plasmid construction human wt Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    ATCC 293t cells
    Cripto-1 and LRP6 co-localize on the cell membrane. <t>293T/Cripto-1</t> cells transiently transfected with LRP6-GFP expression vector were stained with anti-LRP6 (green) and anti-Cripto-1 (red) antibodies. Nuclei were visualized by Hoechst 33258 (blue). The merged image shows significant co-localization of Cripto-1 and LRP6 on the cell membrane. The arrows indicate clear examples of co-localization.
    293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 11984 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/293t cells/product/ATCC
    Average 99 stars, based on 11984 article reviews
    Price from $9.99 to $1999.99
    293t cells - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher lipofectamine 2000
    p300 siRNA suppressed UV-induced MMP-1 expression and inhibited UV-enhanced levels of γ-H2AX, p53 and acetyl-H3, and AA inhibited interaction of p300 with γ-H2AX or acetyl-H3 after UV. HDFs were transfected with scrambled control siRNA and p300 siRNA at 100 nM using <t>Lipofectamine</t> as recommended by the manufacturer. A, HDFs were irradiated with UV and incubated at 37°C 24 h after transfection with scrambled control or p300 siRNA. The MMP-1 mRNA level was analyzed by RT-PCR and the amount of MMP-1 protein was analyzed by western blot. B, The effect of p300 siRNA on the protein levels of γ-H2AX, p53, and acetyl-H3 were analyzed by western blotting. C, HDFs were UV-irradiated. After 6 h, whole cell lysates were immunoprecipitated with anti-p300 antibody. D, HDFs were UV-irradiated and post-treated with AA for 6 h. Whole cell lysates were immunoprecipitated with anti-p300 antibody. Proteins were immunoblotted with anti-p300, anti-acetyl-H3, and anti-γ-H2AX antibodies ( upper panels ). Bar graphs ( lower panels ) show quantitative analysis of scanning densitometric values of γ-H2AX, p53, and acetyl-H3 as ratios to β-actin, which was used as a loading control. Data shown are representative of three independent experiments. C: control, UV: UV-irradiated cells, AA: AA treated cells, UV/AA: UV irradiated cells incubated with AA. *P
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 478206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine 2000/product/Thermo Fisher
    Average 99 stars, based on 478206 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 2000 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    ATCC hek293 cells
    SOX9 direct binding to the promoter of miR-130a to regulate its expression (A) si-SOX9 or SOX9 vector was transfected into HeLa and CaSki cells to achieve SOX9 expression, as verified using Western blot assays. (B) miR-130a expression in the indicated cells was determined using real-time PCR assays. (C) A schematic diagram of potential SOX9 binding element (two possible binding sites) in the promoter region of miR-130a predicted by Jaspar database. A wt-miR-130a promoter luciferase reporter vector and a mut-miR-130a promoter luciferase reporter vector were constructed. (D) The indicated vectors were co-transfected into <t>HEK293</t> cells with SOX9 vector; the luciferase activity was determined. (E) The real-time ChIP assay showed that the level of SOX9 antibody binding to miR-130a promoter was much greater than that of IgG. The data are presented as mean ± SD of three independent experiments. * P
    Hek293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 14474 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hek293 cells/product/ATCC
    Average 99 stars, based on 14474 article reviews
    Price from $9.99 to $1999.99
    hek293 cells - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    95
    Stratagene quikchange site directed mutagenesis kit
    SOX9 direct binding to the promoter of miR-130a to regulate its expression (A) si-SOX9 or SOX9 vector was transfected into HeLa and CaSki cells to achieve SOX9 expression, as verified using Western blot assays. (B) miR-130a expression in the indicated cells was determined using real-time PCR assays. (C) A schematic diagram of potential SOX9 binding element (two possible binding sites) in the promoter region of miR-130a predicted by Jaspar database. A wt-miR-130a promoter luciferase reporter vector and a mut-miR-130a promoter luciferase reporter vector were constructed. (D) The indicated vectors were co-transfected into <t>HEK293</t> cells with SOX9 vector; the luciferase activity was determined. (E) The real-time ChIP assay showed that the level of SOX9 antibody binding to miR-130a promoter was much greater than that of IgG. The data are presented as mean ± SD of three independent experiments. * P
    Quikchange Site Directed Mutagenesis Kit, supplied by Stratagene, used in various techniques. Bioz Stars score: 95/100, based on 66977 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quikchange site directed mutagenesis kit/product/Stratagene
    Average 95 stars, based on 66977 article reviews
    Price from $9.99 to $1999.99
    quikchange site directed mutagenesis kit - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcdna3 1
    HSPA1L and GR protein levels in decidualized human endometrial stromal fibroblasts. Cultured ESFs were transfected with WT or Ala268Thr HSPA1L <t>-pcDNA3.1</t> constructs or with empty pcDNA3.1 vector (control). Cells were treated with decidualization media supplemented with 100nM dexamethasone (glucocorticoids) for 72h. Both cytosolic and nuclear protein were extracted, and HSPA1L and GR protein levels were measured by Western blot. Band intensity of HSPA1L or GR was normalized to band intensity of the corresponding β-actin. Cytosolic (A) and nuclear (B) HSPA1L levels as well as cytosolic (C) and nuclear (D) GR levels are shown for control (empty vector), WT and Ala268Thr sample groups. Each experiment was performed as triplicates in three different passages (n = 9 each group, except n = 8 for nuclear control group) and bars represent mean + SEM. Significant p-value
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 49697 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1/product/Thermo Fisher
    Average 99 stars, based on 49697 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    95
    Thermo Fisher pcdna3
    NRF-1 regulates PCFT mRNA levels. A , HeLa cells were stably transfected with one of the following expression vectors: <t>pCDNA3,</t> NRF-1 DN, NRF-1 WT, or NRF-1 VP16 and a stable population was established by drug selection using G418 or hygromycin. PCFT and
    Pcdna3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 45292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3/product/Thermo Fisher
    Average 95 stars, based on 45292 article reviews
    Price from $9.99 to $1999.99
    pcdna3 - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    95
    Promega pgl3 basic vector
    FXR enhances the transcriptional activity of miR-122 promoter. a Potential FXREs in miR-122 promoter region were predicted using an online algorithm (NUBIScan: http://www.nubiscan.unibas.ch/ ). Transcription start sites (TSS, +1) are indicated by an arrow. b Huh7 cells were co-transfected with renilla luciferase expression vector pRL-TK and one of a series of luciferase reporter constructs containing different fragments of miR-122 promoter region. After 6 h incubation, the cells were treated with DMSO or 5 μM GW4064 for 24 h, and then dual luciferase assays were performed. Firefly luciferase activity was normalized to that of renilla luciferase. Data are shown as means ± SD from three assays performed in triplicate. c Huh7 cells were co-transfected with pRL-TK and either <t>pGL3-F4(DR2)-WT</t> (containing the wild-type DR2 element) or pGL3-F4(DR2)-Mut (containing the mutant DR2 element) for 6 h. Subsequent treatment and luciferase assays were as performed in ( b ). * P
    Pgl3 Basic Vector, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 27770 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic vector/product/Promega
    Average 95 stars, based on 27770 article reviews
    Price from $9.99 to $1999.99
    pgl3 basic vector - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    99
    Promega human genomic dna
    MiR-138-5p functionally targeted circ_002136 . ( a ) A schematic representation of how circ_002136 arose from the CDK11A gene as determined by scanning CDK11A genomic <t>DNA</t> and circBase. ( b ) The expression of miR-138-5p was measured after knockdown of circ_002136 by <t>qRT-PCR.</t> Values represent the means ± SD (n = 5, each group). ** P
    Human Genomic Dna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 7262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human genomic dna/product/Promega
    Average 99 stars, based on 7262 article reviews
    Price from $9.99 to $1999.99
    human genomic dna - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    96
    Stratagene site directed mutagenesis
    MiR-138-5p functionally targeted circ_002136 . ( a ) A schematic representation of how circ_002136 arose from the CDK11A gene as determined by scanning CDK11A genomic <t>DNA</t> and circBase. ( b ) The expression of miR-138-5p was measured after knockdown of circ_002136 by <t>qRT-PCR.</t> Values represent the means ± SD (n = 5, each group). ** P
    Site Directed Mutagenesis, supplied by Stratagene, used in various techniques. Bioz Stars score: 96/100, based on 47076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/site directed mutagenesis/product/Stratagene
    Average 96 stars, based on 47076 article reviews
    Price from $9.99 to $1999.99
    site directed mutagenesis - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    99
    Thermo Fisher hek 293 cells
    IRF4 mutations in CLL. (A). Schematic representation of the L116R mutation. (B) Chromatographs of L116R and L116P mutations. (C) Clinical characteristics of patients showing IRF4 mutations. (D) Significantly overexpressed genes in <t>HEK</t> 293 cells transfected
    Hek 293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12989 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hek 293 cells/product/Thermo Fisher
    Average 99 stars, based on 12989 article reviews
    Price from $9.99 to $1999.99
    hek 293 cells - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher lipofectamine 3000
    IRF4 mutations in CLL. (A). Schematic representation of the L116R mutation. (B) Chromatographs of L116R and L116P mutations. (C) Clinical characteristics of patients showing IRF4 mutations. (D) Significantly overexpressed genes in <t>HEK</t> 293 cells transfected
    Lipofectamine 3000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 42769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine 3000/product/Thermo Fisher
    Average 99 stars, based on 42769 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 3000 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    ATCC hela cells
    Cells depleted of both <t>hFis1</t> and Opa1 show apoptosis resistance like hFis1-depleted cells. (A) <t>HeLa</t> cells depleted of hFis1, Opa1, or both of them by RNAi along with control RNAi cells were treated with STS (1 μM; 6 h), Act D (10 μM; 8 h), etoposide (100 μM; 30 h), or anti-Fas antibody (500 ng/ml; 15 h), and the nuclei were stained with Hoechst 33342 (1 μg/ml; 15 min at RT). Normal or apoptotic nuclei of these cells in several fields were counted under the fluorescent microscope (for UV excitation). At least 200 cells altogether in each treatment were counted and plotted as a percentage of cells with apoptotic nuclei among the total cells counted. The data are shown as the mean ± SD of at least three independent experiments. (B) HeLa cells depleted of Opa1, hFis1, or both of them along with control RNAi cells were incubated with anti-Fas antibody (500 ng/ml) for the periods as indicated, and the nuclei were stained and counted as normal or apoptotic nuclei. At least 200 cells altogether were counted in each sample at each time point and plotted as a percentage of cells with apoptotic nuclei among the total cells counted. The data were plotted as the mean ± SD of at least three independent experiments. (C) Bax translocation and cytochrome c release induced by Act D treatment are inhibited in hFis/Opa1 RNAi cells. Left, HeLa cells depleted of both hFis1 and Opa1 by RNAi along with control RNAi cells were treated with Act D (10 μM; 8 h) in the presence of zVAD-fmk (50 μM), fixed, and double stained with anti-Bax (rabbit polyclonal, red) and anti-cytochrome c (mouse monoclonal, green) antibodies. Right, the number of cells displaying Bax translocation or cytochrome c release was counted and plotted as a percentage of the total cells counted in each RNAi cell population that had been treated with Act D and stained with anti-Bax and anti-cytochrome c antibodies (the same samples as shown on the left). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments. (D) hFis1 depletion prevents mitochondrial membrane potential reduction induced by Opa1 depletion. Left, HeLa cells depleted of hFis1 (b), Opa1 (c), or both of them (d) along with control RNAi cells (a) were incubated with 5 μg/ml JC-1 for 20 min and observed by confocal microscopy. JC-1 is a cationic dye that indicates mitochondrial polarization by shifting its fluorescence emission from green to red. Regions of high mitochondrial membrane potential are indicated by red fluorescence, and regions of low mitochondrial membrane potential are indicated by green fluorescence. Right, the number of cells with mitochondria whose membrane potential was lost was counted and plotted as a percentage of the total cells counted in each RNAi cell population.
    Hela Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 24553 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hela cells/product/ATCC
    Average 99 stars, based on 24553 article reviews
    Price from $9.99 to $1999.99
    hela cells - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Promega pgl3 vector
    miR-7 post-transcriptionally downregulates PA28gamma expression by directly targeting its 3′-UTR. ( A ) Putative target genes were predicted by PicTar, Target Scan, and miRanda. Each circle represents one prediction algorithm with the number of predicted genes. The number listed in overlapping areas of the circles was simultaneously predicted by different algorithms. ( B ) The potential binding sequences within the 3′-UTR of PA28gamma for miR-7 is conserved in human (Hsa), chimpanzee (Ptr), rhesus (Mml), mouse (Mmu), rat, hedgehog (Eeu), shrew (Sar), horse (Eca), cow (Eta), and elephant (Dno). Seed sequences are highlighted. ( C ) The PA28gamma 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in the <t>pGL3-con</t> vector. The sequences of the predicted miR-7 binding sites within the PA28gamma 3′-UTR including the wild-type UTR or mutant UTR segments (dotted lines) are shown. ( D ) After co-transfection of miR-7 mimic or miR-NC with the above reporter plasmids and pRL-TK vector in HEK 293T cells, relative luciferase activity was analysed by Dual-Luciferase Reporter Assay. ( E ) A549 and H1299 cells were transfected with miR-7, miR-NC, PA28gamma -siRNA, and miR-7 inhibitor for 48 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). ( F ) H1299 cells were transfected with PA28gamma -siRNA, and/or miR-7 inhibitor for 72 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). These experiments were performed in triplicate and results are shown as the mean±s.e.m. (*** P
    Pgl3 Vector, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 7864 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 vector/product/Promega
    Average 94 stars, based on 7864 article reviews
    Price from $9.99 to $1999.99
    pgl3 vector - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    90
    Addgene inc prk5 ha ubiquitin wt
    miR-7 post-transcriptionally downregulates PA28gamma expression by directly targeting its 3′-UTR. ( A ) Putative target genes were predicted by PicTar, Target Scan, and miRanda. Each circle represents one prediction algorithm with the number of predicted genes. The number listed in overlapping areas of the circles was simultaneously predicted by different algorithms. ( B ) The potential binding sequences within the 3′-UTR of PA28gamma for miR-7 is conserved in human (Hsa), chimpanzee (Ptr), rhesus (Mml), mouse (Mmu), rat, hedgehog (Eeu), shrew (Sar), horse (Eca), cow (Eta), and elephant (Dno). Seed sequences are highlighted. ( C ) The PA28gamma 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in the <t>pGL3-con</t> vector. The sequences of the predicted miR-7 binding sites within the PA28gamma 3′-UTR including the wild-type UTR or mutant UTR segments (dotted lines) are shown. ( D ) After co-transfection of miR-7 mimic or miR-NC with the above reporter plasmids and pRL-TK vector in HEK 293T cells, relative luciferase activity was analysed by Dual-Luciferase Reporter Assay. ( E ) A549 and H1299 cells were transfected with miR-7, miR-NC, PA28gamma -siRNA, and miR-7 inhibitor for 48 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). ( F ) H1299 cells were transfected with PA28gamma -siRNA, and/or miR-7 inhibitor for 72 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). These experiments were performed in triplicate and results are shown as the mean±s.e.m. (*** P
