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Thermo Fisher pcdna to
Pcdna To, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcdna to/product/Thermo Fisher
Average 85 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcdna to - by Bioz Stars, 2020-09
85/100 stars

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Plasmid Preparation:

Article Title: mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity
Article Snippet: .. The enhanced yellow fluorescent protein (eYFP) reporter-based vector pNAS-055 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs. pNAS-055 was generated by inserting the BamHI/NotI fragment derived from the peYFP-N1 vector (Clontech) into the BamHI/NotI site of pcDNA/TO (Invitrogen) bearing the tet-operator. .. The enhanced cyan fluorescent protein (eCFP) and eYFP dual reporter-based vector pNAS-092 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs.

Clone Assay:

Article Title: mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity
Article Snippet: .. The enhanced yellow fluorescent protein (eYFP) reporter-based vector pNAS-055 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs. pNAS-055 was generated by inserting the BamHI/NotI fragment derived from the peYFP-N1 vector (Clontech) into the BamHI/NotI site of pcDNA/TO (Invitrogen) bearing the tet-operator. .. The enhanced cyan fluorescent protein (eCFP) and eYFP dual reporter-based vector pNAS-092 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs.

Generated:

Article Title: mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity
Article Snippet: .. The enhanced yellow fluorescent protein (eYFP) reporter-based vector pNAS-055 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs. pNAS-055 was generated by inserting the BamHI/NotI fragment derived from the peYFP-N1 vector (Clontech) into the BamHI/NotI site of pcDNA/TO (Invitrogen) bearing the tet-operator. .. The enhanced cyan fluorescent protein (eCFP) and eYFP dual reporter-based vector pNAS-092 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs.

Construct:

Article Title: mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity
Article Snippet: .. The enhanced yellow fluorescent protein (eYFP) reporter-based vector pNAS-055 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs. pNAS-055 was generated by inserting the BamHI/NotI fragment derived from the peYFP-N1 vector (Clontech) into the BamHI/NotI site of pcDNA/TO (Invitrogen) bearing the tet-operator. .. The enhanced cyan fluorescent protein (eCFP) and eYFP dual reporter-based vector pNAS-092 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs.

Derivative Assay:

Article Title: mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity
Article Snippet: .. The enhanced yellow fluorescent protein (eYFP) reporter-based vector pNAS-055 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs. pNAS-055 was generated by inserting the BamHI/NotI fragment derived from the peYFP-N1 vector (Clontech) into the BamHI/NotI site of pcDNA/TO (Invitrogen) bearing the tet-operator. .. The enhanced cyan fluorescent protein (eCFP) and eYFP dual reporter-based vector pNAS-092 (Fig. and Table ) was constructed to contain a multiple cloning site after the stop codon of the eYFP for inserting the cDNAs.

