pcdna Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher pcdna3 1 vector
    Plasmid construction and generation of stable cells. ( A ) Schematic representation of miR-34a expression vector. ( B ) Relative miR-34a expression levels in three groups of SOSP-9607 cells (Blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with <t>pcDNA3.1;</t> miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a.). ( C ) Relative miR-34a expression levels in SAOS-2 cells transiently transfected with pcDNA3.1 and pcDNA-miR34a respectively. U24 small nucleolar RNA (RNU24) was used as an internal loading control to normalize the results. Data are presented as means±SD. *P
    Pcdna3 1 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17854 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 vector/product/Thermo Fisher
    Average 99 stars, based on 17854 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 vector - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcdna 3 1
    ERRα antagonist Compound A inhibits constitutive transcriptional activity of ERRα. (A) Plasmids pGL2 Luc ERE (empty vector control) or p3xERE-TK-LUC and phRL-TK renilla plasmid were co-transfected into MCF-7 cells. After indicated cell treatment, cells were harvested and cell lysates were assayed for luciferase activity (renilla normalized). Cells transfected with the p3xERE-TK-Luc showed constitutive activity compared to the empty vector pGL2 Luc treated with vehicle. MCF-7 cells transfected with the p3xERE-TK-Luc and treated with E2 conferred a 6.3-fold increase in transcriptional activity while cells treated with Compound A are significantly repressed, 0.73-fold (or 27% decrease) below basal level (*, P = 0.032) (  Fig. 1A  inset) or when treated with Compound A in combination with E2, Compound A still represses the transcriptional effect conferred by ERRα. ICI - 182,780 also greatly reduces any transactivation. (B) The same experiment (described above) was performed along with either pcDNA 3.1 (empty vector control) or pcDNA3.1 hERRα as indicated. Over-expressing ERRα in MCF-7 cells increases down modulation (relative luciferase activity) within all treatment groups. Cells transfected with the p3xERE-TK-Luc+pcDNA 3.1 hERRα conferred an 114-fold increase in transcriptional activity above cells transfected with pGL2 Luc and treated with vehicle. Cells transfected with the p3xERE-TK-Luc+pcDNA 3.1 hERRα and treated with E2 exhibited a 2.6-fold increase in transcriptional activity in comparison to pGL2 Luc+pcDNA3.1 hERRα. Over expressing ERRα leads to a 45% decrease in transactivation upon treatment with Compound A (P = 0.001) (  Fig. 1B  inset). Similarly, Cmpd A+E2 led to a 33% decrease and over expressing ERRα in MCF-7 cells still led to nearly abolishing transactivation upon treatment with ICI 182,780. Results are expressed as the normalized luciferase activity (mean±SEM) of three independent experiments performed in triplicate. Differences in luciferase activity between vehicle (DMSO) and Cmpd A were measured by ANOVA followed by a student t-test with a 0.05 significance level. *, P = 0.032 and **, P = 0.001.
    Pcdna 3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5455 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna 3 1/product/Thermo Fisher
    Average 99 stars, based on 5455 article reviews
    Price from $9.99 to $1999.99
    pcdna 3 1 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher pcdna
    Co-culture of NSCs with Notch1-expressing cells or Dll1-expressing cells. (a) NSCs were cultured for 2 days on monolayers of Dll1 (2) or Notch1 (3) over-expressing COS7 cells and immunostained with Tuj1, a marker of neurons (red) and the NSC marker Nestin (green). (1): Control experiment with <t>pCDNA-transfected</t> COS7 cells. (b) NSCs were co-cultured for two (black bar: 2 days diff.) or four (white bar: 4 days diff.) days as described above. After immunostaining, Tuj1-positive neurons and Nestin-positive NSCs were counted. Values are means ± S.D. for 10 independent fields.*** P
    Pcdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna/product/Thermo Fisher
    Average 94 stars, based on 5306 article reviews
    Price from $9.99 to $1999.99
    pcdna - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcdna5 frt to vector
    Expression of APP in wild-type or mutant APP -transfected cells. (A) The distribution of APP was similar in APP -transfected cells. The scale bar represents 50 μm. (B) The expression of total APP and β-actin in cell lysates (5 μg protein/lane) was detected by immunoblotting. Full-sized images are available in Supplementary Material. The summed density of the two bands (arrows) was quantified as the amount of total APP and demonstrated in (C). Nc: negative control, Vec: <t>pcDNA5/FRT/TO</t> vector-transfected cells, Wt: wild-type, Sw: Swedish, Du: Dutch, Lo: London-type mutant-transfected cells, respectively, a.u.: arbitrary unit.
    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

    99
    Thermo Fisher pcdna3 1 directional topo expression kit
    Expression of APP in wild-type or mutant APP -transfected cells. (A) The distribution of APP was similar in APP -transfected cells. The scale bar represents 50 μm. (B) The expression of total APP and β-actin in cell lysates (5 μg protein/lane) was detected by immunoblotting. Full-sized images are available in Supplementary Material. The summed density of the two bands (arrows) was quantified as the amount of total APP and demonstrated in (C). Nc: negative control, Vec: <t>pcDNA5/FRT/TO</t> vector-transfected cells, Wt: wild-type, Sw: Swedish, Du: Dutch, Lo: London-type mutant-transfected cells, respectively, a.u.: arbitrary unit.
    Pcdna3 1 Directional Topo Expression Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 directional topo expression kit/product/Thermo Fisher
    Average 99 stars, based on 526 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 directional topo expression kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    90
    Thermo Fisher pcdna 3
    ORF 57 increases ORF 59/58 mRNA and protein levels. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 0.1 μg of pcDNA 3.1 ORF 57. A ds DNA probe specific for ORF 59 was used; bottom, the blot was reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. The ORF 59 protein was detected with a KSHV MAb, and then with a secondary goat anti-mouse conjugated to horseradish peroxidase and ECL (Amersham). Equal amounts (10 μg) of extracts as determined by Bradford assay from each transfection were loaded onto the gel.
    Pcdna 3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 599 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna 3/product/Thermo Fisher
    Average 90 stars, based on 599 article reviews
    Price from $9.99 to $1999.99
    pcdna 3 - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    91
    Thermo Fisher pcdna 6 2 gw emgfp mir vector
    Cloning of amiRNA into <t>pcDNA™6.2-GW/EmGFP-miR</t> vector. (A) Schematic representations of the JEV 3 ′ UTR structure. (B) Schematic illustrations of the three amiRNA designed against the target sites cloned into pcDNA6.2-GW/EmGFP-miR vector. amiRNA, artificial microRNA; JEV, Japanese encephalitis virus; UTR, untranslated region. Color images available online at www.liebertpub.com/nat
    Pcdna 6 2 Gw Emgfp Mir Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna 6 2 gw emgfp mir vector/product/Thermo Fisher
    Average 91 stars, based on 653 article reviews
    Price from $9.99 to $1999.99
    pcdna 6 2 gw emgfp mir vector - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    90
    Thermo Fisher pcdna dest40
    Calibration EYFP-53BP1 reporter system. ( A ) EYFP-53BP1 primary structures. EYFP coding sequence is joined to full-length 53BP1, with Tudor and BRCT domains indicated. EYFP1-53BP coding sequence was inserted into <t>pcDNA-DEST40</t> (Invitrogen) for expression under control of the CMV promoter. ( B ) Dose response to calibrated doses of 137 Cs reference radiation. Cells were co-transfected with EYFP-53BP1 WT and H2B-diHcRed. Live-cell images were collected 30 min post-irradiation. ( C ) Quantification of dose response. Foci were scored using 8–24 individual nuclei at each dose point, and linear regression was performed to obtain the slope of the dose-response curve.
    Pcdna Dest40, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 457 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna dest40/product/Thermo Fisher
    Average 90 stars, based on 457 article reviews
    Price from $9.99 to $1999.99
    pcdna dest40 - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    Image Search Results


    Plasmid construction and generation of stable cells. ( A ) Schematic representation of miR-34a expression vector. ( B ) Relative miR-34a expression levels in three groups of SOSP-9607 cells (Blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a.). ( C ) Relative miR-34a expression levels in SAOS-2 cells transiently transfected with pcDNA3.1 and pcDNA-miR34a respectively. U24 small nucleolar RNA (RNU24) was used as an internal loading control to normalize the results. Data are presented as means±SD. *P

    Journal: PLoS ONE

    Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo

    doi: 10.1371/journal.pone.0033778

    Figure Lengend Snippet: Plasmid construction and generation of stable cells. ( A ) Schematic representation of miR-34a expression vector. ( B ) Relative miR-34a expression levels in three groups of SOSP-9607 cells (Blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a.). ( C ) Relative miR-34a expression levels in SAOS-2 cells transiently transfected with pcDNA3.1 and pcDNA-miR34a respectively. U24 small nucleolar RNA (RNU24) was used as an internal loading control to normalize the results. Data are presented as means±SD. *P

    Article Snippet: The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene.