    Prk5 Ha Ubiquitin Wt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/prk5 ha ubiquitin wt/product/Addgene inc
    Average 90 stars, based on 275 article reviews
    Price from $9.99 to $1999.99
    prk5 ha ubiquitin wt - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    99
    Promega pmirglo dual luciferase mirna target expression vector
    miR-7 post-transcriptionally downregulates PA28gamma expression by directly targeting its 3′-UTR. ( A ) Putative target genes were predicted by PicTar, Target Scan, and miRanda. Each circle represents one prediction algorithm with the number of predicted genes. The number listed in overlapping areas of the circles was simultaneously predicted by different algorithms. ( B ) The potential binding sequences within the 3′-UTR of PA28gamma for miR-7 is conserved in human (Hsa), chimpanzee (Ptr), rhesus (Mml), mouse (Mmu), rat, hedgehog (Eeu), shrew (Sar), horse (Eca), cow (Eta), and elephant (Dno). Seed sequences are highlighted. ( C ) The PA28gamma 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in the <t>pGL3-con</t> vector. The sequences of the predicted miR-7 binding sites within the PA28gamma 3′-UTR including the wild-type UTR or mutant UTR segments (dotted lines) are shown. ( D ) After co-transfection of miR-7 mimic or miR-NC with the above reporter plasmids and pRL-TK vector in HEK 293T cells, relative luciferase activity was analysed by Dual-Luciferase Reporter Assay. ( E ) A549 and H1299 cells were transfected with miR-7, miR-NC, PA28gamma -siRNA, and miR-7 inhibitor for 48 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). ( F ) H1299 cells were transfected with PA28gamma -siRNA, and/or miR-7 inhibitor for 72 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). These experiments were performed in triplicate and results are shown as the mean±s.e.m. (*** P
    Pmirglo Dual Luciferase Mirna Target Expression Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 6909 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmirglo dual luciferase mirna target expression vector/product/Promega
    Average 99 stars, based on 6909 article reviews
    Price from $9.99 to $1999.99
    pmirglo dual luciferase mirna target expression vector - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    95
    Stratagene quickchange site directed mutagenesis kit
    miR-7 post-transcriptionally downregulates PA28gamma expression by directly targeting its 3′-UTR. ( A ) Putative target genes were predicted by PicTar, Target Scan, and miRanda. Each circle represents one prediction algorithm with the number of predicted genes. The number listed in overlapping areas of the circles was simultaneously predicted by different algorithms. ( B ) The potential binding sequences within the 3′-UTR of PA28gamma for miR-7 is conserved in human (Hsa), chimpanzee (Ptr), rhesus (Mml), mouse (Mmu), rat, hedgehog (Eeu), shrew (Sar), horse (Eca), cow (Eta), and elephant (Dno). Seed sequences are highlighted. ( C ) The PA28gamma 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in the <t>pGL3-con</t> vector. The sequences of the predicted miR-7 binding sites within the PA28gamma 3′-UTR including the wild-type UTR or mutant UTR segments (dotted lines) are shown. ( D ) After co-transfection of miR-7 mimic or miR-NC with the above reporter plasmids and pRL-TK vector in HEK 293T cells, relative luciferase activity was analysed by Dual-Luciferase Reporter Assay. ( E ) A549 and H1299 cells were transfected with miR-7, miR-NC, PA28gamma -siRNA, and miR-7 inhibitor for 48 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). ( F ) H1299 cells were transfected with PA28gamma -siRNA, and/or miR-7 inhibitor for 72 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). These experiments were performed in triplicate and results are shown as the mean±s.e.m. (*** P
    Quickchange Site Directed Mutagenesis Kit, supplied by Stratagene, used in various techniques. Bioz Stars score: 95/100, based on 26650 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quickchange site directed mutagenesis kit/product/Stratagene
    Average 95 stars, based on 26650 article reviews
    Price from $9.99 to $1999.99
    quickchange site directed mutagenesis kit - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    95
    Agilent technologies quikchange site directed mutagenesis kit
    miR-7 post-transcriptionally downregulates PA28gamma expression by directly targeting its 3′-UTR. ( A ) Putative target genes were predicted by PicTar, Target Scan, and miRanda. Each circle represents one prediction algorithm with the number of predicted genes. The number listed in overlapping areas of the circles was simultaneously predicted by different algorithms. ( B ) The potential binding sequences within the 3′-UTR of PA28gamma for miR-7 is conserved in human (Hsa), chimpanzee (Ptr), rhesus (Mml), mouse (Mmu), rat, hedgehog (Eeu), shrew (Sar), horse (Eca), cow (Eta), and elephant (Dno). Seed sequences are highlighted. ( C ) The PA28gamma 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in the <t>pGL3-con</t> vector. The sequences of the predicted miR-7 binding sites within the PA28gamma 3′-UTR including the wild-type UTR or mutant UTR segments (dotted lines) are shown. ( D ) After co-transfection of miR-7 mimic or miR-NC with the above reporter plasmids and pRL-TK vector in HEK 293T cells, relative luciferase activity was analysed by Dual-Luciferase Reporter Assay. ( E ) A549 and H1299 cells were transfected with miR-7, miR-NC, PA28gamma -siRNA, and miR-7 inhibitor for 48 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). ( F ) H1299 cells were transfected with PA28gamma -siRNA, and/or miR-7 inhibitor for 72 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). These experiments were performed in triplicate and results are shown as the mean±s.e.m. (*** P
    Quikchange Site Directed Mutagenesis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 95/100, based on 15564 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quikchange site directed mutagenesis kit/product/Agilent technologies
    Average 95 stars, based on 15564 article reviews
    Price from $9.99 to $1999.99
    quikchange site directed mutagenesis kit - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    99
    Thermo Fisher hek293 cells
    TDP2 Excises Cellular TOP3Bccs both from DNA and RNA (A-D) DNA RNA TOP3Bccs detection by RADAR assays. (A) <t>HEK293</t> cells were transfected with the indicated siRNA: siTDP1, siTDP2 or both siTDP1 and siTDP2 and co-transfected with R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and detected with anti-FLAG antibody. The figure is representative of two independent experiments. (B) Quantitation of TOP3Bcc of RADAR assays as shown in panel A. TOP3Bccs were measured by densitometric analyses of slot-blot signals and plotted as a function of total nucleic acid (DNA and RNA) concentration. Two independent experiments are plotted. (C) Wild-type or TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay and slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (D) Quantitation of TOP3Bcc formation in RADAR assays as shown in panel C. Three independent experiments are plotted. (E-H) DNA RNA TOP3Bccs detection by ICE assays. (E) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, ICE bioassay was performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (F) Quantitation of TOP3Bcc in the DNA and RNA fractions as shown in panel E. Three independent experiments are plotted. (G) HCT116 WT and TDP2KO cells were transfected either with wild-type TOP3B or R338W TOP3B or a combination of R338W TOP3B and siTDP2 constructs as indicated. After 72 h, ICE bioassays were performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (H) Quantitation of TOP3Bcc formation in ICE assays as shown in panel G. Three independent experiments are plotted. (I-L) Detection of RNA TOP3Bccs. (I) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (J) Quantitation of TOP3Bcc in RNA as shown in panel I. Three independent experiments are plotted. (K) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (L) Quantitation of RNA TOP3Bcc in RNA as shown in panel K. Three independent experiments are plotted. (M-N) Ectopic expression of TDP2 reduces TOP3Bccs. (M) Wild-type (WT) and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (N) Quantitation of TOP3Bcc formation using the RADAR assays as shown in panel M. Three independent experiments are plotted.
    Hek293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 36683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hek293 cells/product/Thermo Fisher
    Average 99 stars, based on 36683 article reviews
    Price from $9.99 to $1999.99
    hek293 cells - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Vazyme Biotech Co clonexpress ii one step cloning kit
    TDP2 Excises Cellular TOP3Bccs both from DNA and RNA (A-D) DNA RNA TOP3Bccs detection by RADAR assays. (A) <t>HEK293</t> cells were transfected with the indicated siRNA: siTDP1, siTDP2 or both siTDP1 and siTDP2 and co-transfected with R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and detected with anti-FLAG antibody. The figure is representative of two independent experiments. (B) Quantitation of TOP3Bcc of RADAR assays as shown in panel A. TOP3Bccs were measured by densitometric analyses of slot-blot signals and plotted as a function of total nucleic acid (DNA and RNA) concentration. Two independent experiments are plotted. (C) Wild-type or TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay and slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (D) Quantitation of TOP3Bcc formation in RADAR assays as shown in panel C. Three independent experiments are plotted. (E-H) DNA RNA TOP3Bccs detection by ICE assays. (E) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, ICE bioassay was performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (F) Quantitation of TOP3Bcc in the DNA and RNA fractions as shown in panel E. Three independent experiments are plotted. (G) HCT116 WT and TDP2KO cells were transfected either with wild-type TOP3B or R338W TOP3B or a combination of R338W TOP3B and siTDP2 constructs as indicated. After 72 h, ICE bioassays were performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (H) Quantitation of TOP3Bcc formation in ICE assays as shown in panel G. Three independent experiments are plotted. (I-L) Detection of RNA TOP3Bccs. (I) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (J) Quantitation of TOP3Bcc in RNA as shown in panel I. Three independent experiments are plotted. (K) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (L) Quantitation of RNA TOP3Bcc in RNA as shown in panel K. Three independent experiments are plotted. (M-N) Ectopic expression of TDP2 reduces TOP3Bccs. (M) Wild-type (WT) and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (N) Quantitation of TOP3Bcc formation using the RADAR assays as shown in panel M. Three independent experiments are plotted.
    Clonexpress Ii One Step Cloning Kit, supplied by Vazyme Biotech Co, used in various techniques. Bioz Stars score: 93/100, based on 2319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/clonexpress ii one step cloning kit/product/Vazyme Biotech Co
    Average 93 stars, based on 2319 article reviews
    Price from $9.99 to $1999.99
    clonexpress ii one step cloning kit - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    94
    Thermo Fisher pcdna3 vector
    T835M-UNC5C-induced cell death is mediated by ASK1 and JNK. A and B, F11 cells were co-transfected with the empty pHA vector ( vector ) or pRK5-T835M-UNC5C together with the <t>pcDNA3</t> vector ( vector ), pcDNA3-dominant-negative ASK1 ( dnASK1 ), or pcDNA3-dominant-negative JNK ( dnJNK ). At 72 h, the cells were harvested for trypan blue exclusion assays ( A ). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA ( B ). ***, p
    Pcdna3 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 9899 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 vector/product/Thermo Fisher
    Average 94 stars, based on 9899 article reviews
    Price from $9.99 to $1999.99
    pcdna3 vector - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    99
    Thermo Fisher fetal bovine serum
    T835M-UNC5C-induced cell death is mediated by ASK1 and JNK. A and B, F11 cells were co-transfected with the empty pHA vector ( vector ) or pRK5-T835M-UNC5C together with the <t>pcDNA3</t> vector ( vector ), pcDNA3-dominant-negative ASK1 ( dnASK1 ), or pcDNA3-dominant-negative JNK ( dnJNK ). At 72 h, the cells were harvested for trypan blue exclusion assays ( A ). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA ( B ). ***, p
    Fetal Bovine Serum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 225914 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fetal bovine serum/product/Thermo Fisher
    Average 99 stars, based on 225914 article reviews
    Price from $9.99 to $1999.99
    fetal bovine serum - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcdna5 frt to vector
    Transiently transfected 293 FT cells show a strongly reduced NMD efficiency in comparison to stably transfected cells. ( a ) Schematic representation of the experimental setup. The reporter constructs are generated by cloning the gene of interest into the <t>pcDNA5/FRT/TO</t> vector. The blue bar represents the coding sequence of triosephosphate isomerase (TPI) or β-globin with or without a premature termination codon (PTC) and with a normal stop codon (stop). Downstream, a viral XRN1-resistant sequence (xrRNA) is present that allows the detection of decay intermediates resulting from 5′–3′ exonucleolytic decay. The grey boxes correspond to four repeats of a northern blot probe binding site. Cells are transfected with these constructs using either a stable or transient transfection system. Flp-In T-REx (FT) cells constitutively express a Tet repressor (TetR) that blocks transcription of the reporter by binding to Tet operators (TetO 2 ) present downstream of the cytomegalovirus (CMV) promoter in the reporter construct. Therefore, FT cells are induced with tetracycline(Tet)/doxycycline. In non-FT cells, the reporter is always expressed. Unstable reporter mRNAs are degraded with participation of the cellular 5′–3′ exonuclease XRN1. Full-length reporter levels and decay fragment (xrFrag) caused by stalling of XRN1 at the xrRNA are detected via northern blotting. p(A): poly(A) tail. ( b , c ) Expression of reporter mRNAs in HeLa FT ( b ) and 293 FT ( c ) stable cell lines was induced with doxycycline for 24 h and the reporter levels detected by northern blotting. The lower band (xrFrag) corresponds to a 5′–3′ exonucleolytic decay intermediate. Endogenous 7SL RNA levels were detected and quantified on the same blot and are shown as control. The mean values ± SD (n = 3) for relative transcript levels were quantified and the PTC values normalized to the wild-type control. The xrRNA/reporter ratio is indicated below the graph. ( d , e ) HeLa FT ( d ) and 293 FT ( e ) cells were transiently transfected with 3 µg plasmid DNA per 6-well and transcription was induced with doxycycline for 24 h. Reporter mRNA levels were analyzed by northern blotting. LacZ was co-expressed and serves as a transfection control. The mean values ± SD (n = 3) were quantified and the PTC values normalized to the wild-type control. The xrRNA/reporter ratio is shown below the graph.
    Pcdna5 Frt To Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1923 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna5 frt to vector/product/Thermo Fisher
    Average 99 stars, based on 1923 article reviews
    Price from $9.99 to $1999.99
    pcdna5 frt to vector - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Cripto-1 and LRP6 co-localize on the cell membrane. 293T/Cripto-1 cells transiently transfected with LRP6-GFP expression vector were stained with anti-LRP6 (green) and anti-Cripto-1 (red) antibodies. Nuclei were visualized by Hoechst 33258 (blue). The merged image shows significant co-localization of Cripto-1 and LRP6 on the cell membrane. The arrows indicate clear examples of co-localization.

