were pre-incubated with Lipofectamine (Invitrogen) for 15 minutes prior to injection into tumors in a final volume of 60µl of OPTI-EM (Invitrogen).

    Techniques: Transfection, Luciferase, Activity Assay, Microscopy, Staining

    YY1 represses miR-29b/c through binding to a conserved regulatory region (A) An rVISTA schematic showing the degree of sequence conservation between human and mouse chromosome (Chr) 1 in a region upstream of the miR-29b/c cluster. Predicted YY1, MyoD, myogenin, SRF, and Mef2 sites are displayed. (B) EMSA performed from C2C12 MB or MT with probes corresponding to YY1 sites A-D. With MB extracts, a supershift EMSA was performed using YY1 antisera. Arrows denote YY1/DNA bound complexes. (C) ChIP assays for YY1 was performed with chromatin from C2C12 MB or MT. Precipitated DNA was amplified with oligonucleotides spanning regions A-D. Total inputs are indicated. (D) ChIPs as in (C) were repeated for Ezh2, H3K27, HDAC-1, SRF or Mef2. (E) MB were transfected with either an YY1 expression plasmid (pCMV-YY1) or YY1 siRNA oligos and then induced to differentiate for 24h, at which time miR-29b and miR-29c were measured by qRT-PCR and normalized to U6. Fold changes are shown with respect to control siRNA transfected cells where miR-29 levels were set to a value of 1. (F) MB were transfected with a miR-29b/c-enhancer-Luc reporter and maintained as MB or differentiated in MT for 48h, at which time luciferase activities were determined. (G) MB were transfected with a miR-29b/c-enhancer-Luc reporter, along with plasmids for YY1, SRF or Mef2. Cells were then switched differentiation conditions and luciferase activity was subsequently measured. (H) MB were transfected with the miR-29b/cenhancer- Luc reporter (YY1 wt) or with an enhancer reporter containing a deletion mutation in the YY1 “D” site (YY1 mut), and subsequently differentiated for 48h (MT) at which time luciferase activity was determined (left graph). Separate transfections were performed with YY1 wt and YY1 mut reporters along with an YY1 expression plasmid (pCMV-YY1) or YY1 siRNA. Cells were subsequently differentiated for 48h at which time luciferase was determined. Vector siRNA oligo transfected cells were used as a control (right graph). All luciferase data were normalized to β-galactosidase protein and represent the average of three independent experiments ± S.D.

    Journal: Cancer cell

    Article Title: NF-?B-YY1-miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

    doi: 10.1016/j.ccr.2008.10.006

    Figure Lengend Snippet: YY1 represses miR-29b/c through binding to a conserved regulatory region (A) An rVISTA schematic showing the degree of sequence conservation between human and mouse chromosome (Chr) 1 in a region upstream of the miR-29b/c cluster. Predicted YY1, MyoD, myogenin, SRF, and Mef2 sites are displayed. (B) EMSA performed from C2C12 MB or MT with probes corresponding to YY1 sites A-D. With MB extracts, a supershift EMSA was performed using YY1 antisera. Arrows denote YY1/DNA bound complexes. (C) ChIP assays for YY1 was performed with chromatin from C2C12 MB or MT. Precipitated DNA was amplified with oligonucleotides spanning regions A-D. Total inputs are indicated. (D) ChIPs as in (C) were repeated for Ezh2, H3K27, HDAC-1, SRF or Mef2. (E) MB were transfected with either an YY1 expression plasmid (pCMV-YY1) or YY1 siRNA oligos and then induced to differentiate for 24h, at which time miR-29b and miR-29c were measured by qRT-PCR and normalized to U6. Fold changes are shown with respect to control siRNA transfected cells where miR-29 levels were set to a value of 1. (F) MB were transfected with a miR-29b/c-enhancer-Luc reporter and maintained as MB or differentiated in MT for 48h, at which time luciferase activities were determined. (G) MB were transfected with a miR-29b/c-enhancer-Luc reporter, along with plasmids for YY1, SRF or Mef2. Cells were then switched differentiation conditions and luciferase activity was subsequently measured. (H) MB were transfected with the miR-29b/cenhancer- Luc reporter (YY1 wt) or with an enhancer reporter containing a deletion mutation in the YY1 “D” site (YY1 mut), and subsequently differentiated for 48h (MT) at which time luciferase activity was determined (left graph). Separate transfections were performed with YY1 wt and YY1 mut reporters along with an YY1 expression plasmid (pCMV-YY1) or YY1 siRNA. Cells were subsequently differentiated for 48h at which time luciferase was determined. Vector siRNA oligo transfected cells were used as a control (right graph). All luciferase data were normalized to β-galactosidase protein and represent the average of three independent experiments ± S.D.

    Article Snippet: 5 µM of pre-miR oligos or siRNA oligos were pre-incubated with Lipofectamine (Invitrogen) for 15 minutes prior to injection into tumors in a final volume of 60µl of OPTI-EM (Invitrogen).