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  • 93
    Thermo Fisher nf1 nanoluc pcdna expression plasmid
    AMG-510 prevents KRAS-G12C from interacting with <t>NF1</t> and CRAF. ( A ) SW48 isogenic cell lines, SW837 and SW1463 were treated with increasing doses of AMG-510 for 48 hours and cell viability was measured with the MTT assay. Data points represent the mean of eight biological replicates. Results are representative of three separate experiments. ( B ) HEK293T cells were co-transfected with KRAS-GFP and <t>NF1-NanoLuc</t> with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( C ) HEK293T cells were co-transfected with KRAS-GFP and CRAF-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( D ). HEK293T cells were transfected with either KRAS-G12C-GFP and NF1, NF1 alone, or mock transfection. Cells were then treated with either vehicle or 500 nM AMG-510 for 24 hours. Results are representative of an individual experiment from three separate experiments. ( E ) Mean KRAS G12C pull-down for the three independent NF1-coIP experiments. Statistical analysis was performed with unpaired t-test and P-values are indicated. Error bars in all panels represent standard deviation.
    Nf1 Nanoluc Pcdna Expression Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nf1 nanoluc pcdna expression plasmid/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nf1 nanoluc pcdna expression plasmid - by Bioz Stars, 2020-09
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    85
    Thermo Fisher pcdna myc vector
    Expression of <t>YES1</t> attenuated the inhibition of cell growth by miR-133 in TNBC cells. A, Cells were transfected with the overexpression plasmid of <t>pcDNA-3Myc-YES1,</t> and the ectopic expression of YES1 was confirmed by Western blot with anti-Myc antibody. B and C, Inhibition of miR-133 in the proliferation of TNBC cells was reversed with YES1 overexpression. D, Restoration of YES1 attenuated the suppressive role of miR-133 in the colony formation of TNBC cells. ns indicates no significance; TNBC, triple-negative breast cancer.
    Pcdna Myc Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna myc vector/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna myc vector - by Bioz Stars, 2020-09
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    84
    Thermo Fisher pcdna trub1
    <t>TruB1</t> selectively promotes the expression of let-7 family miRNAs (A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. MiRNAs significantly suppressed are enclosed by the red square. (B). Northern blotting for let-7a and RNU6B in HEK293FT cells with TruB1 KD or ctrl (siRNA). (C). Hybridization intensities of (B) were quantified and normalized to ctrl. (D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. (E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. All experiments were performed in triplicate. Error bars show SD; n=3.
    Pcdna Trub1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher pcdna kcnq1ot1
    Effect of <t>KCNQ1OT1</t> on β-catenin in mMSCs differentiation. a The mRNA and protein expression levels of β-catenin in mMSCs transfected with <t>pcDNA-KCNQ1OT1,</t> sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. b ALP activity in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. c The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 and siRNA-β-catenin before the addition of PMMA particles. d Osteoblastic differentiation in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. e The protein and mRNA levels of β-catenin in KCNQ1OT1 pulled down pellet using IgG antibody as negative control by RNA pull-down assay. *P
    Pcdna Kcnq1ot1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    AMG-510 prevents KRAS-G12C from interacting with NF1 and CRAF. ( A ) SW48 isogenic cell lines, SW837 and SW1463 were treated with increasing doses of AMG-510 for 48 hours and cell viability was measured with the MTT assay. Data points represent the mean of eight biological replicates. Results are representative of three separate experiments. ( B ) HEK293T cells were co-transfected with KRAS-GFP and NF1-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( C ) HEK293T cells were co-transfected with KRAS-GFP and CRAF-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( D ). HEK293T cells were transfected with either KRAS-G12C-GFP and NF1, NF1 alone, or mock transfection. Cells were then treated with either vehicle or 500 nM AMG-510 for 24 hours. Results are representative of an individual experiment from three separate experiments. ( E ) Mean KRAS G12C pull-down for the three independent NF1-coIP experiments. Statistical analysis was performed with unpaired t-test and P-values are indicated. Error bars in all panels represent standard deviation.

    Journal: bioRxiv

    Article Title: Inhibition of both mutant and wild-type RAS-GTP in KRAS G12C colorectal cancer through cotreatment with G12C and EGFR inhibitors

    doi: 10.1101/845263

    Figure Lengend Snippet: AMG-510 prevents KRAS-G12C from interacting with NF1 and CRAF. ( A ) SW48 isogenic cell lines, SW837 and SW1463 were treated with increasing doses of AMG-510 for 48 hours and cell viability was measured with the MTT assay. Data points represent the mean of eight biological replicates. Results are representative of three separate experiments. ( B ) HEK293T cells were co-transfected with KRAS-GFP and NF1-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( C ) HEK293T cells were co-transfected with KRAS-GFP and CRAF-NanoLuc with and without 500nM AMG-510 for 24 hours and signal is represented as a BRET ratio for both sample groups. Bars represent the mean of eight biological replicates. Results are representative of an individual experiment from three separate experiments. ( D ). HEK293T cells were transfected with either KRAS-G12C-GFP and NF1, NF1 alone, or mock transfection. Cells were then treated with either vehicle or 500 nM AMG-510 for 24 hours. Results are representative of an individual experiment from three separate experiments. ( E ) Mean KRAS G12C pull-down for the three independent NF1-coIP experiments. Statistical analysis was performed with unpaired t-test and P-values are indicated. Error bars in all panels represent standard deviation.