    Journal: Cellular signalling

    Article Title: Cripto-1 enhances the canonical Wnt/?-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors

    doi: 10.1016/j.cellsig.2012.09.024

    Figure Lengend Snippet: Cripto-1 and LRP6 co-localize on the cell membrane. 293T/Cripto-1 cells transiently transfected with LRP6-GFP expression vector were stained with anti-LRP6 (green) and anti-Cripto-1 (red) antibodies. Nuclei were visualized by Hoechst 33258 (blue). The merged image shows significant co-localization of Cripto-1 and LRP6 on the cell membrane. The arrows indicate clear examples of co-localization.

    Article Snippet: A soluble form of Cripto-1, lacking the GPI anchor, which tethers Cripto-1 to the cell membrane , was unable to potentiate Wnt3a induction of a SuperTOPFLASH luciferase reporter construct in 293T cells, indicating that Cripto-1 attachment to the cell membrane is required for its modulation of a canonical Wnt/β-catenin signaling pathway in 293T cells ( ).

    Techniques: Transfection, Expressing, Plasmid Preparation, Staining

    Cripto-1 enhances Wnt3a induced SuperTOPFLASH luciferase activity in 293T cells. (A) Full-length (CR-1 WT) and deletion constructs (CR-1 ΔEGF lacking the EGF-like domain, CR-1 ΔCFC lacking the CFC domain, CR-1 ΔEGF ΔCFC lacking both EGF and CFC domains) of human Cripto-1 in a 3×-FLAG expression vector. (B) SuperTOPFLASH luciferase reporter assay in 293T cells transiently transfected with different concentrations of full-length 3×-FLAG CR-1 WT plasmid (1, 10 and 100 ng) or with 3×-FLAG CR-1 ΔEGF, 3×-FLAG CR-1 ΔCFC, 3×-FLAG CR-1 ΔEGF ΔCFC deletion constructs (10 and 100 ng) together with 50 ng of SuperTOPFLASH luciferase vector. Cells were subsequently treated with rhWnt3a protein (50 ng/ml) for 16-20 hr. Cells were also transfected with 50 ng of SuperFOPFLASH luciferase vector and subsequently stimulated with rhWnt3a. These results are the mean ±SD of triplicates from one of three separate experiments.

    Journal: Cellular signalling

    Article Title: Cripto-1 enhances the canonical Wnt/?-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors

    doi: 10.1016/j.cellsig.2012.09.024

    Figure Lengend Snippet: Cripto-1 enhances Wnt3a induced SuperTOPFLASH luciferase activity in 293T cells. (A) Full-length (CR-1 WT) and deletion constructs (CR-1 ΔEGF lacking the EGF-like domain, CR-1 ΔCFC lacking the CFC domain, CR-1 ΔEGF ΔCFC lacking both EGF and CFC domains) of human Cripto-1 in a 3×-FLAG expression vector. (B) SuperTOPFLASH luciferase reporter assay in 293T cells transiently transfected with different concentrations of full-length 3×-FLAG CR-1 WT plasmid (1, 10 and 100 ng) or with 3×-FLAG CR-1 ΔEGF, 3×-FLAG CR-1 ΔCFC, 3×-FLAG CR-1 ΔEGF ΔCFC deletion constructs (10 and 100 ng) together with 50 ng of SuperTOPFLASH luciferase vector. Cells were subsequently treated with rhWnt3a protein (50 ng/ml) for 16-20 hr. Cells were also transfected with 50 ng of SuperFOPFLASH luciferase vector and subsequently stimulated with rhWnt3a. These results are the mean ±SD of triplicates from one of three separate experiments.

    Article Snippet: A soluble form of Cripto-1, lacking the GPI anchor, which tethers Cripto-1 to the cell membrane , was unable to potentiate Wnt3a induction of a SuperTOPFLASH luciferase reporter construct in 293T cells, indicating that Cripto-1 attachment to the cell membrane is required for its modulation of a canonical Wnt/β-catenin signaling pathway in 293T cells ( ).

    Techniques: Luciferase, Activity Assay, Construct, Expressing, Plasmid Preparation, Reporter Assay, Transfection

    Cripto-1 lacking GPI-anchor does not enhance the canonical Wnt/β-catenin signaling pathway stimulated by Wnt3a in 293T cells. (A) Full-length (CR-1 WT) and GPI-anchor signal sequence deletion mutant (CR-1 ΔC) of human Cripto-1 in a 3×FLAG expression vector. (B) SuperTOPFLASH luciferase assay in 293T cells transiently transfected with different concentrations of 3×FLAG-CR-1 WT or 3×FLAG-CR-1 ΔC (10 and 100 ng) together with 50 ng of SuperTOPFLASH luciferase vector. Cells were subsequently treated with 50 ng/ml of rhWnt3a protein for 16-20 hr. These results are the mean ±SD of triplicates from one of three separate experiments.

    Journal: Cellular signalling

    Article Title: Cripto-1 enhances the canonical Wnt/?-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors

    doi: 10.1016/j.cellsig.2012.09.024

    Figure Lengend Snippet: Cripto-1 lacking GPI-anchor does not enhance the canonical Wnt/β-catenin signaling pathway stimulated by Wnt3a in 293T cells. (A) Full-length (CR-1 WT) and GPI-anchor signal sequence deletion mutant (CR-1 ΔC) of human Cripto-1 in a 3×FLAG expression vector. (B) SuperTOPFLASH luciferase assay in 293T cells transiently transfected with different concentrations of 3×FLAG-CR-1 WT or 3×FLAG-CR-1 ΔC (10 and 100 ng) together with 50 ng of SuperTOPFLASH luciferase vector. Cells were subsequently treated with 50 ng/ml of rhWnt3a protein for 16-20 hr. These results are the mean ±SD of triplicates from one of three separate experiments.

    Article Snippet: A soluble form of Cripto-1, lacking the GPI anchor, which tethers Cripto-1 to the cell membrane , was unable to potentiate Wnt3a induction of a SuperTOPFLASH luciferase reporter construct in 293T cells, indicating that Cripto-1 attachment to the cell membrane is required for its modulation of a canonical Wnt/β-catenin signaling pathway in 293T cells ( ).

    Techniques: Sequencing, Mutagenesis, Expressing, Plasmid Preparation, Luciferase, Transfection

    Interaction between CR-1 and Wnt ligand or Wnt receptors. Mouse Wnt3a-HA, human Fz8 CRD-Fc, human LRP5ΔC-myc and full-length 3×FLAG-CR-1 WT expression plasmids were co-transfected into 293T cells. After 24 h, cells were harvested and lysed in RIPA buffer. The cell lysates were treated with 10 μl of anti-FLAG M2 affinity gel for 2 h at 4 °C. The Affinity gels were washed with RIPA buffer for 4 times. Proteins were then eluted by 2× Laemmli SDS sample buffer and separated by SDS-PAGE. Western blotting was carried out using following antibodies: anti-FLAG, anti-myc, anti-HA and anti-human IgG-HRP.

    Journal: Cellular signalling

    Article Title: Cripto-1 enhances the canonical Wnt/?-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors

    doi: 10.1016/j.cellsig.2012.09.024

    Figure Lengend Snippet: Interaction between CR-1 and Wnt ligand or Wnt receptors. Mouse Wnt3a-HA, human Fz8 CRD-Fc, human LRP5ΔC-myc and full-length 3×FLAG-CR-1 WT expression plasmids were co-transfected into 293T cells. After 24 h, cells were harvested and lysed in RIPA buffer. The cell lysates were treated with 10 μl of anti-FLAG M2 affinity gel for 2 h at 4 °C. The Affinity gels were washed with RIPA buffer for 4 times. Proteins were then eluted by 2× Laemmli SDS sample buffer and separated by SDS-PAGE. Western blotting was carried out using following antibodies: anti-FLAG, anti-myc, anti-HA and anti-human IgG-HRP.

    Article Snippet: A soluble form of Cripto-1, lacking the GPI anchor, which tethers Cripto-1 to the cell membrane , was unable to potentiate Wnt3a induction of a SuperTOPFLASH luciferase reporter construct in 293T cells, indicating that Cripto-1 attachment to the cell membrane is required for its modulation of a canonical Wnt/β-catenin signaling pathway in 293T cells ( ).

    Techniques: Expressing, Transfection, SDS Page, Western Blot

    Cripto-1 binds to LRP5 and LRP6 in 293T cells. 293T cells were co-transfected with LRP-5-tGFP or LRP6-GFP expression vectors together with Cripto-1 full-length or deleted expression plasmids. 3×-FLAG Cripto-1 was immunoprecipated using an anti-FLAG mouse monoclonal antibody and the immunoprecipitated proteins were analyzed by Western blot with anti-tGFP, anti-GFP and anti-FLAG mouse monoclonal antibodies (A and C). LRP5-tGFP or LRP6-GFP were immunoprecipitated with anti-tGFP or anti-GFP mouse monoclonal antibody and immunoprecipitated proteins were analyzed by Western blot with anti-tGFP, anti-GFP and anti-FLAG mouse monoclonal antibodies (B and D). Cell lysates of 293T cells transiently transfected with various expression vectors as described above were analyzed by Western blot for Cripto-1 using an anti-FLAG antibody, for LRP5 using anti-tGFP antibody and for LRP6 using anti-GFP antibody (A through D).

    Journal: Cellular signalling

    Article Title: Cripto-1 enhances the canonical Wnt/?-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors

    doi: 10.1016/j.cellsig.2012.09.024

    Figure Lengend Snippet: Cripto-1 binds to LRP5 and LRP6 in 293T cells. 293T cells were co-transfected with LRP-5-tGFP or LRP6-GFP expression vectors together with Cripto-1 full-length or deleted expression plasmids. 3×-FLAG Cripto-1 was immunoprecipated using an anti-FLAG mouse monoclonal antibody and the immunoprecipitated proteins were analyzed by Western blot with anti-tGFP, anti-GFP and anti-FLAG mouse monoclonal antibodies (A and C). LRP5-tGFP or LRP6-GFP were immunoprecipitated with anti-tGFP or anti-GFP mouse monoclonal antibody and immunoprecipitated proteins were analyzed by Western blot with anti-tGFP, anti-GFP and anti-FLAG mouse monoclonal antibodies (B and D). Cell lysates of 293T cells transiently transfected with various expression vectors as described above were analyzed by Western blot for Cripto-1 using an anti-FLAG antibody, for LRP5 using anti-tGFP antibody and for LRP6 using anti-GFP antibody (A through D).

    Article Snippet: A soluble form of Cripto-1, lacking the GPI anchor, which tethers Cripto-1 to the cell membrane , was unable to potentiate Wnt3a induction of a SuperTOPFLASH luciferase reporter construct in 293T cells, indicating that Cripto-1 attachment to the cell membrane is required for its modulation of a canonical Wnt/β-catenin signaling pathway in 293T cells ( ).

    Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

    Cripto-1 binds to LRP5 and LRP6 co-receptors on the cell surface. 293T cells transiently transfected with Cripto-1, LRP5 and/or LRP6 expression plasmids were incubated with 2mM NHS-PEG 4 -Biotin and then lysed using RIPA buffer. Following immunoprecipitation with anti-FLAG or anti-GFP monoclonal antibodies, cell surface biotinylated proteins were isolated by adding streptavidin agarose beads to the immunoprecipitated proteins for 3 h at 4 °C. A and C show co-immunoprecipitation of LRP5-tGFP (A) or LRP6-GFP (C) with 3×-FLAG CR-1 WT only in the presence of biotin. B and D show co-immunoprecipitation of 3×-FLAG CR-1 WT with LRP5-tGFP (B) or LRP6-GFP (D) only in the presence of biotin. Cell lysates of immunoprecipitated samples were also analyzed by Western blot to ensure expression of transfected plasmids. (E) Endogenous Cripto-1 and LRP6 co-immunoprecipitate in NCCIT human embryonal carcinoma cells. NCCIT protein lysates were immunoprecipitated with an anti-Cripto-1 (V-17) rabbit polyclonal antibody and probed with an LRP6 monoclonal antibody (E) or immunoprecipitated with an anti-LRP6 monoclonal antibody and probed with and anti-Cripto-1 rabbit polyclonal antibody (F). * indicates the immunoglobulin light chain.

    Journal: Cellular signalling

    Article Title: Cripto-1 enhances the canonical Wnt/?-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors

    doi: 10.1016/j.cellsig.2012.09.024

    Figure Lengend Snippet: Cripto-1 binds to LRP5 and LRP6 co-receptors on the cell surface. 293T cells transiently transfected with Cripto-1, LRP5 and/or LRP6 expression plasmids were incubated with 2mM NHS-PEG 4 -Biotin and then lysed using RIPA buffer. Following immunoprecipitation with anti-FLAG or anti-GFP monoclonal antibodies, cell surface biotinylated proteins were isolated by adding streptavidin agarose beads to the immunoprecipitated proteins for 3 h at 4 °C. A and C show co-immunoprecipitation of LRP5-tGFP (A) or LRP6-GFP (C) with 3×-FLAG CR-1 WT only in the presence of biotin. B and D show co-immunoprecipitation of 3×-FLAG CR-1 WT with LRP5-tGFP (B) or LRP6-GFP (D) only in the presence of biotin. Cell lysates of immunoprecipitated samples were also analyzed by Western blot to ensure expression of transfected plasmids. (E) Endogenous Cripto-1 and LRP6 co-immunoprecipitate in NCCIT human embryonal carcinoma cells. NCCIT protein lysates were immunoprecipitated with an anti-Cripto-1 (V-17) rabbit polyclonal antibody and probed with an LRP6 monoclonal antibody (E) or immunoprecipitated with an anti-LRP6 monoclonal antibody and probed with and anti-Cripto-1 rabbit polyclonal antibody (F). * indicates the immunoglobulin light chain.

    Article Snippet: A soluble form of Cripto-1, lacking the GPI anchor, which tethers Cripto-1 to the cell membrane , was unable to potentiate Wnt3a induction of a SuperTOPFLASH luciferase reporter construct in 293T cells, indicating that Cripto-1 attachment to the cell membrane is required for its modulation of a canonical Wnt/β-catenin signaling pathway in 293T cells ( ).

    Techniques: Transfection, Expressing, Incubation, Immunoprecipitation, Isolation, Western Blot

    Pregnancy-upregulated nonubiquitous calmodulin kinase (Pnck) inhibits EGF-induced MAP kinase activation in human embryonic kidney (HEK)-293T cells. A : inhibition of EGF-induced MAP kinase activity by wild-type (WT) Pnck. Subconfluent HEK-293T cells were

    Journal:

    Article Title: Pregnancy-upregulated nonubiquitous calmodulin kinase induces ligand-independent EGFR degradation

    doi: 10.1152/ajpcell.00449.2007

    Figure Lengend Snippet: Pregnancy-upregulated nonubiquitous calmodulin kinase (Pnck) inhibits EGF-induced MAP kinase activation in human embryonic kidney (HEK)-293T cells. A : inhibition of EGF-induced MAP kinase activity by wild-type (WT) Pnck. Subconfluent HEK-293T cells were

    Article Snippet: Upon stimulation of serum-starved, subconfluent control cells with a panel of ligands (EGF, IGF-I, IGF-II, and insulin), we observed that EGF was the only ligand able to stimulate MAP kinase activation in HEK-293T cells.