    Techniques: Plasmid Preparation, Expressing, Transfection

    c-Met is a target of miR-34a, and regulates the migration and invasion of osteosarcoma cells. ( A ) Western blotting analysis of c-Met protein expression. ( B ) qRT-PCR analysis of c-Met mRNA expression. The mRNA levels of c-Met in three groups of SOSP-9607 cells (blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a.) were normalized against that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which served as an internal control. ( C ) The result from luciferase assay showed that the luciferase activity of the pmiR-Met UTR-Wt construct was significantly inhibited after the introduction of miR-34a mimics. Meanwhile, mutations of the two c-Met 3′UTR-binding sites abolished the ability of miR-34a to regulate luciferase expression. ( D, E ) SOSP-9607 cells were transiently transfected with 100 nM of c-Met siRNA or inhibitor NC (Genepharma, China), respectively. 48 h later, the migration and invasion assay were performed. Quantitative results for the effects of c-Met siRNA on the migration and invasion ability of SOSP-9607 cells were shown as migrated and invaded cell number. The results are presented as means±SD. *P

    Journal: PLoS ONE

    Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo

    doi: 10.1371/journal.pone.0033778

    Figure Lengend Snippet: c-Met is a target of miR-34a, and regulates the migration and invasion of osteosarcoma cells. ( A ) Western blotting analysis of c-Met protein expression. ( B ) qRT-PCR analysis of c-Met mRNA expression. The mRNA levels of c-Met in three groups of SOSP-9607 cells (blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a.) were normalized against that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which served as an internal control. ( C ) The result from luciferase assay showed that the luciferase activity of the pmiR-Met UTR-Wt construct was significantly inhibited after the introduction of miR-34a mimics. Meanwhile, mutations of the two c-Met 3′UTR-binding sites abolished the ability of miR-34a to regulate luciferase expression. ( D, E ) SOSP-9607 cells were transiently transfected with 100 nM of c-Met siRNA or inhibitor NC (Genepharma, China), respectively. 48 h later, the migration and invasion assay were performed. Quantitative results for the effects of c-Met siRNA on the migration and invasion ability of SOSP-9607 cells were shown as migrated and invaded cell number. The results are presented as means±SD. *P

    Article Snippet: The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene.

    Techniques: Migration, Western Blot, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Activity Assay, Construct, Binding Assay, Invasion Assay

    miR-34a inhibits migration and invasion of osteosarcoma in vitro. Representative photographs of migrated and invaded SOSP-9607 cells (Blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a) on the membrane at a magnification of 100× ( A, B ). Quantitative results for the migration and invasion ability of each group of SOSP-9607 cells were shown as migrated and invaded cell number, 16 h after incubation ( C, D ). Representative photographs of migrated and invaded SAOS-2 cells (pcDNA3.1, SAOS-2 cells transiently transfected with pcDNA3.1; pcDNA-miR34a, SAOS-2 cells transiently transfected with pcDNA-miR34a) on the membrane at a magnification of 100× ( E, F ). Quantitative results for the migration and invasion ability of SAOS-2 cells were shown as migrated and invaded cell number, 16 h after incubation ( G, H ). Data are expressed as means±SD. *P

    Journal: PLoS ONE

    Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo

    doi: 10.1371/journal.pone.0033778

    Figure Lengend Snippet: miR-34a inhibits migration and invasion of osteosarcoma in vitro. Representative photographs of migrated and invaded SOSP-9607 cells (Blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a) on the membrane at a magnification of 100× ( A, B ). Quantitative results for the migration and invasion ability of each group of SOSP-9607 cells were shown as migrated and invaded cell number, 16 h after incubation ( C, D ). Representative photographs of migrated and invaded SAOS-2 cells (pcDNA3.1, SAOS-2 cells transiently transfected with pcDNA3.1; pcDNA-miR34a, SAOS-2 cells transiently transfected with pcDNA-miR34a) on the membrane at a magnification of 100× ( E, F ). Quantitative results for the migration and invasion ability of SAOS-2 cells were shown as migrated and invaded cell number, 16 h after incubation ( G, H ). Data are expressed as means±SD. *P

    Article Snippet: The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene.

    Techniques: Migration, In Vitro, Transfection, Incubation

    miR-34a inhibits proliferation of osteosarcoma in vitro. Every 24 h, MTT assay was performed on three groups of SOSP-9607 cells (Blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a) and SAOS-2 cells (pcDNA3.1, SAOS-2 cells transiently transfected with pcDNA3.1; pcDNA-miR34a, SAOS-2 cells transiently transfected with pcDNA-miR34a) respectively ( A, B ). The viable cell number was evaluated as the value of the absorbance at 490 nm with a reference wavelength of 630 nm. Values of optical density (OD) are expressed as means±SD. *P

    Journal: PLoS ONE

    Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo

    doi: 10.1371/journal.pone.0033778

    Figure Lengend Snippet: miR-34a inhibits proliferation of osteosarcoma in vitro. Every 24 h, MTT assay was performed on three groups of SOSP-9607 cells (Blank, SOSP-9607 cells; control, stable SOSP-9607 cells transfected with pcDNA3.1; miR-34a, stable SOSP-9607 cells transfected with pcDNA-miR34a) and SAOS-2 cells (pcDNA3.1, SAOS-2 cells transiently transfected with pcDNA3.1; pcDNA-miR34a, SAOS-2 cells transiently transfected with pcDNA-miR34a) respectively ( A, B ). The viable cell number was evaluated as the value of the absorbance at 490 nm with a reference wavelength of 630 nm. Values of optical density (OD) are expressed as means±SD. *P

    Article Snippet: The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene.

    Techniques: In Vitro, MTT Assay, Transfection

    miR-34a inhibits pulmonary metastasis of osteosarcoma in vivo. ( A ) Representative macroscopic pictures of mouse lungs, 42 days after inoculation. ( B ) Graph displaying the total number of tumor nodules per lung in control group (stable SOSP-9607 cells transfected with pcDNA3.1) and miR-34a group (stable SOSP-9607 cells transfected with pcDNA-miR34a.). Data are presented as means±SD. *P

    Journal: PLoS ONE

    Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo

    doi: 10.1371/journal.pone.0033778

    Figure Lengend Snippet: miR-34a inhibits pulmonary metastasis of osteosarcoma in vivo. ( A ) Representative macroscopic pictures of mouse lungs, 42 days after inoculation. ( B ) Graph displaying the total number of tumor nodules per lung in control group (stable SOSP-9607 cells transfected with pcDNA3.1) and miR-34a group (stable SOSP-9607 cells transfected with pcDNA-miR34a.). Data are presented as means±SD. *P

    Article Snippet: The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene.