    Article Snippet: Twenty-four hours after seeding, cells were co-transfected with either a constant concentration of 0.1 μg of NF1-NanoLuc pcDNA expression plasmid or CRAF RBD-NanoLuc pcDNA expression plasmid with increasing concentrations of GFP-tagged KRAS (WT or Mutant) with 0.25 μl of Lipofectamine 2000 per well following the manufacturer’s protocol (Thermo Fisher Scientific).

    Techniques: MTT Assay, Transfection, Bioluminescence Resonance Energy Transfer, Co-Immunoprecipitation Assay, Standard Deviation

    KRAS-G12C binds less well to NF1 and to CRAF. ( A ) HEK293T cells were transfected with increasing amount of KRAS-GFP expression plasmid and a constant concentration of NF1-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with eight biological replicates, and were repeated three times. ( B ) Average BRET ratio from the three experiments at a RAS:NF1 ratio of 4:1. ( C ) HEK293T cells were transfected with either WT-KRAS-GFP, G12V-KRAS-GFP, G12C-KRAS GFP or NF1-FLAG expression plasmids. The NF1 co-IP mixing assay was performed and IP product and input lysate were probed by western blot for NF1, KRAS, and GFP. Results are representative of three independent experiments. ( D ) Average intensity of NF1 co-IP from the three independent experiments. ( E ) Isoelectric focused whole cell lysates from SW48 G12C, SW48 G12V and SW48 WT isogenic cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( F ) Average proportions of KRAS, NRAS, and HRAS bound to RBD and in whole cell lysates from three separate IEF experiments. ( G ) Isoelectric focused whole cell lysates from heterozygous mutant KRAS G12C SW837 and homozygous mutant KRAS G12C SW1463 cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( H ) Average proportions of KRAS, NRAS, and HRAS bound to and in whole cell lysates from three separate IEF experiments. ( I ) HEK293T cells were transfected with increasing concentrations of KRAS-GFP expression plasmids and a constant concentration of CRAF-RBD-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with three experimental replicates, each containing eight biological replicates. ( J ) Average BRET ratio from the three experiments at a RAS:RBD ratio of 2:1. ( K ) SW48 Isogenic cells were harvested and prepared for RAF-RBD pulldown assay, one set was not exposed to γ-S-GTP and the other was incubated in γ-S-GTP for 20 minutes. IP-product and input were probed by western blot for KRAS and GAPDH. Results are representative of three independent experiments. ( L ) Average quantity of KRAS WT, KRAS G12C, and KRAS G12C pulled down by RAF-RBD from three independent experiments. All experiments were performed three times. Treatment groups were analyzed by one-way ANOVA followed by post-hoc Tukey’s test for multiple comparisons and P-values are indicated. Error bars in all panels represent standard deviation.

    Journal: bioRxiv

    Article Title: Inhibition of both mutant and wild-type RAS-GTP in KRAS G12C colorectal cancer through cotreatment with G12C and EGFR inhibitors