    Techniques: Activation Assay, Inhibition, Activity Assay

    p300 siRNA suppressed UV-induced MMP-1 expression and inhibited UV-enhanced levels of γ-H2AX, p53 and acetyl-H3, and AA inhibited interaction of p300 with γ-H2AX or acetyl-H3 after UV. HDFs were transfected with scrambled control siRNA and p300 siRNA at 100 nM using Lipofectamine as recommended by the manufacturer. A, HDFs were irradiated with UV and incubated at 37°C 24 h after transfection with scrambled control or p300 siRNA. The MMP-1 mRNA level was analyzed by RT-PCR and the amount of MMP-1 protein was analyzed by western blot. B, The effect of p300 siRNA on the protein levels of γ-H2AX, p53, and acetyl-H3 were analyzed by western blotting. C, HDFs were UV-irradiated. After 6 h, whole cell lysates were immunoprecipitated with anti-p300 antibody. D, HDFs were UV-irradiated and post-treated with AA for 6 h. Whole cell lysates were immunoprecipitated with anti-p300 antibody. Proteins were immunoblotted with anti-p300, anti-acetyl-H3, and anti-γ-H2AX antibodies ( upper panels ). Bar graphs ( lower panels ) show quantitative analysis of scanning densitometric values of γ-H2AX, p53, and acetyl-H3 as ratios to β-actin, which was used as a loading control. Data shown are representative of three independent experiments. C: control, UV: UV-irradiated cells, AA: AA treated cells, UV/AA: UV irradiated cells incubated with AA. *P

    Journal: PLoS ONE

    Article Title: The Role of p300 Histone Acetyltransferase in UV-Induced Histone Modifications and MMP-1 Gene Transcription

    doi: 10.1371/journal.pone.0004864

    Figure Lengend Snippet: p300 siRNA suppressed UV-induced MMP-1 expression and inhibited UV-enhanced levels of γ-H2AX, p53 and acetyl-H3, and AA inhibited interaction of p300 with γ-H2AX or acetyl-H3 after UV. HDFs were transfected with scrambled control siRNA and p300 siRNA at 100 nM using Lipofectamine as recommended by the manufacturer. A, HDFs were irradiated with UV and incubated at 37°C 24 h after transfection with scrambled control or p300 siRNA. The MMP-1 mRNA level was analyzed by RT-PCR and the amount of MMP-1 protein was analyzed by western blot. B, The effect of p300 siRNA on the protein levels of γ-H2AX, p53, and acetyl-H3 were analyzed by western blotting. C, HDFs were UV-irradiated. After 6 h, whole cell lysates were immunoprecipitated with anti-p300 antibody. D, HDFs were UV-irradiated and post-treated with AA for 6 h. Whole cell lysates were immunoprecipitated with anti-p300 antibody. Proteins were immunoblotted with anti-p300, anti-acetyl-H3, and anti-γ-H2AX antibodies ( upper panels ). Bar graphs ( lower panels ) show quantitative analysis of scanning densitometric values of γ-H2AX, p53, and acetyl-H3 as ratios to β-actin, which was used as a loading control. Data shown are representative of three independent experiments. C: control, UV: UV-irradiated cells, AA: AA treated cells, UV/AA: UV irradiated cells incubated with AA. *P

    Article Snippet: For luciferase assays, HDFs were cultured in 12-well plates for 2 days before transfection. pCMV-E1A, pCMV-p300, and MMP1-Luc were transiently co-transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

    Techniques: Expressing, Transfection, Irradiation, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation

    SOX9 direct binding to the promoter of miR-130a to regulate its expression (A) si-SOX9 or SOX9 vector was transfected into HeLa and CaSki cells to achieve SOX9 expression, as verified using Western blot assays. (B) miR-130a expression in the indicated cells was determined using real-time PCR assays. (C) A schematic diagram of potential SOX9 binding element (two possible binding sites) in the promoter region of miR-130a predicted by Jaspar database. A wt-miR-130a promoter luciferase reporter vector and a mut-miR-130a promoter luciferase reporter vector were constructed. (D) The indicated vectors were co-transfected into HEK293 cells with SOX9 vector; the luciferase activity was determined. (E) The real-time ChIP assay showed that the level of SOX9 antibody binding to miR-130a promoter was much greater than that of IgG. The data are presented as mean ± SD of three independent experiments. * P

    Journal: Cell Cycle

    Article Title: SOX9/miR-130a/CTR1 axis modulates DDP-resistance of cervical cancer cell

    doi: 10.1080/15384101.2017.1395533

    Figure Lengend Snippet: SOX9 direct binding to the promoter of miR-130a to regulate its expression (A) si-SOX9 or SOX9 vector was transfected into HeLa and CaSki cells to achieve SOX9 expression, as verified using Western blot assays. (B) miR-130a expression in the indicated cells was determined using real-time PCR assays. (C) A schematic diagram of potential SOX9 binding element (two possible binding sites) in the promoter region of miR-130a predicted by Jaspar database. A wt-miR-130a promoter luciferase reporter vector and a mut-miR-130a promoter luciferase reporter vector were constructed. (D) The indicated vectors were co-transfected into HEK293 cells with SOX9 vector; the luciferase activity was determined. (E) The real-time ChIP assay showed that the level of SOX9 antibody binding to miR-130a promoter was much greater than that of IgG. The data are presented as mean ± SD of three independent experiments. * P

    Article Snippet: HEK293 cells (ATCC) were cultured overnight after being seeded into a 24-well plate.

    Techniques: Binding Assay, Expressing, Plasmid Preparation, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, Construct, Activity Assay, Chromatin Immunoprecipitation

    MiR-130a direct binding to the 3′UTR of SCL31A1 (A) HeLa and CaSki cells were transfected with miR-130a mimics or miR-130a inhibitor; the CTR1 protein levels in the indicated cells were determined using Western blot assays. (B) A wild-type and mutated SLC31A1 3′UTR luciferase reporter gene vector (wt- SLC31A1 3′UTR and mut- SLC31A1 3′UTR containing a 7 bp mutation in two predicted binding sites of miR-130a) was constructed. (C) The indicated vectors were co-transfected with miR-130a mimics or miR-130a inhibitor into HEK293 cells; the luciferase activity was then determined using dual luciferase assays. (D)-(E) Association of miR-130a and SOX9 with AGO2. HeLa cellular lysates were used for RNA immunoprecipitation with AGO2 antibody. Detection of AGO2 and IgG using Western blot (up), and detection of miR-130a or SOX9 using qRT-PCR (low). All data of SOX9 expression were normalized to β-actin mRNA expression levels. MiR-130a expression data was normalized to U6 small RNA expression. The data are presented as mean ± SD of three independent experiments. * P

    Journal: Cell Cycle

    Article Title: SOX9/miR-130a/CTR1 axis modulates DDP-resistance of cervical cancer cell

    doi: 10.1080/15384101.2017.1395533

    Figure Lengend Snippet: MiR-130a direct binding to the 3′UTR of SCL31A1 (A) HeLa and CaSki cells were transfected with miR-130a mimics or miR-130a inhibitor; the CTR1 protein levels in the indicated cells were determined using Western blot assays. (B) A wild-type and mutated SLC31A1 3′UTR luciferase reporter gene vector (wt- SLC31A1 3′UTR and mut- SLC31A1 3′UTR containing a 7 bp mutation in two predicted binding sites of miR-130a) was constructed. (C) The indicated vectors were co-transfected with miR-130a mimics or miR-130a inhibitor into HEK293 cells; the luciferase activity was then determined using dual luciferase assays. (D)-(E) Association of miR-130a and SOX9 with AGO2. HeLa cellular lysates were used for RNA immunoprecipitation with AGO2 antibody. Detection of AGO2 and IgG using Western blot (up), and detection of miR-130a or SOX9 using qRT-PCR (low). All data of SOX9 expression were normalized to β-actin mRNA expression levels. MiR-130a expression data was normalized to U6 small RNA expression. The data are presented as mean ± SD of three independent experiments. * P

    Article Snippet: HEK293 cells (ATCC) were cultured overnight after being seeded into a 24-well plate.

    Techniques: Binding Assay, Transfection, Western Blot, Luciferase, Plasmid Preparation, Mutagenesis, Construct, Activity Assay, Immunoprecipitation, Quantitative RT-PCR, Expressing, RNA Expression

    HSPA1L and GR protein levels in decidualized human endometrial stromal fibroblasts. Cultured ESFs were transfected with WT or Ala268Thr HSPA1L -pcDNA3.1 constructs or with empty pcDNA3.1 vector (control). Cells were treated with decidualization media supplemented with 100nM dexamethasone (glucocorticoids) for 72h. Both cytosolic and nuclear protein were extracted, and HSPA1L and GR protein levels were measured by Western blot. Band intensity of HSPA1L or GR was normalized to band intensity of the corresponding β-actin. Cytosolic (A) and nuclear (B) HSPA1L levels as well as cytosolic (C) and nuclear (D) GR levels are shown for control (empty vector), WT and Ala268Thr sample groups. Each experiment was performed as triplicates in three different passages (n = 9 each group, except n = 8 for nuclear control group) and bars represent mean + SEM. Significant p-value

    Journal: PLoS Genetics

    Article Title: Whole exome sequencing reveals HSPA1L as a genetic risk factor for spontaneous preterm birth

    doi: 10.1371/journal.pgen.1007394

    Figure Lengend Snippet: HSPA1L and GR protein levels in decidualized human endometrial stromal fibroblasts. Cultured ESFs were transfected with WT or Ala268Thr HSPA1L -pcDNA3.1 constructs or with empty pcDNA3.1 vector (control). Cells were treated with decidualization media supplemented with 100nM dexamethasone (glucocorticoids) for 72h. Both cytosolic and nuclear protein were extracted, and HSPA1L and GR protein levels were measured by Western blot. Band intensity of HSPA1L or GR was normalized to band intensity of the corresponding β-actin. Cytosolic (A) and nuclear (B) HSPA1L levels as well as cytosolic (C) and nuclear (D) GR levels are shown for control (empty vector), WT and Ala268Thr sample groups. Each experiment was performed as triplicates in three different passages (n = 9 each group, except n = 8 for nuclear control group) and bars represent mean + SEM. Significant p-value

    Article Snippet: The HSPA1L sequences were subcloned into pcDNA3.1(+) vector (Invitrogen) by ligating into the BamHI/NotI sites using Quick ligase (New England Biolabs).

    Techniques: Cell Culture, Transfection, Construct, Plasmid Preparation, Western Blot

    NRF-1 regulates PCFT mRNA levels. A , HeLa cells were stably transfected with one of the following expression vectors: pCDNA3, NRF-1 DN, NRF-1 WT, or NRF-1 VP16 and a stable population was established by drug selection using G418 or hygromycin. PCFT and

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M110.135640

    Figure Lengend Snippet: NRF-1 regulates PCFT mRNA levels. A , HeLa cells were stably transfected with one of the following expression vectors: pCDNA3, NRF-1 DN, NRF-1 WT, or NRF-1 VP16 and a stable population was established by drug selection using G418 or hygromycin. PCFT and

    Article Snippet: Construction and cloning of the following reporter plasmids harboring the PCFT promoter pGL3-3.1kb, pGL3-271bp, and pGL3-157bp was previously described ( ). pCDNA3 was purchased from Invitrogen, whereas pCDNA3-Flag-NRF-1 WT, which contains the full-length human NRF-1 and a dominant-negative pCDNA3-Flag-NRF-1 DN construct, which expresses amino acids 1–342 of NRF-1 but lacks the transactivation domain, were a generous gift from Prof. Kimitoshi Kohno (University of Occupational and Environment Health, Fukuoka, Japan). pCDNA 3.1 hygro NRF-1 VP16 encodes for a constitutively active fusion protein, consisting of the full-length human NRF-1 and the herpes simplex virus VP16 transactivation domain, was kindly provided by Dr. T. Gulick (Sanford-Burnham Medical Research Institute, CA). pCDNA3-HA-hPGC-1α was a gift from Dr. A. Kralli (The Scripps Research Institute, CA).

    Techniques: Stable Transfection, Transfection, Expressing, Selection

    FXR enhances the transcriptional activity of miR-122 promoter. a Potential FXREs in miR-122 promoter region were predicted using an online algorithm (NUBIScan: http://www.nubiscan.unibas.ch/ ). Transcription start sites (TSS, +1) are indicated by an arrow. b Huh7 cells were co-transfected with renilla luciferase expression vector pRL-TK and one of a series of luciferase reporter constructs containing different fragments of miR-122 promoter region. After 6 h incubation, the cells were treated with DMSO or 5 μM GW4064 for 24 h, and then dual luciferase assays were performed. Firefly luciferase activity was normalized to that of renilla luciferase. Data are shown as means ± SD from three assays performed in triplicate. c Huh7 cells were co-transfected with pRL-TK and either pGL3-F4(DR2)-WT (containing the wild-type DR2 element) or pGL3-F4(DR2)-Mut (containing the mutant DR2 element) for 6 h. Subsequent treatment and luciferase assays were as performed in ( b ). * P

    Journal: Molecular Cancer

    Article Title: Upregulation of microRNA-122 by farnesoid X receptor suppresses the growth of hepatocellular carcinoma cells

    doi: 10.1186/s12943-015-0427-9

    Figure Lengend Snippet: FXR enhances the transcriptional activity of miR-122 promoter. a Potential FXREs in miR-122 promoter region were predicted using an online algorithm (NUBIScan: http://www.nubiscan.unibas.ch/ ). Transcription start sites (TSS, +1) are indicated by an arrow. b Huh7 cells were co-transfected with renilla luciferase expression vector pRL-TK and one of a series of luciferase reporter constructs containing different fragments of miR-122 promoter region. After 6 h incubation, the cells were treated with DMSO or 5 μM GW4064 for 24 h, and then dual luciferase assays were performed. Firefly luciferase activity was normalized to that of renilla luciferase. Data are shown as means ± SD from three assays performed in triplicate. c Huh7 cells were co-transfected with pRL-TK and either pGL3-F4(DR2)-WT (containing the wild-type DR2 element) or pGL3-F4(DR2)-Mut (containing the mutant DR2 element) for 6 h. Subsequent treatment and luciferase assays were as performed in ( b ). * P

    Article Snippet: The fragments were then separately inserted between Kpn I and Hin dIII sites of the pGL3-basic vector (Promega), and the resulting plasmids were named as follows with the fragment of miR-122 promoter region specified: pGL3-F1 (−1100 to +130), pGL3-F2 (−1000 to +130), pGL3-F3 (−900 to +130), pGL3-F4 (−400 to +130) (also named pGL3-F4(DR2)-WT), pGL3-F5 (−200 to +130), pGL3-F6 (−150 to +130), pGL3-F7 (−50 to +130) and pGL3-F8 (+5 to +130). pGL3-F4(DR2)-Mut, derived from pGL3-F4(DR2)-WT, contained mutations in the DR2 element (A ATC GACCAG ACT A, the mutated bases are underlined).

    Techniques: Activity Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Construct, Incubation, Mutagenesis

    MiR-138-5p functionally targeted circ_002136 . ( a ) A schematic representation of how circ_002136 arose from the CDK11A gene as determined by scanning CDK11A genomic DNA and circBase. ( b ) The expression of miR-138-5p was measured after knockdown of circ_002136 by qRT-PCR. Values represent the means ± SD (n = 5, each group). ** P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: FUS/circ_002136/miR-138-5p/SOX13 feedback loop regulates angiogenesis in Glioma

    doi: 10.1186/s13046-019-1065-7

    Figure Lengend Snippet: MiR-138-5p functionally targeted circ_002136 . ( a ) A schematic representation of how circ_002136 arose from the CDK11A gene as determined by scanning CDK11A genomic DNA and circBase. ( b ) The expression of miR-138-5p was measured after knockdown of circ_002136 by qRT-PCR. Values represent the means ± SD (n = 5, each group). ** P

    Article Snippet: For the SOX13 -SPON2 reporter constructs, the SPON2 promoter region (− 1, 000 to + 200 bp) was amplified from human genomic DNA by PCR and then subcloned into pGL3-Basic-Luciferase vector (Promega) containing a firefly luciferase reporter gene, yielding the wide-type plasmid (SPON2 -Wt).