    Techniques: In Vivo, Transfection

    miR-34a inhibits tumor growth of osteosarcoma in vivo. The results showed that the growth velocities of orthotopic tumors in miR-34a group (stable SOSP-9607 cells transfected with pcDNA-miR34a.) decreased as compared with control group (stable SOSP-9607 cells transfected with pcDNA3.1). ( A ) Representative photographs of tumors (arrows) on the left legs of mouse. ( B ) Representative photographs of orthotopic tumors harvested 42 days after inoculation. ( C ) Tumor growth curves measured after the inoculation. The length (L) and width (W) of tumor measured every 7 days after inoculation, and the volume of tumor was calculated according to the formula: volume = 1/2×L×W 2 . ( D ) Orthotopic tumor weights 42 days after inoculation. Data are presented as means±SD. ( E ) 42 days after inoculation, miR-34a expression levels in Orthotopic tumors were tested and showed in relative miR-34a levels. *P

    Journal: PLoS ONE

    Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo

    doi: 10.1371/journal.pone.0033778

    Figure Lengend Snippet: miR-34a inhibits tumor growth of osteosarcoma in vivo. The results showed that the growth velocities of orthotopic tumors in miR-34a group (stable SOSP-9607 cells transfected with pcDNA-miR34a.) decreased as compared with control group (stable SOSP-9607 cells transfected with pcDNA3.1). ( A ) Representative photographs of tumors (arrows) on the left legs of mouse. ( B ) Representative photographs of orthotopic tumors harvested 42 days after inoculation. ( C ) Tumor growth curves measured after the inoculation. The length (L) and width (W) of tumor measured every 7 days after inoculation, and the volume of tumor was calculated according to the formula: volume = 1/2×L×W 2 . ( D ) Orthotopic tumor weights 42 days after inoculation. Data are presented as means±SD. ( E ) 42 days after inoculation, miR-34a expression levels in Orthotopic tumors were tested and showed in relative miR-34a levels. *P

    Article Snippet: The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene.

    Techniques: In Vivo, Transfection, Expressing

    Effect of GPC3 on cell proliferation. Cell viability of ACHN and 786-O cells was measured 24 h, 48 h, 72 h, 96 h and 120 h post-transfection with pcDNA3.1 (empty vector) and pcDNA3.1 /GPC3 by MTT assay. Data are presented as the mean of two independent experiments ± SEM. Group comparisons were carried out using Student’s t test. A) ACHN cells overexpressing GPC3 experienced a significant reduction in proliferation rate after 48 h (**p

    Journal: BMC Cancer

    Article Title: GPC3 reduces cell proliferation in renal carcinoma cell lines

    doi: 10.1186/1471-2407-14-631

    Figure Lengend Snippet: Effect of GPC3 on cell proliferation. Cell viability of ACHN and 786-O cells was measured 24 h, 48 h, 72 h, 96 h and 120 h post-transfection with pcDNA3.1 (empty vector) and pcDNA3.1 /GPC3 by MTT assay. Data are presented as the mean of two independent experiments ± SEM. Group comparisons were carried out using Student’s t test. A) ACHN cells overexpressing GPC3 experienced a significant reduction in proliferation rate after 48 h (**p

    Article Snippet: Transfection The pcDNA3.1/GPC3 expression vector and pcDNA3.1 (empty vector) were transfected into ACHN and 786-O cell lines using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual.

    Techniques: Transfection, Plasmid Preparation, MTT Assay

    GPC3 expression in renal cell carcinoma cell lines. Restoration of GPC3 expression after transfection with the plasmid pcDNA3.1/ GPC3 . ACHN and 786-O cells were transiently transfected with pcDNA3.1 (empty vector) or pcDNA3.1/ GPC3 and re-expression of GPC3 was confirmed 48 h post-transfection by qRT-PCR and immunocytochemistry. A) Quantitative mRNA expression of the GPC3 gene in renal cell carcinoma cell lines after 48 h of transfection with the pcDNA3.1/ GPC3 plasmid is shown as fold change (log2) relative to expression of GPC3 in cell lines after 48 h of transfection with pcDNA3.1 (empty vector). B) Immunolocalization of the GPC3 protein in ACHN and 786-O cell lines after 48 h of transfection with the plasmids pcDNA3.1-GPC3 and pcDNA3.1 (empty vector). Bars = 20 μm. C) Densitometry graphic of GPC3 in ACHN and 786-O cell lines (***p

    Journal: BMC Cancer

    Article Title: GPC3 reduces cell proliferation in renal carcinoma cell lines

    doi: 10.1186/1471-2407-14-631

    Figure Lengend Snippet: GPC3 expression in renal cell carcinoma cell lines. Restoration of GPC3 expression after transfection with the plasmid pcDNA3.1/ GPC3 . ACHN and 786-O cells were transiently transfected with pcDNA3.1 (empty vector) or pcDNA3.1/ GPC3 and re-expression of GPC3 was confirmed 48 h post-transfection by qRT-PCR and immunocytochemistry. A) Quantitative mRNA expression of the GPC3 gene in renal cell carcinoma cell lines after 48 h of transfection with the pcDNA3.1/ GPC3 plasmid is shown as fold change (log2) relative to expression of GPC3 in cell lines after 48 h of transfection with pcDNA3.1 (empty vector). B) Immunolocalization of the GPC3 protein in ACHN and 786-O cell lines after 48 h of transfection with the plasmids pcDNA3.1-GPC3 and pcDNA3.1 (empty vector). Bars = 20 μm. C) Densitometry graphic of GPC3 in ACHN and 786-O cell lines (***p

    Article Snippet: Transfection The pcDNA3.1/GPC3 expression vector and pcDNA3.1 (empty vector) were transfected into ACHN and 786-O cell lines using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual.

    Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunocytochemistry

    Effect of GPC3 on colony formation. The GPC3 gene suppresses the colony formation in ACHN (A) and 786-O (B) cell lines. Tumor cell proliferation was assessed in vitro . Cells were transiently transfected with pcDNA3.1 (empty vector) or pcDNA3.1/ GPC3 and replated 24 h post-transfection for selection with Geneticin/G418. After 14 days of selection, colonies were stained with Giemsa and counted. Data are presented as the mean of two independent experiments ± SEM. Group comparisons were carried out using Student’s t test, *p

    Journal: BMC Cancer

    Article Title: GPC3 reduces cell proliferation in renal carcinoma cell lines

    doi: 10.1186/1471-2407-14-631

    Figure Lengend Snippet: Effect of GPC3 on colony formation. The GPC3 gene suppresses the colony formation in ACHN (A) and 786-O (B) cell lines. Tumor cell proliferation was assessed in vitro . Cells were transiently transfected with pcDNA3.1 (empty vector) or pcDNA3.1/ GPC3 and replated 24 h post-transfection for selection with Geneticin/G418. After 14 days of selection, colonies were stained with Giemsa and counted. Data are presented as the mean of two independent experiments ± SEM. Group comparisons were carried out using Student’s t test, *p

    Article Snippet: Transfection The pcDNA3.1/GPC3 expression vector and pcDNA3.1 (empty vector) were transfected into ACHN and 786-O cell lines using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual.

    Techniques: In Vitro, Transfection, Plasmid Preparation, Selection, Staining

    Effect of GPC3 on cell cycle in cell lines. A) ACHN cell cycle histogram. B) 786-O cell cycle histogram. C) The number of ACHN pcDNA3.1/ GPC3 cells was significantly increased during the G1 phase (24 h and 48 h ***p

    Journal: BMC Cancer

    Article Title: GPC3 reduces cell proliferation in renal carcinoma cell lines

    doi: 10.1186/1471-2407-14-631

    Figure Lengend Snippet: Effect of GPC3 on cell cycle in cell lines. A) ACHN cell cycle histogram. B) 786-O cell cycle histogram. C) The number of ACHN pcDNA3.1/ GPC3 cells was significantly increased during the G1 phase (24 h and 48 h ***p

    Article Snippet: Transfection The pcDNA3.1/GPC3 expression vector and pcDNA3.1 (empty vector) were transfected into ACHN and 786-O cell lines using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual.