    doi: 10.1101/845263

    Figure Lengend Snippet: KRAS-G12C binds less well to NF1 and to CRAF. ( A ) HEK293T cells were transfected with increasing amount of KRAS-GFP expression plasmid and a constant concentration of NF1-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with eight biological replicates, and were repeated three times. ( B ) Average BRET ratio from the three experiments at a RAS:NF1 ratio of 4:1. ( C ) HEK293T cells were transfected with either WT-KRAS-GFP, G12V-KRAS-GFP, G12C-KRAS GFP or NF1-FLAG expression plasmids. The NF1 co-IP mixing assay was performed and IP product and input lysate were probed by western blot for NF1, KRAS, and GFP. Results are representative of three independent experiments. ( D ) Average intensity of NF1 co-IP from the three independent experiments. ( E ) Isoelectric focused whole cell lysates from SW48 G12C, SW48 G12V and SW48 WT isogenic cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( F ) Average proportions of KRAS, NRAS, and HRAS bound to RBD and in whole cell lysates from three separate IEF experiments. ( G ) Isoelectric focused whole cell lysates from heterozygous mutant KRAS G12C SW837 and homozygous mutant KRAS G12C SW1463 cells, probed with a pan-RAS antibody. Results are representative of three independent experiments. ( H ) Average proportions of KRAS, NRAS, and HRAS bound to and in whole cell lysates from three separate IEF experiments. ( I ) HEK293T cells were transfected with increasing concentrations of KRAS-GFP expression plasmids and a constant concentration of CRAF-RBD-NanoLuc. 24 hours post-transfection cells were treated with Nano-Glo Live Cell Reagent. BRET ratio was plotted as a function of RAS/NF1 expression plasmid ratio. BRET assays were performed with three experimental replicates, each containing eight biological replicates. ( J ) Average BRET ratio from the three experiments at a RAS:RBD ratio of 2:1. ( K ) SW48 Isogenic cells were harvested and prepared for RAF-RBD pulldown assay, one set was not exposed to γ-S-GTP and the other was incubated in γ-S-GTP for 20 minutes. IP-product and input were probed by western blot for KRAS and GAPDH. Results are representative of three independent experiments. ( L ) Average quantity of KRAS WT, KRAS G12C, and KRAS G12C pulled down by RAF-RBD from three independent experiments. All experiments were performed three times. Treatment groups were analyzed by one-way ANOVA followed by post-hoc Tukey’s test for multiple comparisons and P-values are indicated. Error bars in all panels represent standard deviation.

    Article Snippet: Twenty-four hours after seeding, cells were co-transfected with either a constant concentration of 0.1 μg of NF1-NanoLuc pcDNA expression plasmid or CRAF RBD-NanoLuc pcDNA expression plasmid with increasing concentrations of GFP-tagged KRAS (WT or Mutant) with 0.25 μl of Lipofectamine 2000 per well following the manufacturer’s protocol (Thermo Fisher Scientific).

    Techniques: Transfection, Expressing, Plasmid Preparation, Concentration Assay, Bioluminescence Resonance Energy Transfer, Co-Immunoprecipitation Assay, Western Blot, Electrofocusing, Mutagenesis, Incubation, Standard Deviation

    Expression of YES1 attenuated the inhibition of cell growth by miR-133 in TNBC cells. A, Cells were transfected with the overexpression plasmid of pcDNA-3Myc-YES1, and the ectopic expression of YES1 was confirmed by Western blot with anti-Myc antibody. B and C, Inhibition of miR-133 in the proliferation of TNBC cells was reversed with YES1 overexpression. D, Restoration of YES1 attenuated the suppressive role of miR-133 in the colony formation of TNBC cells. ns indicates no significance; TNBC, triple-negative breast cancer.

    Journal: Technology in Cancer Research & Treatment

    Article Title: MiR-133 Targets YES1 and Inhibits the Growth of Triple-Negative Breast Cancer Cells

    doi: 10.1177/1533033820927011

    Figure Lengend Snippet: Expression of YES1 attenuated the inhibition of cell growth by miR-133 in TNBC cells. A, Cells were transfected with the overexpression plasmid of pcDNA-3Myc-YES1, and the ectopic expression of YES1 was confirmed by Western blot with anti-Myc antibody. B and C, Inhibition of miR-133 in the proliferation of TNBC cells was reversed with YES1 overexpression. D, Restoration of YES1 attenuated the suppressive role of miR-133 in the colony formation of TNBC cells. ns indicates no significance; TNBC, triple-negative breast cancer.

    Article Snippet: The expression plasmid of pcDNA-Myc-YES1 was constructed by amplifying the complementary DNA (cDNA) fragment of YES1 via polymerase chain reaction (PCR) and inserted into the backbone of pcDNA-Myc vector.