    Techniques: Expressing, Quantitative RT-PCR

    IRF4 mutations in CLL. (A). Schematic representation of the L116R mutation. (B) Chromatographs of L116R and L116P mutations. (C) Clinical characteristics of patients showing IRF4 mutations. (D) Significantly overexpressed genes in HEK 293 cells transfected

    Journal: Blood

    Article Title: IRF4 mutations in chronic lymphocytic leukemia

    doi: 10.1182/blood-2011-04-350579

    Figure Lengend Snippet: IRF4 mutations in CLL. (A). Schematic representation of the L116R mutation. (B) Chromatographs of L116R and L116P mutations. (C) Clinical characteristics of patients showing IRF4 mutations. (D) Significantly overexpressed genes in HEK 293 cells transfected

    Article Snippet: HEK 293 cells were transfected with 1 μg of the WT/mutant plasmids using lipofectamine 2000 (Invitrogen).

    Techniques: Mutagenesis, Transfection

    Cells depleted of both hFis1 and Opa1 show apoptosis resistance like hFis1-depleted cells. (A) HeLa cells depleted of hFis1, Opa1, or both of them by RNAi along with control RNAi cells were treated with STS (1 μM; 6 h), Act D (10 μM; 8 h), etoposide (100 μM; 30 h), or anti-Fas antibody (500 ng/ml; 15 h), and the nuclei were stained with Hoechst 33342 (1 μg/ml; 15 min at RT). Normal or apoptotic nuclei of these cells in several fields were counted under the fluorescent microscope (for UV excitation). At least 200 cells altogether in each treatment were counted and plotted as a percentage of cells with apoptotic nuclei among the total cells counted. The data are shown as the mean ± SD of at least three independent experiments. (B) HeLa cells depleted of Opa1, hFis1, or both of them along with control RNAi cells were incubated with anti-Fas antibody (500 ng/ml) for the periods as indicated, and the nuclei were stained and counted as normal or apoptotic nuclei. At least 200 cells altogether were counted in each sample at each time point and plotted as a percentage of cells with apoptotic nuclei among the total cells counted. The data were plotted as the mean ± SD of at least three independent experiments. (C) Bax translocation and cytochrome c release induced by Act D treatment are inhibited in hFis/Opa1 RNAi cells. Left, HeLa cells depleted of both hFis1 and Opa1 by RNAi along with control RNAi cells were treated with Act D (10 μM; 8 h) in the presence of zVAD-fmk (50 μM), fixed, and double stained with anti-Bax (rabbit polyclonal, red) and anti-cytochrome c (mouse monoclonal, green) antibodies. Right, the number of cells displaying Bax translocation or cytochrome c release was counted and plotted as a percentage of the total cells counted in each RNAi cell population that had been treated with Act D and stained with anti-Bax and anti-cytochrome c antibodies (the same samples as shown on the left). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments. (D) hFis1 depletion prevents mitochondrial membrane potential reduction induced by Opa1 depletion. Left, HeLa cells depleted of hFis1 (b), Opa1 (c), or both of them (d) along with control RNAi cells (a) were incubated with 5 μg/ml JC-1 for 20 min and observed by confocal microscopy. JC-1 is a cationic dye that indicates mitochondrial polarization by shifting its fluorescence emission from green to red. Regions of high mitochondrial membrane potential are indicated by red fluorescence, and regions of low mitochondrial membrane potential are indicated by green fluorescence. Right, the number of cells with mitochondria whose membrane potential was lost was counted and plotted as a percentage of the total cells counted in each RNAi cell population.

    Journal: Molecular Biology of the Cell

    Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

    doi: 10.1091/mbc.E04-04-0294

    Figure Lengend Snippet: Cells depleted of both hFis1 and Opa1 show apoptosis resistance like hFis1-depleted cells. (A) HeLa cells depleted of hFis1, Opa1, or both of them by RNAi along with control RNAi cells were treated with STS (1 μM; 6 h), Act D (10 μM; 8 h), etoposide (100 μM; 30 h), or anti-Fas antibody (500 ng/ml; 15 h), and the nuclei were stained with Hoechst 33342 (1 μg/ml; 15 min at RT). Normal or apoptotic nuclei of these cells in several fields were counted under the fluorescent microscope (for UV excitation). At least 200 cells altogether in each treatment were counted and plotted as a percentage of cells with apoptotic nuclei among the total cells counted. The data are shown as the mean ± SD of at least three independent experiments. (B) HeLa cells depleted of Opa1, hFis1, or both of them along with control RNAi cells were incubated with anti-Fas antibody (500 ng/ml) for the periods as indicated, and the nuclei were stained and counted as normal or apoptotic nuclei. At least 200 cells altogether were counted in each sample at each time point and plotted as a percentage of cells with apoptotic nuclei among the total cells counted. The data were plotted as the mean ± SD of at least three independent experiments. (C) Bax translocation and cytochrome c release induced by Act D treatment are inhibited in hFis/Opa1 RNAi cells. Left, HeLa cells depleted of both hFis1 and Opa1 by RNAi along with control RNAi cells were treated with Act D (10 μM; 8 h) in the presence of zVAD-fmk (50 μM), fixed, and double stained with anti-Bax (rabbit polyclonal, red) and anti-cytochrome c (mouse monoclonal, green) antibodies. Right, the number of cells displaying Bax translocation or cytochrome c release was counted and plotted as a percentage of the total cells counted in each RNAi cell population that had been treated with Act D and stained with anti-Bax and anti-cytochrome c antibodies (the same samples as shown on the left). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments. (D) hFis1 depletion prevents mitochondrial membrane potential reduction induced by Opa1 depletion. Left, HeLa cells depleted of hFis1 (b), Opa1 (c), or both of them (d) along with control RNAi cells (a) were incubated with 5 μg/ml JC-1 for 20 min and observed by confocal microscopy. JC-1 is a cationic dye that indicates mitochondrial polarization by shifting its fluorescence emission from green to red. Regions of high mitochondrial membrane potential are indicated by red fluorescence, and regions of low mitochondrial membrane potential are indicated by green fluorescence. Right, the number of cells with mitochondria whose membrane potential was lost was counted and plotted as a percentage of the total cells counted in each RNAi cell population.

    Article Snippet: We cotransfected HeLa cells with cDNA constructs encoding either hFis1 wt or an hFis1ΔC mutant lacking the membrane anchor along with the hFis1 shRNAi construct, selected transfectants with hygromycin, and examined protein expression levels and sensitivity to apoptosis.

    Techniques: Activated Clotting Time Assay, Staining, Microscopy, Incubation, Translocation Assay, Confocal Microscopy, Fluorescence

    Knockdown of hFis1 did not affect the distribution of Drp1 to mitochondria. (A) Control or hFis1-depleted HeLa cells were incubated with Mitotracker CMXRos (red) for 20 min before fixation and stained with mouse monoclonal anti-DLP1/Drp1 followed by anti-mouse Alexa Fluor 488 antibodies (green). Mitochondria and distribution of Drp1 were analyzed by confocal microscopy. Red, mitochondria; green, Drp1. (B) HeLa cells depleted of hFis1 along with control RNAi were fractionated into S and HM (mainly mitochondria) and analyzed for the distribution of Drp1 by Western blotting. Fractionation quality was verified by the distribution of specific subcellular markers: CoxIV for mitochondria and actin for cytosol.

    Journal: Molecular Biology of the Cell

    Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

    doi: 10.1091/mbc.E04-04-0294

    Figure Lengend Snippet: Knockdown of hFis1 did not affect the distribution of Drp1 to mitochondria. (A) Control or hFis1-depleted HeLa cells were incubated with Mitotracker CMXRos (red) for 20 min before fixation and stained with mouse monoclonal anti-DLP1/Drp1 followed by anti-mouse Alexa Fluor 488 antibodies (green). Mitochondria and distribution of Drp1 were analyzed by confocal microscopy. Red, mitochondria; green, Drp1. (B) HeLa cells depleted of hFis1 along with control RNAi were fractionated into S and HM (mainly mitochondria) and analyzed for the distribution of Drp1 by Western blotting. Fractionation quality was verified by the distribution of specific subcellular markers: CoxIV for mitochondria and actin for cytosol.

    Article Snippet: We cotransfected HeLa cells with cDNA constructs encoding either hFis1 wt or an hFis1ΔC mutant lacking the membrane anchor along with the hFis1 shRNAi construct, selected transfectants with hygromycin, and examined protein expression levels and sensitivity to apoptosis.

    Techniques: Incubation, Staining, Confocal Microscopy, Western Blot, Fractionation

    Mitochondria are extensively fragmented in the cells depleted of both hFis1 and Opa1. HeLa cells were transfected with pREP4 constructs containing shRNA of the target sequence of hFis1, Opa1, or both of them together along with control, and the transfectants were selected by growing in media containing hygromycin B. (A) Total cell lysates from the hFis1 RNAi cells, Opa1 RNAi cells, or both hFis1 and Opa1 RNAi cells along with control RNAi cells were prepared, and the expression levels of hFis1 and Opa1 were analyzed by Western blotting. Actin also was analyzed as a loading control. (B) Left, mitochondria of hFis1/Opa1 RNAi cells were visualized with Mitotracker Red CMXRos and analyzed by confocal microscopy. An enlargement is shown for detailed structure of mitochondria. Right, percentage of cell population with fragmented (dotted), normal (solid), or elongated (striped) mitochondria in hFis1/Opa1 RNAi cell culture. At least 200 cells in several fields were counted in each experiment. Data represent the mean ± SD of at least three independent experiments. (C) The elongated mitochondrial morphology in hFis1-depleted cells was reversed by subsequent depletion of Opa1. Hela cells depleted of hFis1 by RNAi were selected for 5 d and transfected again with Opa1 shRNA- or a control shRNA-construct and examined 2 d later. To identify transfectants, pEYFP vector was cotransfected. Mitochondria were visualized with Mitotracker Red CMXRos and analyzed by confocal microscopy. (D and E) Control RNAi, Opa1 RNAi, and hFis1/Opa1 RNAi double RNAi cells were analyzed for mitochondrial fusion by using mito-PAGF–based fusion assay. Cells were transfected with mito-PAGFP, followed by the photoactivation of the small regions within the cells (white circles in prepanels) and confocal acquisition of z-series covering the entire thickness of the cell. (E) Changes in the fluorescence intensity within activated regions were measured over time.

    Journal: Molecular Biology of the Cell

    Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

    doi: 10.1091/mbc.E04-04-0294

    Figure Lengend Snippet: Mitochondria are extensively fragmented in the cells depleted of both hFis1 and Opa1. HeLa cells were transfected with pREP4 constructs containing shRNA of the target sequence of hFis1, Opa1, or both of them together along with control, and the transfectants were selected by growing in media containing hygromycin B. (A) Total cell lysates from the hFis1 RNAi cells, Opa1 RNAi cells, or both hFis1 and Opa1 RNAi cells along with control RNAi cells were prepared, and the expression levels of hFis1 and Opa1 were analyzed by Western blotting. Actin also was analyzed as a loading control. (B) Left, mitochondria of hFis1/Opa1 RNAi cells were visualized with Mitotracker Red CMXRos and analyzed by confocal microscopy. An enlargement is shown for detailed structure of mitochondria. Right, percentage of cell population with fragmented (dotted), normal (solid), or elongated (striped) mitochondria in hFis1/Opa1 RNAi cell culture. At least 200 cells in several fields were counted in each experiment. Data represent the mean ± SD of at least three independent experiments. (C) The elongated mitochondrial morphology in hFis1-depleted cells was reversed by subsequent depletion of Opa1. Hela cells depleted of hFis1 by RNAi were selected for 5 d and transfected again with Opa1 shRNA- or a control shRNA-construct and examined 2 d later. To identify transfectants, pEYFP vector was cotransfected. Mitochondria were visualized with Mitotracker Red CMXRos and analyzed by confocal microscopy. (D and E) Control RNAi, Opa1 RNAi, and hFis1/Opa1 RNAi double RNAi cells were analyzed for mitochondrial fusion by using mito-PAGF–based fusion assay. Cells were transfected with mito-PAGFP, followed by the photoactivation of the small regions within the cells (white circles in prepanels) and confocal acquisition of z-series covering the entire thickness of the cell. (E) Changes in the fluorescence intensity within activated regions were measured over time.

    Article Snippet: We cotransfected HeLa cells with cDNA constructs encoding either hFis1 wt or an hFis1ΔC mutant lacking the membrane anchor along with the hFis1 shRNAi construct, selected transfectants with hygromycin, and examined protein expression levels and sensitivity to apoptosis.

    Techniques: Transfection, Construct, shRNA, Sequencing, Expressing, Western Blot, Confocal Microscopy, Cell Culture, Plasmid Preparation, Single Vesicle Fusion Assay, Fluorescence

    Overexpression of hFis1 wild-type (hFis1 wt) but not C terminus-truncated mutant (hFis1 ΔC) reverts apoptosis-resistant hFis1 RNAi cells. HeLa cells were transfected with control shRNA/pREP4 (a), hFis1 shRNA/pREP4 (b), hFis1 shRNA/pREP4 plus hFis1wt cDNA/pcDNA3.1 (c), or hFis1 shRNA/pREP4 plus hFis1ΔC cDNA/pcDNA3.1 (d), and transfectants were selected as described in Materials and Methods . (A) Transfectants of each group (a–d) were treated with or without STS (1 μM) for 6 h, lysed, and analyzed for hFis1 expression levels and PARP cleavage by Western blotting. Actin also was analyzed as a loading control. (B) Transfectants of each group (a–d) were treated with STS (1 μM; 6 h), and the nuclei were stained with Hoechst 33342 (1 μg/ml; 15 min at RT). Normal or apoptotic nuclei of these cells in several fields were counted under the fluorescent microscope (for UV excitation). At least 200 cells altogether in each treatment were counted and shown as a percentage of cells with apoptotic nuclei among the total cells counted. The data are plotted as the mean ± SD of at least three independent experiments. (C) Transfectants of each group (a–d) were treated with Act D (10 μM; 8 h) in the presence of zVAD-fmk (50 μM), fixed, and stained with anti-cytochrome c (mouse monoclonal, green) antibodies, and images were captured by confocal microscopy. (D) The number of cells whose cytochrome c was released was counted and plotted as a percentage of the total cells counted in each group (a–d) of transfectants that had been treated with Act D and stained with anti-cytochrome c (the same samples as shown in C). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

    doi: 10.1091/mbc.E04-04-0294

    Figure Lengend Snippet: Overexpression of hFis1 wild-type (hFis1 wt) but not C terminus-truncated mutant (hFis1 ΔC) reverts apoptosis-resistant hFis1 RNAi cells. HeLa cells were transfected with control shRNA/pREP4 (a), hFis1 shRNA/pREP4 (b), hFis1 shRNA/pREP4 plus hFis1wt cDNA/pcDNA3.1 (c), or hFis1 shRNA/pREP4 plus hFis1ΔC cDNA/pcDNA3.1 (d), and transfectants were selected as described in Materials and Methods . (A) Transfectants of each group (a–d) were treated with or without STS (1 μM) for 6 h, lysed, and analyzed for hFis1 expression levels and PARP cleavage by Western blotting. Actin also was analyzed as a loading control. (B) Transfectants of each group (a–d) were treated with STS (1 μM; 6 h), and the nuclei were stained with Hoechst 33342 (1 μg/ml; 15 min at RT). Normal or apoptotic nuclei of these cells in several fields were counted under the fluorescent microscope (for UV excitation). At least 200 cells altogether in each treatment were counted and shown as a percentage of cells with apoptotic nuclei among the total cells counted. The data are plotted as the mean ± SD of at least three independent experiments. (C) Transfectants of each group (a–d) were treated with Act D (10 μM; 8 h) in the presence of zVAD-fmk (50 μM), fixed, and stained with anti-cytochrome c (mouse monoclonal, green) antibodies, and images were captured by confocal microscopy. (D) The number of cells whose cytochrome c was released was counted and plotted as a percentage of the total cells counted in each group (a–d) of transfectants that had been treated with Act D and stained with anti-cytochrome c (the same samples as shown in C). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments.