    Techniques:

    UBIAD1 inhibits the Ras/ERK signaling pathway. a UBIAD1 reduced cell viability in T24 bladder cancer cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell viability was detected by the MTT assay. *** p

    Journal: Cell Death & Disease

    Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras

    doi: 10.1038/s41419-018-1215-4

    Figure Lengend Snippet: UBIAD1 inhibits the Ras/ERK signaling pathway. a UBIAD1 reduced cell viability in T24 bladder cancer cells. T24 cells were transiently transfected with pcDNA3.1-UBIAD1 plasmid. Twenty-four hours after transfection, cell viability was detected by the MTT assay. *** p

    Article Snippet: Enhanced green fluorescent protein (EGFP) vectors pEGFP-N1, pEGFP-C1, pCasper3-BG (TAGBFP-GFP), mammalian expression vector pcDNA3.1 and TAGBFP were obtained from Invitrogen. pDsRed-Golgi vector was obtained from Clontech, previously described .

    Techniques: Transfection, Plasmid Preparation, MTT Assay

    Activation of KSHV lytic cycle genes by KSHV/Rta. ( A ) KSHV/Rta stimulates expression of PAN RNA. Replicate aliquots of 2.5 × 10 7 ). RNA prepared at the times indicated after transfection was analyzed by Northern blotting by using probes specific for PAN RNA and the H1 component of RNase P. ( B ) KSHV/Rta stimulates expression of KSHV/K8 and KSHV/sVCA (ORF 65). The analysis of KSHV/K8 was carried out in BC-1 cells; RNA was harvested 48 h after transfection. The analysis of KSHV/sVCA was carried out in BCBL-1 cells; the RNA was prepared 96 h after transfection. ( C ) KSHV/Rta cDNA activates expression of viral IL-6. BC-1 cells were transfected with plasmids containing KSHV/Rta in the genomic configuration (g), in the cDNA configuration (c), in the genomic configuration in the reverse orientation (rev), or in the genomic configuration with a stop codon mutation (mut). The expression plasmids pRTS and pcDNA3.1 were used as negative controls. RNA prepared 24 h after transfection was analyzed by Northern blotting with probes specific for viral IL-6, PAN RNA and RNase P. ( D ) Response of PAN RNA expression following transfection of different amounts of KSHV/gRta expression plasmid into HH-B2 cells.

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

    Article Title: A viral gene that activates lytic cycle expression of Kaposi's sarcoma-associated herpesvirus

    doi:

    Figure Lengend Snippet: Activation of KSHV lytic cycle genes by KSHV/Rta. ( A ) KSHV/Rta stimulates expression of PAN RNA. Replicate aliquots of 2.5 × 10 7 ). RNA prepared at the times indicated after transfection was analyzed by Northern blotting by using probes specific for PAN RNA and the H1 component of RNase P. ( B ) KSHV/Rta stimulates expression of KSHV/K8 and KSHV/sVCA (ORF 65). The analysis of KSHV/K8 was carried out in BC-1 cells; RNA was harvested 48 h after transfection. The analysis of KSHV/sVCA was carried out in BCBL-1 cells; the RNA was prepared 96 h after transfection. ( C ) KSHV/Rta cDNA activates expression of viral IL-6. BC-1 cells were transfected with plasmids containing KSHV/Rta in the genomic configuration (g), in the cDNA configuration (c), in the genomic configuration in the reverse orientation (rev), or in the genomic configuration with a stop codon mutation (mut). The expression plasmids pRTS and pcDNA3.1 were used as negative controls. RNA prepared 24 h after transfection was analyzed by Northern blotting with probes specific for viral IL-6, PAN RNA and RNase P. ( D ) Response of PAN RNA expression following transfection of different amounts of KSHV/gRta expression plasmid into HH-B2 cells.

    Article Snippet: These PCR fragments were cloned into the pcDNA3.1 expression vector (Invitrogen) and designated as KSHV/gRta.

    Techniques: Activation Assay, Expressing, Transfection, Northern Blot, Mutagenesis, RNA Expression, Plasmid Preparation

    Adenovirus-mediated gene transfer. Duplicate monolayers of CHO-hCAR, CHO-tailless CAR, CHO-GPI-CAR, or CHO-pcDNA3.1 were exposed to Ad.CMV.β-gal (0, 20, or 200 PFU per cell) for 1 h at room temperature and then washed. After incubation for 2 days at 37°C, β-galactosidase activity was detected by in situ staining with X-Gal.

    Journal: Journal of Virology

    Article Title: Coxsackievirus and Adenovirus Receptor Cytoplasmic and Transmembrane Domains Are Not Essential for Coxsackievirus and Adenovirus Infection

    doi:

    Figure Lengend Snippet: Adenovirus-mediated gene transfer. Duplicate monolayers of CHO-hCAR, CHO-tailless CAR, CHO-GPI-CAR, or CHO-pcDNA3.1 were exposed to Ad.CMV.β-gal (0, 20, or 200 PFU per cell) for 1 h at room temperature and then washed. After incubation for 2 days at 37°C, β-galactosidase activity was detected by in situ staining with X-Gal.

    Article Snippet: This construct is referred to as GPI-CAR.), cleavage of the signal peptide occurs between amino acids 19 and 20 (numbered as in reference Wild-type and truncated CAR cDNAs were inserted into the Eco RI and Xba I sites of the expression vector pcDNA3.1 (Invitrogen, Carlsbad, Calif.).

    Techniques: Incubation, Activity Assay, In Situ, Staining

    Expression of CAR on transfected CHO cells. (A) Flow cytometry. CHO cells transfected with cDNA encoding wild-type hCAR or truncated CAR (tailless and GPI), and control cells transfected with the empty pcDNA3.1 vector, were incubated first with MAb RmcB (heavy line) or with a control antibody (MOPC 195 [thin line]) and then with fluorescein isothiocyanate-conjugated goat antibody to mouse immunoglobulin. All panels are shown on the same scale. (B) Flow cytometry after PIPLC treatment. Transfected CHO cells were incubated for 30 min at 37°C in RPMI 1640 supplemented with 0.2% bovine serum albumin, 50 μM 2-mercaptoethanol, 10 mM HEPES (pH 7.0), and 0.1% sodium azide, with or without the addition of PIPLC (0.4 U per million cells; Sigma). Cells were then washed and stained with MAb RmcB, MOPC 195, or MAb P1F6, which recognizes the integrin αvβ5. All panels are shown on the same scale. CAR expression on GPI-CHO cells was reduced 30-fold after PIPLC treatment.

    Journal: Journal of Virology

    Article Title: Coxsackievirus and Adenovirus Receptor Cytoplasmic and Transmembrane Domains Are Not Essential for Coxsackievirus and Adenovirus Infection

    doi:

    Figure Lengend Snippet: Expression of CAR on transfected CHO cells. (A) Flow cytometry. CHO cells transfected with cDNA encoding wild-type hCAR or truncated CAR (tailless and GPI), and control cells transfected with the empty pcDNA3.1 vector, were incubated first with MAb RmcB (heavy line) or with a control antibody (MOPC 195 [thin line]) and then with fluorescein isothiocyanate-conjugated goat antibody to mouse immunoglobulin. All panels are shown on the same scale. (B) Flow cytometry after PIPLC treatment. Transfected CHO cells were incubated for 30 min at 37°C in RPMI 1640 supplemented with 0.2% bovine serum albumin, 50 μM 2-mercaptoethanol, 10 mM HEPES (pH 7.0), and 0.1% sodium azide, with or without the addition of PIPLC (0.4 U per million cells; Sigma). Cells were then washed and stained with MAb RmcB, MOPC 195, or MAb P1F6, which recognizes the integrin αvβ5. All panels are shown on the same scale. CAR expression on GPI-CHO cells was reduced 30-fold after PIPLC treatment.