    Techniques: Binding Assay, Sequencing, Chromatin Immunoprecipitation, Amplification, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Luciferase, Activity Assay, Mutagenesis

    Rottlerin and IL-1β-induced COX-2 expression is independent of PKC δ inhibition. (A) MDA-MB-231 cells were transfected with PKC δ siRNA or control siRNA. After transfection, cells were treated with or without the indicated concentrations

    Journal: Experimental & Molecular Medicine

    Article Title: Rottlerin enhances IL-1?-induced COX-2 expression through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells

    doi: 10.3858/emm.2011.43.12.077

    Figure Lengend Snippet: Rottlerin and IL-1β-induced COX-2 expression is independent of PKC δ inhibition. (A) MDA-MB-231 cells were transfected with PKC δ siRNA or control siRNA. After transfection, cells were treated with or without the indicated concentrations

    Article Snippet: Cells were transfected with siRNA oligonucleotides using Oligofectamine™ Reagent (Invitrogen).

    Techniques: Expressing, Inhibition, Multiple Displacement Amplification, Transfection

    Combined detection of miRNAs and mRNAs by in situ HCR for cell identification. ( a–e ) miR-182 expression overlaps with Rhodopsin (Rho) expression in mouse rods. ( f – j ) miR-182 expression also overlaps with Pcp2, a marker of rod and ON cone bipolar cells. ( k – o ) miR-124 is absent in Müller glial cells expressing Rlbp1. ( e , j , o ) show enlargements of the indicated regions in b, g, l. Magenta: Cy3, green: Cy5, blue: DNA. Scale bars: 25 µm ( a – d , g – i , k–m ); 10 µm ( e , j , k ).

    Journal: Scientific Reports

    Article Title: Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction

    doi: 10.1038/s41598-019-57194-0

    Figure Lengend Snippet: Combined detection of miRNAs and mRNAs by in situ HCR for cell identification. ( a–e ) miR-182 expression overlaps with Rhodopsin (Rho) expression in mouse rods. ( f – j ) miR-182 expression also overlaps with Pcp2, a marker of rod and ON cone bipolar cells. ( k – o ) miR-124 is absent in Müller glial cells expressing Rlbp1. ( e , j , o ) show enlargements of the indicated regions in b, g, l. Magenta: Cy3, green: Cy5, blue: DNA. Scale bars: 25 µm ( a – d , g – i , k–m ); 10 µm ( e , j , k ).

    Article Snippet: HCR in situ probes and hairpin amplifiers DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT); sequences are in Supplementary Tables – .

    Techniques: In Situ, Expressing, Marker

    Characterization of miR-181a expressing retinal cells. ( a , b ) miR-181a is highly expressed in inner INL of in adult mouse retinas. ( c , d ) High level miR-181a and miR-182 expression does not overlap, although lower level signal for miR-181a overlaps with miR-182 in the INL. ( e – h ) miR-181a detected by in situ HCR and the amacrine cell specific protein AP2α detected by indirect immunofluorescence in P7 retina sections. Cells with strongest miR-181a signal overlap AP2α. ( i – l ) AP2α protein and strong expression of miR-181a overlap at E18.5 in forming amacrine layer, but both are lost in Ptf1a −/− retinas, although lower level signal for miR-181a persists in the GCL and INL. ( m , n ) Combined detection of miR-181a with Gad1 mRNA in GABAergic amacrine cells. ( o , p ) Combined detection of miR-181a and Slc6a9a mRNA in glycinergic amacrine cells, using split initiator HCR probes for Slc6a9 mRNA in situ HCR. Complete sets of panels for m-p are shown in Supplementary Figs. S2 and S3 . Examples of overlapping cells in n and p indicated by cyan asterisks. Magenta; Cy3, green: Alexa-647 ( e – l ) or Cy5 ( m , n ) or Alexa-488 ( o , p ), blue: DNA. Scale bar: 25 µm ( a – p ).

    Journal: Scientific Reports

    Article Title: Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction

    doi: 10.1038/s41598-019-57194-0

    Figure Lengend Snippet: Characterization of miR-181a expressing retinal cells. ( a , b ) miR-181a is highly expressed in inner INL of in adult mouse retinas. ( c , d ) High level miR-181a and miR-182 expression does not overlap, although lower level signal for miR-181a overlaps with miR-182 in the INL. ( e – h ) miR-181a detected by in situ HCR and the amacrine cell specific protein AP2α detected by indirect immunofluorescence in P7 retina sections. Cells with strongest miR-181a signal overlap AP2α. ( i – l ) AP2α protein and strong expression of miR-181a overlap at E18.5 in forming amacrine layer, but both are lost in Ptf1a −/− retinas, although lower level signal for miR-181a persists in the GCL and INL. ( m , n ) Combined detection of miR-181a with Gad1 mRNA in GABAergic amacrine cells. ( o , p ) Combined detection of miR-181a and Slc6a9a mRNA in glycinergic amacrine cells, using split initiator HCR probes for Slc6a9 mRNA in situ HCR. Complete sets of panels for m-p are shown in Supplementary Figs. S2 and S3 . Examples of overlapping cells in n and p indicated by cyan asterisks. Magenta; Cy3, green: Alexa-647 ( e – l ) or Cy5 ( m , n ) or Alexa-488 ( o , p ), blue: DNA. Scale bar: 25 µm ( a – p ).