    Article Snippet: We cotransfected HeLa cells with cDNA constructs encoding either hFis1 wt or an hFis1ΔC mutant lacking the membrane anchor along with the hFis1 shRNAi construct, selected transfectants with hygromycin, and examined protein expression levels and sensitivity to apoptosis.

    Techniques: Over Expression, Mutagenesis, Transfection, shRNA, Expressing, Western Blot, Staining, Microscopy, Activated Clotting Time Assay, Confocal Microscopy

    hFis1 depletion produces greater resistance to apoptosis than depletion of Drp1. (A) HeLa cells depleted of hFis1 or Drp1 by RNAi along with control RNAi cells were treated with STS (1 μM; 6 h), Act D (10 μM; 8 h), etoposide (100 μM; 30 h), or anti-Fas antibody (500 ng/ml, 15 h), and the nuclei were stained with Hoechst 33342 (1 μg/ml; 15 min at RT). Normal or apoptotic nuclei of these cells in several fields were counted under the fluorescent microscope (for UV excitation). At least 200 cells altogether in each treatment were counted and shown as a percentage of cells with apoptotic nuclei among total cells counted. The data are plotted as the mean ± SD of at least three independent experiments. (B) PARP cleavage was analyzed in the total extracts from HeLa cells depleted of hFis1, Drp1, and control, which were treated with STS (1 μM; 0, 3, 6 h), Act D (10 μM; 0, 4, 8 h), or anti-Fas (500 ng/ml; 0, 6, 15 h). The figure is a representative of at least three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

    doi: 10.1091/mbc.E04-04-0294

    Figure Lengend Snippet: hFis1 depletion produces greater resistance to apoptosis than depletion of Drp1. (A) HeLa cells depleted of hFis1 or Drp1 by RNAi along with control RNAi cells were treated with STS (1 μM; 6 h), Act D (10 μM; 8 h), etoposide (100 μM; 30 h), or anti-Fas antibody (500 ng/ml, 15 h), and the nuclei were stained with Hoechst 33342 (1 μg/ml; 15 min at RT). Normal or apoptotic nuclei of these cells in several fields were counted under the fluorescent microscope (for UV excitation). At least 200 cells altogether in each treatment were counted and shown as a percentage of cells with apoptotic nuclei among total cells counted. The data are plotted as the mean ± SD of at least three independent experiments. (B) PARP cleavage was analyzed in the total extracts from HeLa cells depleted of hFis1, Drp1, and control, which were treated with STS (1 μM; 0, 3, 6 h), Act D (10 μM; 0, 4, 8 h), or anti-Fas (500 ng/ml; 0, 6, 15 h). The figure is a representative of at least three independent experiments.

    Article Snippet: We cotransfected HeLa cells with cDNA constructs encoding either hFis1 wt or an hFis1ΔC mutant lacking the membrane anchor along with the hFis1 shRNAi construct, selected transfectants with hygromycin, and examined protein expression levels and sensitivity to apoptosis.

    Techniques: Activated Clotting Time Assay, Staining, Microscopy

    Both Bax translocation and cytochrome c release induced by actinomycin D are inhibited in hFis1-depleted HeLa cells, whereas cytochrome c release, but not Bax translocation, is inhibited in Drp1-depleted cells. (A) HeLa cells depleted of hFis1 or Drp1 by RNAi along with control RNAi cells were treated with Act D (10 μM; 8 h) in the presence of zVAD-fmk (50 μM), fixed, and double stained with anti-Bax (rabbit polyclonal, red) and anti-cytochrome c (mouse monoclonal, green) antibodies. (B) The number of cells displaying Bax translocation or cytochrome c release was counted and plotted as a percentage of total cells counted in each RNAi cell population that had been treated with Act D and stained with anti-Bax and anti-cytochrome c (the same samples as shown in A). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments. (C) A unique group of cells (Bax had translocated but cytochrome c was not released) was seen in Drp1 RNAi cells. Four groups of cells were counted in each RNAi cell population that had been treated with Act D (the same samples as in A): i) Bax did not translocate and cytochrome c was not released; ii) Bax translocated, but cytochrome c is not released; iii) Bax not translocated, but cytochrome c is released; and iv) Bax translocated and cytochrome c is released were plotted as a percentage of total cells counted in each RNAi cell population. There were no cells categorized as iii, so only i, ii, and iv were plotted. Bars are labeled solid or striped, as in B. Data were plotted as the mean ± SD of at least three independent experiments. (D) HeLa cells depleted of Drp1 or hFis1 along with control RNAi were incubated with or without Act D (10 μM) for 8 h, fractionated into S and HM (mainly mitochondria), and analyzed for the distribution of Bax and cytochrome c by Western blotting. Fractionation quality was verified by the distribution of specific subcellular markers: CoxIV for mitochondria and actin for cytosol. To confirm the depletion of proteins (hFis1 and Drp1) and their localizations, hFis1 and Drp1 also were examined. The figure shown is representative of three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

    doi: 10.1091/mbc.E04-04-0294

    Figure Lengend Snippet: Both Bax translocation and cytochrome c release induced by actinomycin D are inhibited in hFis1-depleted HeLa cells, whereas cytochrome c release, but not Bax translocation, is inhibited in Drp1-depleted cells. (A) HeLa cells depleted of hFis1 or Drp1 by RNAi along with control RNAi cells were treated with Act D (10 μM; 8 h) in the presence of zVAD-fmk (50 μM), fixed, and double stained with anti-Bax (rabbit polyclonal, red) and anti-cytochrome c (mouse monoclonal, green) antibodies. (B) The number of cells displaying Bax translocation or cytochrome c release was counted and plotted as a percentage of total cells counted in each RNAi cell population that had been treated with Act D and stained with anti-Bax and anti-cytochrome c (the same samples as shown in A). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments. (C) A unique group of cells (Bax had translocated but cytochrome c was not released) was seen in Drp1 RNAi cells. Four groups of cells were counted in each RNAi cell population that had been treated with Act D (the same samples as in A): i) Bax did not translocate and cytochrome c was not released; ii) Bax translocated, but cytochrome c is not released; iii) Bax not translocated, but cytochrome c is released; and iv) Bax translocated and cytochrome c is released were plotted as a percentage of total cells counted in each RNAi cell population. There were no cells categorized as iii, so only i, ii, and iv were plotted. Bars are labeled solid or striped, as in B. Data were plotted as the mean ± SD of at least three independent experiments. (D) HeLa cells depleted of Drp1 or hFis1 along with control RNAi were incubated with or without Act D (10 μM) for 8 h, fractionated into S and HM (mainly mitochondria), and analyzed for the distribution of Bax and cytochrome c by Western blotting. Fractionation quality was verified by the distribution of specific subcellular markers: CoxIV for mitochondria and actin for cytosol. To confirm the depletion of proteins (hFis1 and Drp1) and their localizations, hFis1 and Drp1 also were examined. The figure shown is representative of three independent experiments.

    Article Snippet: We cotransfected HeLa cells with cDNA constructs encoding either hFis1 wt or an hFis1ΔC mutant lacking the membrane anchor along with the hFis1 shRNAi construct, selected transfectants with hygromycin, and examined protein expression levels and sensitivity to apoptosis.

    Techniques: Translocation Assay, Activated Clotting Time Assay, Staining, Labeling, Incubation, Western Blot, Fractionation

    Knockdown of hFis1 or Drp1 expression induces mitochondrial fusion. HeLa cells were transfected with pREP4 constructs containing shRNA of the target sequence of hFis1, Drp1, or control, and the transfectants were selected by growth in media containing hygromycin B. (A) Total cell lysates from the hFis1 RNAi cells, Drp1 RNAi cells, and control RNAi cells were prepared, and the expression levels of hFis1 and Drp1 were analyzed by Western blotting. Actin level also was analyzed for a loading control. (B) Mitochondria of control RNAi cells, hFis1 RNAi cells, or Drp1 RNAi cells were visualized with Mitotracker Red CMXRos and analyzed by confocal microscopy. Enlargements are shown for detailed structure of mitochondria. An arrow shows a typical balloon-like structure in Drp1 RNAi cells. (C) Percentage of cell population with fragmented (dotted), normal (solid), or elongated (striped) mitochondria in control RNAi, hFis1 RNAi, or Drp1 RNAi culture. At least 200 cells in several fields were counted in each experiment. Data represent the mean ± SD of at least three independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

    doi: 10.1091/mbc.E04-04-0294

    Figure Lengend Snippet: Knockdown of hFis1 or Drp1 expression induces mitochondrial fusion. HeLa cells were transfected with pREP4 constructs containing shRNA of the target sequence of hFis1, Drp1, or control, and the transfectants were selected by growth in media containing hygromycin B. (A) Total cell lysates from the hFis1 RNAi cells, Drp1 RNAi cells, and control RNAi cells were prepared, and the expression levels of hFis1 and Drp1 were analyzed by Western blotting. Actin level also was analyzed for a loading control. (B) Mitochondria of control RNAi cells, hFis1 RNAi cells, or Drp1 RNAi cells were visualized with Mitotracker Red CMXRos and analyzed by confocal microscopy. Enlargements are shown for detailed structure of mitochondria. An arrow shows a typical balloon-like structure in Drp1 RNAi cells. (C) Percentage of cell population with fragmented (dotted), normal (solid), or elongated (striped) mitochondria in control RNAi, hFis1 RNAi, or Drp1 RNAi culture. At least 200 cells in several fields were counted in each experiment. Data represent the mean ± SD of at least three independent experiments.

    Article Snippet: We cotransfected HeLa cells with cDNA constructs encoding either hFis1 wt or an hFis1ΔC mutant lacking the membrane anchor along with the hFis1 shRNAi construct, selected transfectants with hygromycin, and examined protein expression levels and sensitivity to apoptosis.

    Techniques: Expressing, Transfection, Construct, shRNA, Sequencing, Western Blot, Confocal Microscopy

    miR-7 post-transcriptionally downregulates PA28gamma expression by directly targeting its 3′-UTR. ( A ) Putative target genes were predicted by PicTar, Target Scan, and miRanda. Each circle represents one prediction algorithm with the number of predicted genes. The number listed in overlapping areas of the circles was simultaneously predicted by different algorithms. ( B ) The potential binding sequences within the 3′-UTR of PA28gamma for miR-7 is conserved in human (Hsa), chimpanzee (Ptr), rhesus (Mml), mouse (Mmu), rat, hedgehog (Eeu), shrew (Sar), horse (Eca), cow (Eta), and elephant (Dno). Seed sequences are highlighted. ( C ) The PA28gamma 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in the pGL3-con vector. The sequences of the predicted miR-7 binding sites within the PA28gamma 3′-UTR including the wild-type UTR or mutant UTR segments (dotted lines) are shown. ( D ) After co-transfection of miR-7 mimic or miR-NC with the above reporter plasmids and pRL-TK vector in HEK 293T cells, relative luciferase activity was analysed by Dual-Luciferase Reporter Assay. ( E ) A549 and H1299 cells were transfected with miR-7, miR-NC, PA28gamma -siRNA, and miR-7 inhibitor for 48 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). ( F ) H1299 cells were transfected with PA28gamma -siRNA, and/or miR-7 inhibitor for 72 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). These experiments were performed in triplicate and results are shown as the mean±s.e.m. (*** P

    Journal: British Journal of Cancer

    Article Title: PA28gamma emerges as a novel functional target of tumour suppressor microRNA-7 in non-small-cell lung cancer

    doi: 10.1038/bjc.2013.728

    Figure Lengend Snippet: miR-7 post-transcriptionally downregulates PA28gamma expression by directly targeting its 3′-UTR. ( A ) Putative target genes were predicted by PicTar, Target Scan, and miRanda. Each circle represents one prediction algorithm with the number of predicted genes. The number listed in overlapping areas of the circles was simultaneously predicted by different algorithms. ( B ) The potential binding sequences within the 3′-UTR of PA28gamma for miR-7 is conserved in human (Hsa), chimpanzee (Ptr), rhesus (Mml), mouse (Mmu), rat, hedgehog (Eeu), shrew (Sar), horse (Eca), cow (Eta), and elephant (Dno). Seed sequences are highlighted. ( C ) The PA28gamma 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in the pGL3-con vector. The sequences of the predicted miR-7 binding sites within the PA28gamma 3′-UTR including the wild-type UTR or mutant UTR segments (dotted lines) are shown. ( D ) After co-transfection of miR-7 mimic or miR-NC with the above reporter plasmids and pRL-TK vector in HEK 293T cells, relative luciferase activity was analysed by Dual-Luciferase Reporter Assay. ( E ) A549 and H1299 cells were transfected with miR-7, miR-NC, PA28gamma -siRNA, and miR-7 inhibitor for 48 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). ( F ) H1299 cells were transfected with PA28gamma -siRNA, and/or miR-7 inhibitor for 72 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). These experiments were performed in triplicate and results are shown as the mean±s.e.m. (*** P

    Article Snippet: Vector constructs To construct the wild-type pGL3-con-PA28gamma -UTR-WT plasmid, a wild-type 3′-UTR fragment of the human PA28gamma mRNA (Genbank accession no. NM_005789) containing the putative miR-7 binding sequence (bases 1413–1419) was amplified by PCR and cloned into the XbaI/FseI site of the pGL3-control vector (Promega, Madison, WI, USA), which is downstream of the luciferase reporter gene.