    Article Snippet: This construct is referred to as GPI-CAR.), cleavage of the signal peptide occurs between amino acids 19 and 20 (numbered as in reference Wild-type and truncated CAR cDNAs were inserted into the Eco RI and Xba I sites of the expression vector pcDNA3.1 (Invitrogen, Carlsbad, Calif.).

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Plasmid Preparation, Incubation, Staining

    Functional expression of GFP-tagged GLUT3 protects cells against low-glucose-induced apoptotic death ( a ) RT–PCR analyses of HEK-293T cells reveal high expression of GLUT1 glucose transporter and low/negligible GLUT3 or GLUT4 transporters (754, 856 and 802 bp cDNA fragments respectively). ( b ) Northern-blot analyses of HEK-293T cells confirm high GLUT1 mRNA and low GLUT3 mRNA expression. Transfection of these cells with the plasmid constructions encoding GLUT3-GFP or GLUT1-GPF (pEGFP.G3 or pEGFP.G1) fusion proteins confirms the expression of the corresponding transcripts, at the expected size, when compared with the cells transfected with the vector alone (pEGFP). ( c ) Western-blot analyses (anti-GFP) of transfected HEK-293T cells show the translational products of the plasmid vectors (pEGFP, pEGFP.G3 and pEGFP.G1) at the expected size. ( d ) Western-blot analysis of untreated or N-glycosidase-F-treated extracts obtained from cells expressing pEGFPG3 or pEGFP.G1 reveals localization of N-glycosylated GLUT3 and GLUT1 proteins at the PM. The fluorescence images of HEK-293T cells transfected with pEGFP reveal a diffused localization of GFP, PM localization of GLUT3-GFP (pEGFP.G3) and both cytosolic and PM localization of GLUT1-GFP (pEGFP.G1). ( e ) Expression of pEGFP.G3 afforded HEK-293T cells with higher rate of 2-deoxyglucose uptake than cells transfected with either pEGFP.G1 or pEGFP. ( f ) HEK-293T cells transfected with pEGFP.G3 or pEGFP.G1 took up 2-deoxyglucose (at 150 μM glucose) at a rate that was similar to those observed with pcDNA3-G3 or pcDNA3-G1, when compared with cells transfected with equivalent amounts of their respective empty vectors (pEFGFP or pcDNA3 respectively). ( g ) Glucose deprivation increased the proportion of apoptotic cells in a time- and concentration-dependent fashion (as assessed by annexin V + /7-AAD − by flow cytometry). ( h ) Overexpression of GLUT3 (pEGFP.G3) or GLUT1 (pEGFP.G1) in HEK-293T cells significantly reduced the apoptotic death caused by glucose deprivation when compared with the cells transfected with the empty vector (pEGFP). * P

    Journal: Biochemical Journal

    Article Title: Inhibition of mitochondrial respiration by nitric oxide rapidly stimulates cytoprotective GLUT3-mediated glucose uptake through 5?-AMP-activated protein kinase

    doi: 10.1042/BJ20040886

    Figure Lengend Snippet: Functional expression of GFP-tagged GLUT3 protects cells against low-glucose-induced apoptotic death ( a ) RT–PCR analyses of HEK-293T cells reveal high expression of GLUT1 glucose transporter and low/negligible GLUT3 or GLUT4 transporters (754, 856 and 802 bp cDNA fragments respectively). ( b ) Northern-blot analyses of HEK-293T cells confirm high GLUT1 mRNA and low GLUT3 mRNA expression. Transfection of these cells with the plasmid constructions encoding GLUT3-GFP or GLUT1-GPF (pEGFP.G3 or pEGFP.G1) fusion proteins confirms the expression of the corresponding transcripts, at the expected size, when compared with the cells transfected with the vector alone (pEGFP). ( c ) Western-blot analyses (anti-GFP) of transfected HEK-293T cells show the translational products of the plasmid vectors (pEGFP, pEGFP.G3 and pEGFP.G1) at the expected size. ( d ) Western-blot analysis of untreated or N-glycosidase-F-treated extracts obtained from cells expressing pEGFPG3 or pEGFP.G1 reveals localization of N-glycosylated GLUT3 and GLUT1 proteins at the PM. The fluorescence images of HEK-293T cells transfected with pEGFP reveal a diffused localization of GFP, PM localization of GLUT3-GFP (pEGFP.G3) and both cytosolic and PM localization of GLUT1-GFP (pEGFP.G1). ( e ) Expression of pEGFP.G3 afforded HEK-293T cells with higher rate of 2-deoxyglucose uptake than cells transfected with either pEGFP.G1 or pEGFP. ( f ) HEK-293T cells transfected with pEGFP.G3 or pEGFP.G1 took up 2-deoxyglucose (at 150 μM glucose) at a rate that was similar to those observed with pcDNA3-G3 or pcDNA3-G1, when compared with cells transfected with equivalent amounts of their respective empty vectors (pEFGFP or pcDNA3 respectively). ( g ) Glucose deprivation increased the proportion of apoptotic cells in a time- and concentration-dependent fashion (as assessed by annexin V + /7-AAD − by flow cytometry). ( h ) Overexpression of GLUT3 (pEGFP.G3) or GLUT1 (pEGFP.G1) in HEK-293T cells significantly reduced the apoptotic death caused by glucose deprivation when compared with the cells transfected with the empty vector (pEGFP). * P

    Article Snippet: Full-length GLUT3 or GLUT1 cDNAs, digested from pBS-rG3, were also subcloned into the Eco RI site of the pcDNA3 mammalian expression vector (Invitrogen, Madrid, Spain; pcDNA3-G3).

    Techniques: Functional Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Transfection, Plasmid Preparation, Western Blot, Fluorescence, Concentration Assay, Flow Cytometry, Cytometry, Over Expression

    Tumor-suppressive effect of miR-770 is mediated by targeting of STMN1 mRNA. Both MDA-MB-468 and MDA-MB-231 cells were transfected with miR-NC or miR-770 together with either STMN1 or pcDNA3.1. a mRNA and protein level were detected for verification. And IC50 assay b and cytotoxicity test c were performed. The migration d and invasion ability of TNBC cells. e Quantification of migration and invasion of cells. Data represent means ± S.D. of at least three independent experiments. * P

    Journal: Cell Death & Disease

    Article Title: MiR-770 suppresses the chemo-resistance and metastasis of triple negative breast cancer via direct targeting of STMN1

    doi: 10.1038/s41419-017-0030-7

    Figure Lengend Snippet: Tumor-suppressive effect of miR-770 is mediated by targeting of STMN1 mRNA. Both MDA-MB-468 and MDA-MB-231 cells were transfected with miR-NC or miR-770 together with either STMN1 or pcDNA3.1. a mRNA and protein level were detected for verification. And IC50 assay b and cytotoxicity test c were performed. The migration d and invasion ability of TNBC cells. e Quantification of migration and invasion of cells. Data represent means ± S.D. of at least three independent experiments. * P

    Article Snippet: Briefly, STMN1 cDNA was cloned into the multiple cloning sites of vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), and the resultant expression vector and empty vector were transfected into MDA-MB-468 and MDA-MB-231 cells to establish STMN1 overexpressing and control cell lines, respectively.

    Techniques: Multiple Displacement Amplification, Transfection, Migration

    Functional analysis of SAP30 mutants analyzed by transcription repression of a 14D luciferase reporter.  HEK293 cells were transfected with Gal4DBD fusion plasmids along with the luciferase reporter and pcDNA 3.1.-LacZ control plasmids. The  bars  in the

    Journal: The Journal of Biological Chemistry

    Article Title: Structure of the 30-kDa Sin3-associated Protein (SAP30) in Complex with the Mammalian Sin3A Corepressor and Its Role in Nucleic Acid Binding *

    doi: 10.1074/jbc.M111.252494

    Figure Lengend Snippet: Functional analysis of SAP30 mutants analyzed by transcription repression of a 14D luciferase reporter. HEK293 cells were transfected with Gal4DBD fusion plasmids along with the luciferase reporter and pcDNA 3.1.-LacZ control plasmids. The bars in the

    Article Snippet: LacZ in pcDNA 3.1 plasmid (Invitrogen) was used as a transfection control in repression experiments.