    Techniques: Expressing, Inhibition, Transfection, Over Expression, Plasmid Preparation, Western Blot

    TruB1 selectively promotes the expression of let-7 family miRNAs (A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. MiRNAs significantly suppressed are enclosed by the red square. (B). Northern blotting for let-7a and RNU6B in HEK293FT cells with TruB1 KD or ctrl (siRNA). (C). Hybridization intensities of (B) were quantified and normalized to ctrl. (D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. (E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. All experiments were performed in triplicate. Error bars show SD; n=3.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 selectively promotes the expression of let-7 family miRNAs (A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. MiRNAs significantly suppressed are enclosed by the red square. (B). Northern blotting for let-7a and RNU6B in HEK293FT cells with TruB1 KD or ctrl (siRNA). (C). Hybridization intensities of (B) were quantified and normalized to ctrl. (D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. (E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. All experiments were performed in triplicate. Error bars show SD; n=3.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Expressing, Northern Blot, Hybridization, Quantitative RT-PCR

    TruB1 promotes the microprocessing of primary let-7 and enhances binding of the microprocesser to primary let-7 (A). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). (A). RI intensities of (B) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. (C). RIP assay of pri-let-7a1 and DGCR-8 from HEK-293FT cells. Western blotting for input or immunoprecipitate (IP) using anti-DGCR-8 antibody and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). (D). RIP assay of pri-let-7a1 and TruB1 from HEK-293FT cells with Lin28B KD or ctrl (siRNA). Western blotting for input or IP material using anti-Flag antibody, anti-Lin28B antibody, and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). All experiments were performed in triplicate. Error bars show SD; n=3.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 promotes the microprocessing of primary let-7 and enhances binding of the microprocesser to primary let-7 (A). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). (A). RI intensities of (B) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. (C). RIP assay of pri-let-7a1 and DGCR-8 from HEK-293FT cells. Western blotting for input or immunoprecipitate (IP) using anti-DGCR-8 antibody and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). (D). RIP assay of pri-let-7a1 and TruB1 from HEK-293FT cells with Lin28B KD or ctrl (siRNA). Western blotting for input or IP material using anti-Flag antibody, anti-Lin28B antibody, and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). All experiments were performed in triplicate. Error bars show SD; n=3.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Binding Assay, In Vitro, Transfection, Over Expression, Western Blot, Quantitative RT-PCR

    TruB1 promotes let-7 processing independently of its enzymatic activity. (A). Amino acid sequences encoding for the enzyme activity and RNA binding ability in E.coli TruB and human TruB1 (Top). Design of mutant 1 (mt1) and mutant 2 (mt2) (bottom). (B). In vitro enzyme activity assay. 32 p-UTP-labelled tRNA phe were treated with recombinant TruB1, mt1, or mt2. The strong upper bands represent UTP, and the lower weaker bands represent pseudouridine (Ψ) on autoradiographs of the TLC plate. (C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. (D). Northern blotting for let-7a and RNU6B in HEK-293 cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment. (E). Hybridization intensities of (D) were quantified and normalized to ctrl (GFP). (F). Pseudouridylation activity of TruB1 for tRNA and pri-miRNAs. 32 p-UTP-labelled tRNA phe , pri-let-7a1 or pri-miR10a were treated with recombinant TruB1. Upper bands represent UTP, lower bands represent pseudouridine (Ψ) in autoradiographs of the TLC plate. (G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNA phe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows. All experiments were performed in triplicate. Error bars show SD; n=3.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 promotes let-7 processing independently of its enzymatic activity. (A). Amino acid sequences encoding for the enzyme activity and RNA binding ability in E.coli TruB and human TruB1 (Top). Design of mutant 1 (mt1) and mutant 2 (mt2) (bottom). (B). In vitro enzyme activity assay. 32 p-UTP-labelled tRNA phe were treated with recombinant TruB1, mt1, or mt2. The strong upper bands represent UTP, and the lower weaker bands represent pseudouridine (Ψ) on autoradiographs of the TLC plate. (C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. (D). Northern blotting for let-7a and RNU6B in HEK-293 cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment. (E). Hybridization intensities of (D) were quantified and normalized to ctrl (GFP). (F). Pseudouridylation activity of TruB1 for tRNA and pri-miRNAs. 32 p-UTP-labelled tRNA phe , pri-let-7a1 or pri-miR10a were treated with recombinant TruB1. Upper bands represent UTP, lower bands represent pseudouridine (Ψ) in autoradiographs of the TLC plate. (G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNA phe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows. All experiments were performed in triplicate. Error bars show SD; n=3.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Activity Assay, RNA Binding Assay, Mutagenesis, In Vitro, Enzyme Activity Assay, Recombinant, Thin Layer Chromatography, Expressing, Infection, Quantitative RT-PCR, Northern Blot, Hybridization, Purification, Sequencing