    Techniques: Expressing, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Cotransfection, Activity Assay, Reporter Assay, Transfection, Western Blot

    TDP2 Excises Cellular TOP3Bccs both from DNA and RNA (A-D) DNA RNA TOP3Bccs detection by RADAR assays. (A) HEK293 cells were transfected with the indicated siRNA: siTDP1, siTDP2 or both siTDP1 and siTDP2 and co-transfected with R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and detected with anti-FLAG antibody. The figure is representative of two independent experiments. (B) Quantitation of TOP3Bcc of RADAR assays as shown in panel A. TOP3Bccs were measured by densitometric analyses of slot-blot signals and plotted as a function of total nucleic acid (DNA and RNA) concentration. Two independent experiments are plotted. (C) Wild-type or TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay and slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (D) Quantitation of TOP3Bcc formation in RADAR assays as shown in panel C. Three independent experiments are plotted. (E-H) DNA RNA TOP3Bccs detection by ICE assays. (E) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, ICE bioassay was performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (F) Quantitation of TOP3Bcc in the DNA and RNA fractions as shown in panel E. Three independent experiments are plotted. (G) HCT116 WT and TDP2KO cells were transfected either with wild-type TOP3B or R338W TOP3B or a combination of R338W TOP3B and siTDP2 constructs as indicated. After 72 h, ICE bioassays were performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (H) Quantitation of TOP3Bcc formation in ICE assays as shown in panel G. Three independent experiments are plotted. (I-L) Detection of RNA TOP3Bccs. (I) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (J) Quantitation of TOP3Bcc in RNA as shown in panel I. Three independent experiments are plotted. (K) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (L) Quantitation of RNA TOP3Bcc in RNA as shown in panel K. Three independent experiments are plotted. (M-N) Ectopic expression of TDP2 reduces TOP3Bccs. (M) Wild-type (WT) and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (N) Quantitation of TOP3Bcc formation using the RADAR assays as shown in panel M. Three independent experiments are plotted.

    Journal: bioRxiv

    Article Title: Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks

    doi: 10.1101/2020.03.22.002691

    Figure Lengend Snippet: TDP2 Excises Cellular TOP3Bccs both from DNA and RNA (A-D) DNA RNA TOP3Bccs detection by RADAR assays. (A) HEK293 cells were transfected with the indicated siRNA: siTDP1, siTDP2 or both siTDP1 and siTDP2 and co-transfected with R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and detected with anti-FLAG antibody. The figure is representative of two independent experiments. (B) Quantitation of TOP3Bcc of RADAR assays as shown in panel A. TOP3Bccs were measured by densitometric analyses of slot-blot signals and plotted as a function of total nucleic acid (DNA and RNA) concentration. Two independent experiments are plotted. (C) Wild-type or TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay and slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (D) Quantitation of TOP3Bcc formation in RADAR assays as shown in panel C. Three independent experiments are plotted. (E-H) DNA RNA TOP3Bccs detection by ICE assays. (E) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, ICE bioassay was performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (F) Quantitation of TOP3Bcc in the DNA and RNA fractions as shown in panel E. Three independent experiments are plotted. (G) HCT116 WT and TDP2KO cells were transfected either with wild-type TOP3B or R338W TOP3B or a combination of R338W TOP3B and siTDP2 constructs as indicated. After 72 h, ICE bioassays were performed to separate DNA and RNA from free proteins. DNA and RNA fractions were slot-blotted and TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (H) Quantitation of TOP3Bcc formation in ICE assays as shown in panel G. Three independent experiments are plotted. (I-L) Detection of RNA TOP3Bccs. (I) HEK293 cells were transfected with R338W TOP3B alone or co-transfected with siTDP2. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (J) Quantitation of TOP3Bcc in RNA as shown in panel I. Three independent experiments are plotted. (K) Wild-type and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B. After 72 h, covalent protein-RNA adducts were isolated using TRIzol ® , slot-blotted and TOP3Bccs were detected using anti-FLAG Antibody. The figure is representative of three independent experiments. (L) Quantitation of RNA TOP3Bcc in RNA as shown in panel K. Three independent experiments are plotted. (M-N) Ectopic expression of TDP2 reduces TOP3Bccs. (M) Wild-type (WT) and TDP2KO HCT116 cells were transfected with FLAG-tagged R338W TOP3B alone or co-transfected with HA-tagged TDP2. After 72 h, nucleic acids and protein-nucleic acid adducts were isolated by RADAR assay, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (N) Quantitation of TOP3Bcc formation using the RADAR assays as shown in panel M. Three independent experiments are plotted.

    Article Snippet: Plasmids were transfected in HCT116 and HEK293 cells using Lipofectamine 3000 Reagent (CAT#: L3000015, ThermoFisher Scientific) according to the manufacturer’s protocol for 48–72 h.

    Techniques: Transfection, Isolation, Quantitation Assay, Dot Blot, Concentration Assay, Construct, Expressing

    Recombinant Human TDP2 Only Unhooks Denatured but Not Native TOP3B from TOP3Bcc (A) Recombinant human TDP2 does not excise native TOP3Bccs. A 69-mer single-stranded DNA oligonucleotide was designed to study the processing of suicidal TOP3Bccs by TDP2. The oligonucleotide (300 nM)was incubated with purified recombinant human TOP3B (4 uM). Irreversible (suicidal) TOP3Bccs result in slower migrating DNA (Lane 2). Positions of free TOP3B and TOP3Bccs are indicated. Suicidal TOP3Bccs were incubated with increasing concentration (1 or 3 μM) of recombinant TDP1 (Lanes 7 and 8) or TDP2 (1 or 3 μM, Lanes 5 and 6). Benzonase (3 or 9 Units, Lanes 3 and 4) was used as positive control for degradation of the oligonucleotide and release of TOP3B. Samples were resolved by SDS-PAGE and immunoblotted with anti-TOP3B antibody. (B-C) Recombinant human TDP2 excises denatured cellular DNA and RNA TOP3Bccs. (B) HEK293 cells were transfected with FLAG-tagged R338W TOP3B and nucleic acids and protein-nucleic acid adducts were recovered by RADAR assay. After incubation with increasing concentrations of recombinant TDP2 (1, 2, 3 and 6 μM, lanes 4-7), micrococcal nuclease (MNase, 300 Units/reaction, lane 3) or benzonase (250 Units/reaction, lane 2), reaction mixtures were analyzed by immunoblotting with anti-FLAG antibody after SDS-PAGE. Benzonase and MNase were used as controls for complete degradation of DNA and RNA in TOP3Bccs. (C) HEK293 cells were transfected with R338W TOP3B. Covalent protein-RNA adducts were isolated 72 hs later using TRIzol® reagent. After incubation with increasing concentrations of recombinant TDP2 (1, 2, 3 and 6 μM, lanes 2-5), excess amount of RNase A (200 μg/mL) and RNase T1 (200 Units/ml) mix (Lane 6) or benzonase (250 Units/ reaction, lane 7), reaction mixtures were analyzed by immunoblotting with anti-FLAG antibody after SDS-PAGE. Benzonase and RNase A and RNase T1 mix were used as controls for complete degradation of RNA covalently attached to TOP3Bccs.

    Journal: bioRxiv

    Article Title: Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks

    doi: 10.1101/2020.03.22.002691

    Figure Lengend Snippet: Recombinant Human TDP2 Only Unhooks Denatured but Not Native TOP3B from TOP3Bcc (A) Recombinant human TDP2 does not excise native TOP3Bccs. A 69-mer single-stranded DNA oligonucleotide was designed to study the processing of suicidal TOP3Bccs by TDP2. The oligonucleotide (300 nM)was incubated with purified recombinant human TOP3B (4 uM). Irreversible (suicidal) TOP3Bccs result in slower migrating DNA (Lane 2). Positions of free TOP3B and TOP3Bccs are indicated. Suicidal TOP3Bccs were incubated with increasing concentration (1 or 3 μM) of recombinant TDP1 (Lanes 7 and 8) or TDP2 (1 or 3 μM, Lanes 5 and 6). Benzonase (3 or 9 Units, Lanes 3 and 4) was used as positive control for degradation of the oligonucleotide and release of TOP3B. Samples were resolved by SDS-PAGE and immunoblotted with anti-TOP3B antibody. (B-C) Recombinant human TDP2 excises denatured cellular DNA and RNA TOP3Bccs. (B) HEK293 cells were transfected with FLAG-tagged R338W TOP3B and nucleic acids and protein-nucleic acid adducts were recovered by RADAR assay. After incubation with increasing concentrations of recombinant TDP2 (1, 2, 3 and 6 μM, lanes 4-7), micrococcal nuclease (MNase, 300 Units/reaction, lane 3) or benzonase (250 Units/reaction, lane 2), reaction mixtures were analyzed by immunoblotting with anti-FLAG antibody after SDS-PAGE. Benzonase and MNase were used as controls for complete degradation of DNA and RNA in TOP3Bccs. (C) HEK293 cells were transfected with R338W TOP3B. Covalent protein-RNA adducts were isolated 72 hs later using TRIzol® reagent. After incubation with increasing concentrations of recombinant TDP2 (1, 2, 3 and 6 μM, lanes 2-5), excess amount of RNase A (200 μg/mL) and RNase T1 (200 Units/ml) mix (Lane 6) or benzonase (250 Units/ reaction, lane 7), reaction mixtures were analyzed by immunoblotting with anti-FLAG antibody after SDS-PAGE. Benzonase and RNase A and RNase T1 mix were used as controls for complete degradation of RNA covalently attached to TOP3Bccs.

    Article Snippet: Plasmids were transfected in HCT116 and HEK293 cells using Lipofectamine 3000 Reagent (CAT#: L3000015, ThermoFisher Scientific) according to the manufacturer’s protocol for 48–72 h.

    Techniques: Recombinant, Incubation, Purification, Concentration Assay, Positive Control, SDS Page, Transfection, Isolation

    Cellular TOP3Bccs are Ubiquitylated and Degraded by the Proteasomal Pathway (A-D) Proteasome inhibition enhances cellular TOP3Bccs. HEK293 (A) and HCT116 cells (B) were transfected with FLAG-tagged wild-type TOP3B and R338W TOP3B for 72 h. Before harvest, cells were treated with MG132 (10 µM, 2 h). TOP3Bccs were detected by RADAR assays using anti-FLAG antibody. The figure is representative of two independent experiments. (C-D) Quantitation of TOP3Bccs in two independent RADAR assays as shown in panels A and B. Two independent experiments are plotted for each panels. (E-H) Ubiquitylation inhibition enhances cellular TOP3Bccs. HEK293 (E) and HCT116 cells (G) were transfected with FLAG-tagged R338W TOP3B for 72 h. Before harvesting, the cells were treated with the UAE inhibitor TAK-243 (10 µM, 2 h). TOP3Bccs were detected by RADAR assays using anti-FLAG antibody. The figure is representative of three independent experiments. (F H) Quantitation of RADAR assays as shown in panels E and G. Three independent experiments are plotted for each panels. (I) Scheme of the DUST (Detection of Ubiquitylated-SUMOylated TOPccs) assay. Following the isolation of TOP3Bccs by RADAR assay, the covalently attached nucleic acids are digested with micrococcal nuclease (MNase). Ubiquitylated TOP3B can then be detected by immunoblotting following SDS-PAGE. (J) Ubiquitylation of TOP3Bccs in HCT116 cells transfected with FLAG-tagged R338W TOP3B for 72 h, as detected by the DUST Assay. Equal loading was tested by slot-blotting and probing with anti-dsDNA antibody. (K) TOP3Bcc ubiquitylation involves the classical proteasomal-specific linkages to lysines K11, K27, K48 and K63. HCT116 cells were co-transfected with FLAG-tagged R338W TOP3B plasmid construct and HA-tagged wild-type or mutant ubiquitin constructs. DUST assays were performed after 72 h. (L) Inhibition of TOP3Bcc ubiquitylation by the UBE1 inhibitor TAK-243 and enhancement by the proteasome inhibitor MG132. HCT116 cells transfected with FLAG-tagged R338W TOP3B for 72 h were treated with either MG132 (10 µM, 2 h) or TAK-243 (10 µM, 2 h), as detected by the DUST Assay.

    Journal: bioRxiv

    Article Title: Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks

    doi: 10.1101/2020.03.22.002691

    Figure Lengend Snippet: Cellular TOP3Bccs are Ubiquitylated and Degraded by the Proteasomal Pathway (A-D) Proteasome inhibition enhances cellular TOP3Bccs. HEK293 (A) and HCT116 cells (B) were transfected with FLAG-tagged wild-type TOP3B and R338W TOP3B for 72 h. Before harvest, cells were treated with MG132 (10 µM, 2 h). TOP3Bccs were detected by RADAR assays using anti-FLAG antibody. The figure is representative of two independent experiments. (C-D) Quantitation of TOP3Bccs in two independent RADAR assays as shown in panels A and B. Two independent experiments are plotted for each panels. (E-H) Ubiquitylation inhibition enhances cellular TOP3Bccs. HEK293 (E) and HCT116 cells (G) were transfected with FLAG-tagged R338W TOP3B for 72 h. Before harvesting, the cells were treated with the UAE inhibitor TAK-243 (10 µM, 2 h). TOP3Bccs were detected by RADAR assays using anti-FLAG antibody. The figure is representative of three independent experiments. (F H) Quantitation of RADAR assays as shown in panels E and G. Three independent experiments are plotted for each panels. (I) Scheme of the DUST (Detection of Ubiquitylated-SUMOylated TOPccs) assay. Following the isolation of TOP3Bccs by RADAR assay, the covalently attached nucleic acids are digested with micrococcal nuclease (MNase). Ubiquitylated TOP3B can then be detected by immunoblotting following SDS-PAGE. (J) Ubiquitylation of TOP3Bccs in HCT116 cells transfected with FLAG-tagged R338W TOP3B for 72 h, as detected by the DUST Assay. Equal loading was tested by slot-blotting and probing with anti-dsDNA antibody. (K) TOP3Bcc ubiquitylation involves the classical proteasomal-specific linkages to lysines K11, K27, K48 and K63. HCT116 cells were co-transfected with FLAG-tagged R338W TOP3B plasmid construct and HA-tagged wild-type or mutant ubiquitin constructs. DUST assays were performed after 72 h. (L) Inhibition of TOP3Bcc ubiquitylation by the UBE1 inhibitor TAK-243 and enhancement by the proteasome inhibitor MG132. HCT116 cells transfected with FLAG-tagged R338W TOP3B for 72 h were treated with either MG132 (10 µM, 2 h) or TAK-243 (10 µM, 2 h), as detected by the DUST Assay.

    Article Snippet: Plasmids were transfected in HCT116 and HEK293 cells using Lipofectamine 3000 Reagent (CAT#: L3000015, ThermoFisher Scientific) according to the manufacturer’s protocol for 48–72 h.