    Techniques: Functional Assay, Luciferase, Transfection

    Expression of ADAM17 in gastric cancer cells. (A) western blotting showed the expression of ADAM17 in BGC-823, AGS, KATO III and SGC-7901 cells. (B) ADAM17 expression in mock-transfected cells, pcDNA-3-transfected, or pcDNA-3-ADAM17-transfected BGC-823 cells. (C) ADAM17 expression in mock-transfected, siRNA-NC-transfected or siRNA-ADAM17-transfected SGC-7901 cells. All experiments were repeated three times and data are presented as the mean ± standard deviation. * P

    Journal: International Journal of Molecular Medicine

    Article Title: ADAM17 promotes lymph node metastasis in gastric cancer via activation of the Notch and Wnt signaling pathways

    doi: 10.3892/ijmm.2018.4028

    Figure Lengend Snippet: Expression of ADAM17 in gastric cancer cells. (A) western blotting showed the expression of ADAM17 in BGC-823, AGS, KATO III and SGC-7901 cells. (B) ADAM17 expression in mock-transfected cells, pcDNA-3-transfected, or pcDNA-3-ADAM17-transfected BGC-823 cells. (C) ADAM17 expression in mock-transfected, siRNA-NC-transfected or siRNA-ADAM17-transfected SGC-7901 cells. All experiments were repeated three times and data are presented as the mean ± standard deviation. * P

    Article Snippet: The empty pcDNA-3 vector served as the control.

    Techniques: Expressing, Western Blot, Transfection, Standard Deviation

    ERRα antagonist Compound A inhibits constitutive transcriptional activity of ERRα. (A) Plasmids pGL2 Luc ERE (empty vector control) or p3xERE-TK-LUC and phRL-TK renilla plasmid were co-transfected into MCF-7 cells. After indicated cell treatment, cells were harvested and cell lysates were assayed for luciferase activity (renilla normalized). Cells transfected with the p3xERE-TK-Luc showed constitutive activity compared to the empty vector pGL2 Luc treated with vehicle. MCF-7 cells transfected with the p3xERE-TK-Luc and treated with E2 conferred a 6.3-fold increase in transcriptional activity while cells treated with Compound A are significantly repressed, 0.73-fold (or 27% decrease) below basal level (*, P = 0.032) (  Fig. 1A  inset) or when treated with Compound A in combination with E2, Compound A still represses the transcriptional effect conferred by ERRα. ICI - 182,780 also greatly reduces any transactivation. (B) The same experiment (described above) was performed along with either pcDNA 3.1 (empty vector control) or pcDNA3.1 hERRα as indicated. Over-expressing ERRα in MCF-7 cells increases down modulation (relative luciferase activity) within all treatment groups. Cells transfected with the p3xERE-TK-Luc+pcDNA 3.1 hERRα conferred an 114-fold increase in transcriptional activity above cells transfected with pGL2 Luc and treated with vehicle. Cells transfected with the p3xERE-TK-Luc+pcDNA 3.1 hERRα and treated with E2 exhibited a 2.6-fold increase in transcriptional activity in comparison to pGL2 Luc+pcDNA3.1 hERRα. Over expressing ERRα leads to a 45% decrease in transactivation upon treatment with Compound A (P = 0.001) (  Fig. 1B  inset). Similarly, Cmpd A+E2 led to a 33% decrease and over expressing ERRα in MCF-7 cells still led to nearly abolishing transactivation upon treatment with ICI 182,780. Results are expressed as the normalized luciferase activity (mean±SEM) of three independent experiments performed in triplicate. Differences in luciferase activity between vehicle (DMSO) and Cmpd A were measured by ANOVA followed by a student t-test with a 0.05 significance level. *, P = 0.032 and **, P = 0.001.

    Journal: PLoS ONE

    Article Title: Characterization of a Novel Small Molecule Subtype Specific Estrogen-Related Receptor ? Antagonist in MCF-7 Breast Cancer Cells

    doi: 10.1371/journal.pone.0005624

    Figure Lengend Snippet: ERRα antagonist Compound A inhibits constitutive transcriptional activity of ERRα. (A) Plasmids pGL2 Luc ERE (empty vector control) or p3xERE-TK-LUC and phRL-TK renilla plasmid were co-transfected into MCF-7 cells. After indicated cell treatment, cells were harvested and cell lysates were assayed for luciferase activity (renilla normalized). Cells transfected with the p3xERE-TK-Luc showed constitutive activity compared to the empty vector pGL2 Luc treated with vehicle. MCF-7 cells transfected with the p3xERE-TK-Luc and treated with E2 conferred a 6.3-fold increase in transcriptional activity while cells treated with Compound A are significantly repressed, 0.73-fold (or 27% decrease) below basal level (*, P = 0.032) ( Fig. 1A inset) or when treated with Compound A in combination with E2, Compound A still represses the transcriptional effect conferred by ERRα. ICI - 182,780 also greatly reduces any transactivation. (B) The same experiment (described above) was performed along with either pcDNA 3.1 (empty vector control) or pcDNA3.1 hERRα as indicated. Over-expressing ERRα in MCF-7 cells increases down modulation (relative luciferase activity) within all treatment groups. Cells transfected with the p3xERE-TK-Luc+pcDNA 3.1 hERRα conferred an 114-fold increase in transcriptional activity above cells transfected with pGL2 Luc and treated with vehicle. Cells transfected with the p3xERE-TK-Luc+pcDNA 3.1 hERRα and treated with E2 exhibited a 2.6-fold increase in transcriptional activity in comparison to pGL2 Luc+pcDNA3.1 hERRα. Over expressing ERRα leads to a 45% decrease in transactivation upon treatment with Compound A (P = 0.001) ( Fig. 1B inset). Similarly, Cmpd A+E2 led to a 33% decrease and over expressing ERRα in MCF-7 cells still led to nearly abolishing transactivation upon treatment with ICI 182,780. Results are expressed as the normalized luciferase activity (mean±SEM) of three independent experiments performed in triplicate. Differences in luciferase activity between vehicle (DMSO) and Cmpd A were measured by ANOVA followed by a student t-test with a 0.05 significance level. *, P = 0.032 and **, P = 0.001.

    Article Snippet: 0.125 ug pGL2 Luc (Promega), or 0.125 ug p3xERE-TK-LUC [pGL2 plasmid (Promega, Madison, WI) containing 3 tandem repeats of the estrogen response element (ERE) sequence 5′ – TTTGATC AGGTCA CTG TGACCT CTAGAGT-3′ , placed upstream of a minimal herpes simplex thymidine kinase (TK) promoter directing the expression of the luciferase coding sequence (a generous gift from Tina Chang, Merck Research Laboratories, Rahway, NJ) and 0.0125 ug of phRL-TK renilla plasmid (Promega) were co-transfected in triplicate wells along with (when indicated) either 0.0625 ug pcDNA 3.1 (Invitrogen, Carlsbad, CA) or 0.0625 ug pcDNA3.1 hERRα (described below).