    TruB1 binds to tRNA and primary-let-7 (A). RIP analysis of pri-let-7a1 and Flag-TruB1 from HEK-293FT cells. RNA was extracted from IP material and analyzed by qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. (B). EMSA of 32 p-ATP-labelled pri-let-7a1 or pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes. (C). Design of Flag-labelled TruB1 knock-in (KI) cells. HiBIT and 3 x Flag sequences were inserted into the N-terminus of the TruB1 gene in HEK-293FT cells. (D) Sequencing clusters obtained from HITS-CLIP experiment. Left are for let-7, and right for tRNAs. (E). Tag densities represented as dots. Density of miRNA clusters in HITS-CLIP were normalized by miRNA expression as determined by TaqMan array. (F). The proportions of different types of mapped transcripts from the HITS-CLIP experiment. (G). Sequence motifs from reads mapped to tRNAs from the HITS-CLIP experiment. (H). TruB-modified site in tRNAs. Location of pseudouridine is colored in red. (I). Sequence of the terminal loop in pri-let-7a1. Lin28B binding site is colored in red.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 binds to tRNA and primary-let-7 (A). RIP analysis of pri-let-7a1 and Flag-TruB1 from HEK-293FT cells. RNA was extracted from IP material and analyzed by qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. (B). EMSA of 32 p-ATP-labelled pri-let-7a1 or pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes. (C). Design of Flag-labelled TruB1 knock-in (KI) cells. HiBIT and 3 x Flag sequences were inserted into the N-terminus of the TruB1 gene in HEK-293FT cells. (D) Sequencing clusters obtained from HITS-CLIP experiment. Left are for let-7, and right for tRNAs. (E). Tag densities represented as dots. Density of miRNA clusters in HITS-CLIP were normalized by miRNA expression as determined by TaqMan array. (F). The proportions of different types of mapped transcripts from the HITS-CLIP experiment. (G). Sequence motifs from reads mapped to tRNAs from the HITS-CLIP experiment. (H). TruB-modified site in tRNAs. Location of pseudouridine is colored in red. (I). Sequence of the terminal loop in pri-let-7a1. Lin28B binding site is colored in red.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Quantitative RT-PCR, Infection, Expressing, Recombinant, Knock-In, Sequencing, Cross-linking Immunoprecipitation, Modification, Binding Assay

    TruB1 suppresses cell growth by regulating let-7 (A). Design of luciferase reporter vector comprising the let-7 target region of KRAS mRNA (KRAS reporter). (B). Relative luciferase activity of KRAS reporter or Ctrl (empty) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment. (C). Relative expression of let-7 families determined by qRT-PCR in HEK-293FT cells with KD of let-7 family members or ctrl (using 2’-O-methylated antisense inhibitor). (D). Real-time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1 or GFP, 5 days after doxycycline treatment, with or without KD of let-7 family members. (E). Schematic model for TruB1-dependent induction of let-7. All experiments were performed in triplicate. Error bars show SD; n=3.

    Journal: bioRxiv

    Article Title: The tRNA pseudouridine synthase TruB1 regulates the maturation and function of let-7 miRNA

    doi: 10.1101/2020.02.16.951954

    Figure Lengend Snippet: TruB1 suppresses cell growth by regulating let-7 (A). Design of luciferase reporter vector comprising the let-7 target region of KRAS mRNA (KRAS reporter). (B). Relative luciferase activity of KRAS reporter or Ctrl (empty) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment. (C). Relative expression of let-7 families determined by qRT-PCR in HEK-293FT cells with KD of let-7 family members or ctrl (using 2’-O-methylated antisense inhibitor). (D). Real-time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1 or GFP, 5 days after doxycycline treatment, with or without KD of let-7 family members. (E). Schematic model for TruB1-dependent induction of let-7. All experiments were performed in triplicate. Error bars show SD; n=3.