    Techniques: Inhibition, Transfection, Quantitation Assay, Isolation, SDS Page, Plasmid Preparation, Construct, Mutagenesis

    TOP3B Forms TOP3Bccs both with DNA and RNA in cells Transfected with R338W TOP3B (A) Alignment of the active site regions of E. coli Topo I and Y. pestis Topo I with corresponding region of Human TOP3B. (B) Schematic representation of the domain organization of human TOP3B (1-862 aa) (top) and ribbon representation of human TOP3B (residues 1-612 aa) based on X-ray crystal structure by ( Goto-Ito et al., 2017 )(bottom). Positions of the active site residue (Tyrosine 336) and the self-trapping mutation site (Arginine 338) are indicated. (C) Western Blots showing ectopic expression of wild-type, P337V and R338W TOP3B. HEK293 and HCT116 cells were transfected with the indicated FLAG-tagged TOP3B constructs for 72 h and subjected to Western blotting with anti-FLAG antibody. (D)-(E) Detection of TOP3Bccs by RADAR assay in HEK293 and HCT116 cells transfected with FLAG-tagged wild-type, P337V or R338W TOP3B plasmid constructs for 72 h. Cells were lysed with DNAzol, nucleic acids and protein-nucleic acid adducts were isolated, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (F) Scheme of the modified RADAR assay. Cells transfected with FLAG-tagged TOP3B (blue Circle) form TOP3Bccs in DNA (red) and RNA (green). Nucleic acids containing TOP3Bccs were isolated using the RADAR assay and digested with micrococcal nuclease (MNase) followed by SDS-PAGE and immunoblotting with anti-FLAG antibody. (G) Modified RADAR assay in HEK293 cells transfected with wild-type or R338W TOP3B for 72 h. TOP3B was detected with anti-FLAG antibody. (H) Detection of TOP3Bccs both in DNA and RNA of HEK293 cells transfected for 72 h with wild-type or R338W TOP3B constructs. Equal numbers of cells were lysed in 1% sarkosyl and ICE (In vivo complex of enzymes) bioassay by cesium chloride gradient ultracentrifugation was performed to separate DNA (middle of the gradient) and RNA (bottom of the gradient) from free proteins (top of the gradient). DNA and RNA fractions were then slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (I) Detection of RNA TOP3Bccs. HEK293 cells were transfected with wild-type or R338W TOP3B. 72 h later, covalent protein-RNA adducts were isolated using TRIzol® (Thermo Scientific), slot-blotted and TOP3Bccs were detected with anti-FLAG Antibody. The figure represents three independent experiments.

    Journal: bioRxiv

    Article Title: Topoisomerase 3B (TOP3B) DNA and RNA Cleavage Complexes and Pathway to Repair TOP3B-linked RNA and DNA Breaks

    doi: 10.1101/2020.03.22.002691

    Figure Lengend Snippet: TOP3B Forms TOP3Bccs both with DNA and RNA in cells Transfected with R338W TOP3B (A) Alignment of the active site regions of E. coli Topo I and Y. pestis Topo I with corresponding region of Human TOP3B. (B) Schematic representation of the domain organization of human TOP3B (1-862 aa) (top) and ribbon representation of human TOP3B (residues 1-612 aa) based on X-ray crystal structure by ( Goto-Ito et al., 2017 )(bottom). Positions of the active site residue (Tyrosine 336) and the self-trapping mutation site (Arginine 338) are indicated. (C) Western Blots showing ectopic expression of wild-type, P337V and R338W TOP3B. HEK293 and HCT116 cells were transfected with the indicated FLAG-tagged TOP3B constructs for 72 h and subjected to Western blotting with anti-FLAG antibody. (D)-(E) Detection of TOP3Bccs by RADAR assay in HEK293 and HCT116 cells transfected with FLAG-tagged wild-type, P337V or R338W TOP3B plasmid constructs for 72 h. Cells were lysed with DNAzol, nucleic acids and protein-nucleic acid adducts were isolated, slot-blotted and TOP3Bccs were detected with anti-FLAG antibody. The figure is representative of three independent experiments. (F) Scheme of the modified RADAR assay. Cells transfected with FLAG-tagged TOP3B (blue Circle) form TOP3Bccs in DNA (red) and RNA (green). Nucleic acids containing TOP3Bccs were isolated using the RADAR assay and digested with micrococcal nuclease (MNase) followed by SDS-PAGE and immunoblotting with anti-FLAG antibody. (G) Modified RADAR assay in HEK293 cells transfected with wild-type or R338W TOP3B for 72 h. TOP3B was detected with anti-FLAG antibody. (H) Detection of TOP3Bccs both in DNA and RNA of HEK293 cells transfected for 72 h with wild-type or R338W TOP3B constructs. Equal numbers of cells were lysed in 1% sarkosyl and ICE (In vivo complex of enzymes) bioassay by cesium chloride gradient ultracentrifugation was performed to separate DNA (middle of the gradient) and RNA (bottom of the gradient) from free proteins (top of the gradient). DNA and RNA fractions were then slot-blotted. TOP3Bccs were detected using anti-FLAG antibody. The figure is representative of three independent experiments. (I) Detection of RNA TOP3Bccs. HEK293 cells were transfected with wild-type or R338W TOP3B. 72 h later, covalent protein-RNA adducts were isolated using TRIzol® (Thermo Scientific), slot-blotted and TOP3Bccs were detected with anti-FLAG Antibody. The figure represents three independent experiments.

    Article Snippet: Plasmids were transfected in HCT116 and HEK293 cells using Lipofectamine 3000 Reagent (CAT#: L3000015, ThermoFisher Scientific) according to the manufacturer’s protocol for 48–72 h.

    Techniques: Transfection, Mutagenesis, Western Blot, Expressing, Construct, Plasmid Preparation, Isolation, Modification, SDS Page, In Vivo

    T835M-UNC5C-induced cell death is mediated by ASK1 and JNK. A and B, F11 cells were co-transfected with the empty pHA vector ( vector ) or pRK5-T835M-UNC5C together with the pcDNA3 vector ( vector ), pcDNA3-dominant-negative ASK1 ( dnASK1 ), or pcDNA3-dominant-negative JNK ( dnJNK ). At 72 h, the cells were harvested for trypan blue exclusion assays ( A ). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA ( B ). ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: An Alzheimer Disease-linked Rare Mutation Potentiates Netrin Receptor Uncoordinated-5C-induced Signaling That Merges with Amyloid β Precursor Protein Signaling *

    doi: 10.1074/jbc.M115.698092

    Figure Lengend Snippet: T835M-UNC5C-induced cell death is mediated by ASK1 and JNK. A and B, F11 cells were co-transfected with the empty pHA vector ( vector ) or pRK5-T835M-UNC5C together with the pcDNA3 vector ( vector ), pcDNA3-dominant-negative ASK1 ( dnASK1 ), or pcDNA3-dominant-negative JNK ( dnJNK ). At 72 h, the cells were harvested for trypan blue exclusion assays ( A ). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA ( B ). ***, p

    Article Snippet: HA-tagged human WT-PKD (ID: 10808), constitutively active (S738E/S742E) PKD (ID: 10810), and kinase-dead (K612W) PKD (ID: 10809) in the pcDNA3 vector (Invitrogen) were purchased from Addgene (Tokyo, Japan).

    Techniques: Transfection, Plasmid Preparation, Dominant Negative Mutation, SDS Page

    Constitutively active PKD induces cell death, mediated by ASK1 and JNK. A and B, F11 cells were co-transfected with the empty pHA vector ( vector ) or pcDNA3-constitutively active PKD ( caPKD ) together with the pcDNA3 vector ( vector2 ), pcDNA3-dominant-negative ASK1 ( dnASK1 ), or pcDNA3-dominant-negative JNK ( dnJNK ). At 72 h, the cells were harvested for trypan blue exclusion assays ( A ). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a PKD antibody or a monoclonal antibody against HA ( B ). C, F11 cells were co-transfected with pcDNA3-FLAG-JNK1a1 and the empty pHA vector ( vector ), pRK5-T835M-UNC5C, or pHA-V642I-APP. At 48 h after transfection, the cells were harvested for the preparation of cell lysates. FLAG-JNK1a1 in the lysates was immunoprecipitated with a FLAG antibody (M2) and used for in vitro kinase assays with c-Jun-derived peptide as a substrate. Whole mixtures were subjected to SDS-PAGE and immunoblot analysis with an HA antibody, phospho-c-Jun antibody, phospho-JNK antibody, or FLAG antibody (M2). Similar results were obtained in three independent experiments. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: An Alzheimer Disease-linked Rare Mutation Potentiates Netrin Receptor Uncoordinated-5C-induced Signaling That Merges with Amyloid β Precursor Protein Signaling *

    doi: 10.1074/jbc.M115.698092

    Figure Lengend Snippet: Constitutively active PKD induces cell death, mediated by ASK1 and JNK. A and B, F11 cells were co-transfected with the empty pHA vector ( vector ) or pcDNA3-constitutively active PKD ( caPKD ) together with the pcDNA3 vector ( vector2 ), pcDNA3-dominant-negative ASK1 ( dnASK1 ), or pcDNA3-dominant-negative JNK ( dnJNK ). At 72 h, the cells were harvested for trypan blue exclusion assays ( A ). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a PKD antibody or a monoclonal antibody against HA ( B ). C, F11 cells were co-transfected with pcDNA3-FLAG-JNK1a1 and the empty pHA vector ( vector ), pRK5-T835M-UNC5C, or pHA-V642I-APP. At 48 h after transfection, the cells were harvested for the preparation of cell lysates. FLAG-JNK1a1 in the lysates was immunoprecipitated with a FLAG antibody (M2) and used for in vitro kinase assays with c-Jun-derived peptide as a substrate. Whole mixtures were subjected to SDS-PAGE and immunoblot analysis with an HA antibody, phospho-c-Jun antibody, phospho-JNK antibody, or FLAG antibody (M2). Similar results were obtained in three independent experiments. ***, p

    Article Snippet: HA-tagged human WT-PKD (ID: 10808), constitutively active (S738E/S742E) PKD (ID: 10810), and kinase-dead (K612W) PKD (ID: 10809) in the pcDNA3 vector (Invitrogen) were purchased from Addgene (Tokyo, Japan).

    Techniques: Transfection, Plasmid Preparation, Dominant Negative Mutation, SDS Page, Immunoprecipitation, In Vitro, Derivative Assay

    Transiently transfected 293 FT cells show a strongly reduced NMD efficiency in comparison to stably transfected cells. ( a ) Schematic representation of the experimental setup. The reporter constructs are generated by cloning the gene of interest into the pcDNA5/FRT/TO vector. The blue bar represents the coding sequence of triosephosphate isomerase (TPI) or β-globin with or without a premature termination codon (PTC) and with a normal stop codon (stop). Downstream, a viral XRN1-resistant sequence (xrRNA) is present that allows the detection of decay intermediates resulting from 5′–3′ exonucleolytic decay. The grey boxes correspond to four repeats of a northern blot probe binding site. Cells are transfected with these constructs using either a stable or transient transfection system. Flp-In T-REx (FT) cells constitutively express a Tet repressor (TetR) that blocks transcription of the reporter by binding to Tet operators (TetO 2 ) present downstream of the cytomegalovirus (CMV) promoter in the reporter construct. Therefore, FT cells are induced with tetracycline(Tet)/doxycycline. In non-FT cells, the reporter is always expressed. Unstable reporter mRNAs are degraded with participation of the cellular 5′–3′ exonuclease XRN1. Full-length reporter levels and decay fragment (xrFrag) caused by stalling of XRN1 at the xrRNA are detected via northern blotting. p(A): poly(A) tail. ( b , c ) Expression of reporter mRNAs in HeLa FT ( b ) and 293 FT ( c ) stable cell lines was induced with doxycycline for 24 h and the reporter levels detected by northern blotting. The lower band (xrFrag) corresponds to a 5′–3′ exonucleolytic decay intermediate. Endogenous 7SL RNA levels were detected and quantified on the same blot and are shown as control. The mean values ± SD (n = 3) for relative transcript levels were quantified and the PTC values normalized to the wild-type control. The xrRNA/reporter ratio is indicated below the graph. ( d , e ) HeLa FT ( d ) and 293 FT ( e ) cells were transiently transfected with 3 µg plasmid DNA per 6-well and transcription was induced with doxycycline for 24 h. Reporter mRNA levels were analyzed by northern blotting. LacZ was co-expressed and serves as a transfection control. The mean values ± SD (n = 3) were quantified and the PTC values normalized to the wild-type control. The xrRNA/reporter ratio is shown below the graph.

    Journal: Scientific Reports

    Article Title: Plasmid transfection influences the readout of nonsense-mediated mRNA decay reporter assays in human cells

    doi: 10.1038/s41598-017-10847-4

    Figure Lengend Snippet: Transiently transfected 293 FT cells show a strongly reduced NMD efficiency in comparison to stably transfected cells. ( a ) Schematic representation of the experimental setup. The reporter constructs are generated by cloning the gene of interest into the pcDNA5/FRT/TO vector. The blue bar represents the coding sequence of triosephosphate isomerase (TPI) or β-globin with or without a premature termination codon (PTC) and with a normal stop codon (stop). Downstream, a viral XRN1-resistant sequence (xrRNA) is present that allows the detection of decay intermediates resulting from 5′–3′ exonucleolytic decay. The grey boxes correspond to four repeats of a northern blot probe binding site. Cells are transfected with these constructs using either a stable or transient transfection system. Flp-In T-REx (FT) cells constitutively express a Tet repressor (TetR) that blocks transcription of the reporter by binding to Tet operators (TetO 2 ) present downstream of the cytomegalovirus (CMV) promoter in the reporter construct. Therefore, FT cells are induced with tetracycline(Tet)/doxycycline. In non-FT cells, the reporter is always expressed. Unstable reporter mRNAs are degraded with participation of the cellular 5′–3′ exonuclease XRN1. Full-length reporter levels and decay fragment (xrFrag) caused by stalling of XRN1 at the xrRNA are detected via northern blotting. p(A): poly(A) tail. ( b , c ) Expression of reporter mRNAs in HeLa FT ( b ) and 293 FT ( c ) stable cell lines was induced with doxycycline for 24 h and the reporter levels detected by northern blotting. The lower band (xrFrag) corresponds to a 5′–3′ exonucleolytic decay intermediate. Endogenous 7SL RNA levels were detected and quantified on the same blot and are shown as control. The mean values ± SD (n = 3) for relative transcript levels were quantified and the PTC values normalized to the wild-type control. The xrRNA/reporter ratio is indicated below the graph. ( d , e ) HeLa FT ( d ) and 293 FT ( e ) cells were transiently transfected with 3 µg plasmid DNA per 6-well and transcription was induced with doxycycline for 24 h. Reporter mRNA levels were analyzed by northern blotting. LacZ was co-expressed and serves as a transfection control. The mean values ± SD (n = 3) were quantified and the PTC values normalized to the wild-type control. The xrRNA/reporter ratio is shown below the graph.

    Article Snippet: Plasmid constructs and cell culture The plasmid constructs β-globin-WT and -PTC39; TPI-WT and -PTC160; TPI-RAB7A, -TNF and -IL6 containing an XRN1-resistant structure from the Murray Valley encephalitis virus were generated by cloning the respective DNA fragments into the pcDNA5/FRT/TO vector (Thermo Fisher Scientific).

    Techniques: Transfection, Stable Transfection, Construct, Generated, Clone Assay, Plasmid Preparation, Sequencing, Northern Blot, Binding Assay, Expressing