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

    Purification of USP14 and human proteasomes, preparation of vme-proteasomes, and SELEX for USP14 aptamers. ( A ) Approximately 1 μg of purified recombinant USP14, USP14(C114A), GST-USP14, and GST-USP14(C114A) was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. Bovine serum albumin (BSA) was used as a standard. ( B ) RNA sequences used for  in vitro  selection. The random RNA library contained random 40-nt sequences, flanked by a 3’ region (23 bp) and a sequence containing the T7 promoter (46 bp, underlined). ( C ) Scheme for the SELEX strategy. (1) Purified RNAs were incubated with GST-USP14. (2) USP14-RNA complexes were captured by glutathione agarose beads. (3) Unbound RNA molecules were removed by centrifugation. (4) USP14-RNA complexes were dissociated with elution buffer containing excess imidazole. (5) RNAs bound to USP14 were prepared by phenol:chloroform extraction and ethanol precipitation. Recovered RNAs were reverse transcribed, amplified by polymerase chain reaction (PCR), and  in vitro  transcribed. (6) Following 15 rounds of selection, the resultant cDNA was amplified and cloned into pcDNA 3.1. ( D ) Human proteasomes (PTSM) were purified from β4-tagged HEK293 cell lines. Some of the proteasomes were treated with 1 μM ubiquitin-vinylmethylester (Ub-vme) to yield vme-PTSMs. PTSMs (1 μg) and vme-PTSMs (1 μg) were separated on 12% gradient gels and stained with CBB. ( E ) Western blotting analysis of USP14 and proteasome subunit α3. Approximately 250 ng of PTSMs or vme-PTSMs were subjected to SDS-PAGE. Cropped gels/blots are used in (E).

    Journal: Scientific Reports

    Article Title: Facilitated Tau Degradation by USP14 Aptamers via Enhanced Proteasome Activity

    doi: 10.1038/srep10757

    Figure Lengend Snippet: Purification of USP14 and human proteasomes, preparation of vme-proteasomes, and SELEX for USP14 aptamers. ( A ) Approximately 1 μg of purified recombinant USP14, USP14(C114A), GST-USP14, and GST-USP14(C114A) was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. Bovine serum albumin (BSA) was used as a standard. ( B ) RNA sequences used for in vitro selection. The random RNA library contained random 40-nt sequences, flanked by a 3’ region (23 bp) and a sequence containing the T7 promoter (46 bp, underlined). ( C ) Scheme for the SELEX strategy. (1) Purified RNAs were incubated with GST-USP14. (2) USP14-RNA complexes were captured by glutathione agarose beads. (3) Unbound RNA molecules were removed by centrifugation. (4) USP14-RNA complexes were dissociated with elution buffer containing excess imidazole. (5) RNAs bound to USP14 were prepared by phenol:chloroform extraction and ethanol precipitation. Recovered RNAs were reverse transcribed, amplified by polymerase chain reaction (PCR), and in vitro transcribed. (6) Following 15 rounds of selection, the resultant cDNA was amplified and cloned into pcDNA 3.1. ( D ) Human proteasomes (PTSM) were purified from β4-tagged HEK293 cell lines. Some of the proteasomes were treated with 1 μM ubiquitin-vinylmethylester (Ub-vme) to yield vme-PTSMs. PTSMs (1 μg) and vme-PTSMs (1 μg) were separated on 12% gradient gels and stained with CBB. ( E ) Western blotting analysis of USP14 and proteasome subunit α3. Approximately 250 ng of PTSMs or vme-PTSMs were subjected to SDS-PAGE. Cropped gels/blots are used in (E).

    Article Snippet: Amplicons were digested with Hind III and Eco RI, and cloned into the pcDNA 3.1 vector (Invitrogen) for sequencing.

    Techniques: Purification, Recombinant, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, In Vitro, Selection, Sequencing, Incubation, Centrifugation, Ethanol Precipitation, Amplification, Polymerase Chain Reaction, Clone Assay, Western Blot

    Plot of the current registered versus the [ 125 I]-α-bungarotoxin binding activity for oocytes injected with plasmid expressing CHRNA7 with different variants of CHRFAM7A Oocytes were injected with pcDNA3.1-CHRNA7+pcDNA3.1, pcDNA3.1-CHRNA7+pcDNA3.1-CHRFAM7A, or pcDNA3.1-CHRNA7+pcDNA3.1-CHRFAM7AΔ2bp at a final concentration of 2ng/μl. On 5 to 7 days after the injection, the current generated by an application of 200 μM of ACh for 5s was registered by an homemade automated two electrode voltage clamp. Oocytes generating a current were selected and incubated into OR2-BSA with 10nM [ 125 I]-α-bungarotoxin for 1h. After 3 washes with OR2-BSA, the radioactivity of each oocyte was measured with a scintillation counter and normalized to non-injected oocytes. Fig. 4a, Means of binding in each group. Fig. 4b, Lines represent a linear regression. CHRNA7 + pcDNA3 (Y=-225.93X+249,89 R 2 =0.995), CHRNA7 + CHRFAM7A (Y=-555.45X+207.02 R 2 =0.867), CHRNA7 + CHRFAM7A Δ2bp (Y=-805.79X+244.72 R 2 =0.915).

    Journal: Biochemical pharmacology

    Article Title: The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7*nAChR function

    doi: 10.1016/j.bcp.2011.06.018

    Figure Lengend Snippet: Plot of the current registered versus the [ 125 I]-α-bungarotoxin binding activity for oocytes injected with plasmid expressing CHRNA7 with different variants of CHRFAM7A Oocytes were injected with pcDNA3.1-CHRNA7+pcDNA3.1, pcDNA3.1-CHRNA7+pcDNA3.1-CHRFAM7A, or pcDNA3.1-CHRNA7+pcDNA3.1-CHRFAM7AΔ2bp at a final concentration of 2ng/μl. On 5 to 7 days after the injection, the current generated by an application of 200 μM of ACh for 5s was registered by an homemade automated two electrode voltage clamp. Oocytes generating a current were selected and incubated into OR2-BSA with 10nM [ 125 I]-α-bungarotoxin for 1h. After 3 washes with OR2-BSA, the radioactivity of each oocyte was measured with a scintillation counter and normalized to non-injected oocytes. Fig. 4a, Means of binding in each group. Fig. 4b, Lines represent a linear regression. CHRNA7 + pcDNA3 (Y=-225.93X+249,89 R 2 =0.995), CHRNA7 + CHRFAM7A (Y=-555.45X+207.02 R 2 =0.867), CHRNA7 + CHRFAM7A Δ2bp (Y=-805.79X+244.72 R 2 =0.915).

    Article Snippet: PCR fragments were gel purified and TA cloned into the pcDNA3.1 mammalian expression vector (Invitrogen)(pcDNA3.1-CHRNA7).

    Techniques: Binding Assay, Activity Assay, Injection, Plasmid Preparation, Expressing, Concentration Assay, Generated, Incubation, Radioactivity

    Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using MasR antibodies. Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. In cells transfected with the pcDNA 3.1/c-myc-MasR construct, antibodies NLS-1531 and sc-54682 generated a similar staining pattern to that generated with the anti c-myc antibody. No staining was observed with any of these antibodies in cells transfected with the empty vector pcDNA 3.1. Antibody sc-135063 was capable of staining the plasma membrane of cells overexpressing the MasR but also generated widespread signals in cells transfected with the empty vector. For antibody AAR-013 there was no staining associated with the cell membrane. Intense immunocytochemical staining of identical distribution and intensity was revealed in cells transfected with the empty vector and those transfected with the c-myc-MasR construct. Images are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies

    doi: 10.1371/journal.pone.0183278

    Figure Lengend Snippet: Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using MasR antibodies. Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. In cells transfected with the pcDNA 3.1/c-myc-MasR construct, antibodies NLS-1531 and sc-54682 generated a similar staining pattern to that generated with the anti c-myc antibody. No staining was observed with any of these antibodies in cells transfected with the empty vector pcDNA 3.1. Antibody sc-135063 was capable of staining the plasma membrane of cells overexpressing the MasR but also generated widespread signals in cells transfected with the empty vector. For antibody AAR-013 there was no staining associated with the cell membrane. Intense immunocytochemical staining of identical distribution and intensity was revealed in cells transfected with the empty vector and those transfected with the c-myc-MasR construct. Images are representative of 3 independent experiments.