    Article Snippet: The sequence encoding human TruB1 ORF was cloned by PCR from 293FT cell cDNA. pcDNA-TruB1 was generated by inserting the ORF of human TruB1 with a Flag sequence into pcDNA.3.1(+) (Thermo Fisher Scientific) at the Nhe1 and EcoR1 sites.

    Techniques: Luciferase, Plasmid Preparation, Activity Assay, Infection, Expressing, Quantitative RT-PCR, Methylation, Glo Assay

    Effect of KCNQ1OT1 on β-catenin in mMSCs differentiation. a The mRNA and protein expression levels of β-catenin in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. b ALP activity in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. c The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 and siRNA-β-catenin before the addition of PMMA particles. d Osteoblastic differentiation in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. e The protein and mRNA levels of β-catenin in KCNQ1OT1 pulled down pellet using IgG antibody as negative control by RNA pull-down assay. *P

    Journal: Cell & Bioscience

    Article Title: LncRNA KCNQ1OT1 promotes osteogenic differentiation to relieve osteolysis via Wnt/β-catenin activation

    doi: 10.1186/s13578-018-0216-4

    Figure Lengend Snippet: Effect of KCNQ1OT1 on β-catenin in mMSCs differentiation. a The mRNA and protein expression levels of β-catenin in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. b ALP activity in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. c The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 and siRNA-β-catenin before the addition of PMMA particles. d Osteoblastic differentiation in mMSCs transfected with pcDNA-KCNQ1OT1, sh-NC or pcDNA-KCNQ1OT1 along with siRNA-β-catenin before the addition of PMMA particles. e The protein and mRNA levels of β-catenin in KCNQ1OT1 pulled down pellet using IgG antibody as negative control by RNA pull-down assay. *P

    Article Snippet: Briefly, mMSCs were seeded in antibiotics-free medium in six-well plates at a density of 5 × 105 /mL followed by transfection with pcDNA-KCNQ1OT1 or co-transfection with pcDNA-KCNQ1OT1 and siRNA-β-catenin (Thermo Fisher Scientific, Waltham, MA, USA) along with corresponding negative control (pcDNA) using Lipofectamine 2000 (Invitrogen) and then incubated at 37 °C with 5% CO2 for 24 h. MMSCs were used to detect the protein levels of β-catenin using western blot, ALP activity and the mRNA expression of Runx2, Osterix and OCN using qRT-PCR as well as mMSCs differentiation using ARS staining.

    Techniques: Expressing, Transfection, ALP Assay, Activity Assay, Negative Control, Pull Down Assay

    Effect of KCNQ1OT1 in mMSCs stimulated with PMMA particles. a ALP activity in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles. b The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles. c Osteoblastic differentiation in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles assessed by ARS staining. d The relative expression levels of KCNQ1OT1 in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA. *P

    Journal: Cell & Bioscience

    Article Title: LncRNA KCNQ1OT1 promotes osteogenic differentiation to relieve osteolysis via Wnt/β-catenin activation

    doi: 10.1186/s13578-018-0216-4

    Figure Lengend Snippet: Effect of KCNQ1OT1 in mMSCs stimulated with PMMA particles. a ALP activity in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles. b The mRNA expression levels of Runx2, Osterix and OCN in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles. c Osteoblastic differentiation in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA before the addition of PMMA particles assessed by ARS staining. d The relative expression levels of KCNQ1OT1 in mMSCs transfected with pcDNA KCNQ1OT1 or pcDNA. *P

    Article Snippet: Briefly, mMSCs were seeded in antibiotics-free medium in six-well plates at a density of 5 × 105 /mL followed by transfection with pcDNA-KCNQ1OT1 or co-transfection with pcDNA-KCNQ1OT1 and siRNA-β-catenin (Thermo Fisher Scientific, Waltham, MA, USA) along with corresponding negative control (pcDNA) using Lipofectamine 2000 (Invitrogen) and then incubated at 37 °C with 5% CO2 for 24 h. MMSCs were used to detect the protein levels of β-catenin using western blot, ALP activity and the mRNA expression of Runx2, Osterix and OCN using qRT-PCR as well as mMSCs differentiation using ARS staining.

    Techniques: ALP Assay, Activity Assay, Transfection, Expressing, Staining