    Article Snippet: HCR in situ probes and hairpin amplifiers DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT); sequences are in Supplementary Tables – .

    Techniques: Expressing, In Situ, Immunofluorescence

    miRNA detection by in situ HCR in tissue sections from mouse retinas. ( a ) Schematic of HCR probes for miRNA detection (also see Supplementary Fig. S1 ); initiator sequences are not shown for the mismatch or miR-183 probes. ( b , c ) Non-HCR miRNA in situ hybridization for miR-182 or miR-183, using 20 nt fluorescein-labeled RNA probes detected with alkaline phosphatase (AP) conjugated anti-fluorescein antibody (purple). ( d – f , N) miRNA in situ HCR for miR-182 or miR-183, ( g ) Probe with 3 mismatches to both miR-182 and miR-183, ( h ) Probe containing only two initiator sequences, or ( i ) No probe added. ( j – m , o ) Combined detection of miR-182 and miR-183 by in situ HCR with two different HCR amplifiers. ( n , o ) enlargements of regions indicated by dashed rectangles in ( e , k ). ( p , q ) miRNA in situ HCR for Let-7f or ( r – u ) miR-126-3p. (t , u ) Maximum intensity Z projection of miR-126-3p signal across Z-stack (same retina section), showing labeling of retinal blood vessels and choroid. ( b–q ) adult, ( r – u ) P7 retinas. Magenta: Cy3, green: Cy 5, blue: DNA in nuclei. Scale bars: 25 µm ( d – m ); 10 µm ( n , o ).

    Journal: Scientific Reports

    Article Title: Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction

    doi: 10.1038/s41598-019-57194-0

    Figure Lengend Snippet: miRNA detection by in situ HCR in tissue sections from mouse retinas. ( a ) Schematic of HCR probes for miRNA detection (also see Supplementary Fig. S1 ); initiator sequences are not shown for the mismatch or miR-183 probes. ( b , c ) Non-HCR miRNA in situ hybridization for miR-182 or miR-183, using 20 nt fluorescein-labeled RNA probes detected with alkaline phosphatase (AP) conjugated anti-fluorescein antibody (purple). ( d – f , N) miRNA in situ HCR for miR-182 or miR-183, ( g ) Probe with 3 mismatches to both miR-182 and miR-183, ( h ) Probe containing only two initiator sequences, or ( i ) No probe added. ( j – m , o ) Combined detection of miR-182 and miR-183 by in situ HCR with two different HCR amplifiers. ( n , o ) enlargements of regions indicated by dashed rectangles in ( e , k ). ( p , q ) miRNA in situ HCR for Let-7f or ( r – u ) miR-126-3p. (t , u ) Maximum intensity Z projection of miR-126-3p signal across Z-stack (same retina section), showing labeling of retinal blood vessels and choroid. ( b–q ) adult, ( r – u ) P7 retinas. Magenta: Cy3, green: Cy 5, blue: DNA in nuclei. Scale bars: 25 µm ( d – m ); 10 µm ( n , o ).

    Article Snippet: HCR in situ probes and hairpin amplifiers DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT); sequences are in Supplementary Tables – .

    Techniques: In Situ, In Situ Hybridization, Labeling

    Detection of miR-182 by in situ HCR is abolished in miR-183/96/182 mutant mice. Retina sections from adult wild-type ( a – f ) or mice with disrupted miR-183/96/182 host gene ( g – l ) hybridized with probes for miR-182 and miR-124. ( e , j , k , l) enlargements of regions indicated in b, c, h, i. ( a – d ) Arrowheads indicate overlapping localized miR-124 and miR-182 expression within the IPL; miR-124 expression is detected in the IPL for retinas with the disrupted miR-183/96/182 host gene, but colocalized miR-182 is absent ( g – j ). Magenta: Cy3, green: Cy 5, blue: DNA. Scale bars: 25 µm ( a-d , g – j ); 10 µm ( e , f , k , l ).

    Journal: Scientific Reports

    Article Title: Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction

    doi: 10.1038/s41598-019-57194-0

    Figure Lengend Snippet: Detection of miR-182 by in situ HCR is abolished in miR-183/96/182 mutant mice. Retina sections from adult wild-type ( a – f ) or mice with disrupted miR-183/96/182 host gene ( g – l ) hybridized with probes for miR-182 and miR-124. ( e , j , k , l) enlargements of regions indicated in b, c, h, i. ( a – d ) Arrowheads indicate overlapping localized miR-124 and miR-182 expression within the IPL; miR-124 expression is detected in the IPL for retinas with the disrupted miR-183/96/182 host gene, but colocalized miR-182 is absent ( g – j ). Magenta: Cy3, green: Cy 5, blue: DNA. Scale bars: 25 µm ( a-d , g – j ); 10 µm ( e , f , k , l ).

    Article Snippet: HCR in situ probes and hairpin amplifiers DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT); sequences are in Supplementary Tables – .

    Techniques: In Situ, Mutagenesis, Mouse Assay, Expressing