    Article Snippet: The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR).

    Techniques: Immunofluorescence, Fluorescence, Staining, Transfection, Construct, Generated, Plasmid Preparation

    Western blotting of HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag and three MasR antibodies. Cells were transfected with varying amounts of a plasmid encoding the c-myc-tagged MasR (0.2 and 0.5 μg of pcDNA 3.1/c-myc-MasR).The anti-c-myc antibody detected a band of 37 kDa whose intensity increased as higher amount of DNA was transfected (arrow). Additionally, a ladder of higher molecular weight (MW) immunoreactive bands could be observed (asterisk). sc-54682 antibody reproduced the band pattern obtained with the c-myc antibody (arrow and asterisk). sc-135063 antibody revealed multiple bands close to 37 kDa with one of them of increasing intensity as higher amount of DNA was transfected (arrow) but did not produce a ladder of higher MW immunoreactive bands. High unspecific staining was attained with sc-135063 antibody. AAR-013 antibody revealed a pattern of immunoreactivity not comparable to those obtained with the other antibodies. Detection of β-tubulin was used as a protein loading control. The membranes are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies

    doi: 10.1371/journal.pone.0183278

    Figure Lengend Snippet: Western blotting of HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag and three MasR antibodies. Cells were transfected with varying amounts of a plasmid encoding the c-myc-tagged MasR (0.2 and 0.5 μg of pcDNA 3.1/c-myc-MasR).The anti-c-myc antibody detected a band of 37 kDa whose intensity increased as higher amount of DNA was transfected (arrow). Additionally, a ladder of higher molecular weight (MW) immunoreactive bands could be observed (asterisk). sc-54682 antibody reproduced the band pattern obtained with the c-myc antibody (arrow and asterisk). sc-135063 antibody revealed multiple bands close to 37 kDa with one of them of increasing intensity as higher amount of DNA was transfected (arrow) but did not produce a ladder of higher MW immunoreactive bands. High unspecific staining was attained with sc-135063 antibody. AAR-013 antibody revealed a pattern of immunoreactivity not comparable to those obtained with the other antibodies. Detection of β-tubulin was used as a protein loading control. The membranes are representative of 3 independent experiments.

    Article Snippet: The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Molecular Weight, Staining

    Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag antibody. Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. The intense immunostaining obtained with the anti c-myc tag antibody indicated strong expression of MasR in HEK293T cells after transfection with the pcDNA 3.1/c-myc-MasR construct. A significant amount of the total MasR pool was present at the plasma membrane. No c-myc staining was observed in cells transfected with the empty vector pcDNA 3.1. Images are representative of 3 independent experiments.

    Journal: PLoS ONE

    Article Title: Validation of commercial Mas receptor antibodies for utilization in Western Blotting, immunofluorescence and immunohistochemistry studies

    doi: 10.1371/journal.pone.0183278

    Figure Lengend Snippet: Immunofluorescence studies in HEK293T cells overexpressing c-myc tagged MasR using anti c-myc tag antibody. Images of fluorescence signal corresponding to secondary cyanine (Cy3)-conjugated antibody (A), nuclear Hoechst 33258 staining (B), merged images (C) and bright field (D) are shown. The intense immunostaining obtained with the anti c-myc tag antibody indicated strong expression of MasR in HEK293T cells after transfection with the pcDNA 3.1/c-myc-MasR construct. A significant amount of the total MasR pool was present at the plasma membrane. No c-myc staining was observed in cells transfected with the empty vector pcDNA 3.1. Images are representative of 3 independent experiments.

    Article Snippet: The construct was synthesized and subcloned into the pcDNA 3.1 (+) mammalian expression vector between BamHI and EcoRI restriction sites by Life Technologies (Grand Island, NY) (pcDNA 3.1/c-myc-MasR).

    Techniques: Immunofluorescence, Fluorescence, Staining, Immunostaining, Expressing, Transfection, Construct, Plasmid Preparation

    Binding of VP-14637 to cells expressing the RSV F protein. CEF cells were infected with a modified vaccinia virus expressing T7 polymerase, followed by transfection with pCDNA-F containing a T7 promoter-driven RSV F gene or a control plasmid, pCDNA-3.1. After 24 h, the cells were incubated with 10 nM [ 3 H]VP-14637 (solid bars) or 10 nM [ 3 H]VP-14637 plus 1 μM unlabeled VP-14637 (open bars). The cells were lysed, and measurements were obtained after 6 h.

    Journal: Journal of Virology

    Article Title: Inhibition of Respiratory Syncytial Virus Fusion by the Small Molecule VP-14637 via Specific Interactions with F Protein

    doi: 10.1128/JVI.77.9.5054-5064.2003

    Figure Lengend Snippet: Binding of VP-14637 to cells expressing the RSV F protein. CEF cells were infected with a modified vaccinia virus expressing T7 polymerase, followed by transfection with pCDNA-F containing a T7 promoter-driven RSV F gene or a control plasmid, pCDNA-3.1. After 24 h, the cells were incubated with 10 nM [ 3 H]VP-14637 (solid bars) or 10 nM [ 3 H]VP-14637 plus 1 μM unlabeled VP-14637 (open bars). The cells were lysed, and measurements were obtained after 6 h.

    Article Snippet: To generate the pCDNA-F construct, the F gene from RSV A2 was obtained by reverse transcription-PCR amplification of RNA isolated from RSV-infected Hep-2 cells and cloned into the pCDNA 3.1 expression vector (Invitrogen, Carlsbad, Calif.).

    Techniques: Binding Assay, Expressing, Infection, Modification, Transfection, Plasmid Preparation, Incubation

    hnRNP K Is Involved in Modulating Viral Replication HepG2 cells were co-transfected with full-length replicative HBV clones (indicated by “+”) 1752A, 1752ΔG, 1752ΔT, and 1752ΔC (see   Methods  and   Figure S1 ) with increasing dosages (50, 250, and 1,250 ng/μl) of hnRNP K variant 2 (v2) or variant 3 (v3) as indicated. pcDNA 3.1 serves as a control. Transfections were performed in duplicate; standard deviations are shown.

    Journal: PLoS Medicine

    Article Title: Host Heterogeneous Ribonucleoprotein K (hnRNP K) as a Potential Target to Suppress Hepatitis B Virus ReplicationTargeting of a Host Protein to Suppress Hepatitis B Virus Replication

    doi: 10.1371/journal.pmed.0020163

    Figure Lengend Snippet: hnRNP K Is Involved in Modulating Viral Replication HepG2 cells were co-transfected with full-length replicative HBV clones (indicated by “+”) 1752A, 1752ΔG, 1752ΔT, and 1752ΔC (see Methods and Figure S1 ) with increasing dosages (50, 250, and 1,250 ng/μl) of hnRNP K variant 2 (v2) or variant 3 (v3) as indicated. pcDNA 3.1 serves as a control. Transfections were performed in duplicate; standard deviations are shown.

    Article Snippet: hnRNP K Is an Essential Modulator of Viral Replication To demonstrate the functional connection of hnRNP K to the regulation of HBV viral load, a 1.4-kb RT-PCR fragment coding for the full-length hnRNP K gene from total RNA extracted from HepG2 cells was cloned into the mammalian expression vector pcDNA 3.1 (Invitrogen).

    Techniques: Transfection, Clone Assay, Variant Assay

    TESC mediates the activation of the IGF1R/c-Src/STAT3 signaling pathway. ( A ) The activation of c-Src, FAK, and STAT3 was assayed using western blot analysis in A549 cells transfected with the siRNA and H460 cells transfected with TESC -pcDNA3.1 vector. ( B ) The interactions between TESC, c-Src and IGF1Rβ were determined by immunoprecipitation assays in A549 cells. ( C ) The interactions between TESC, JAK2 and FAK were evaluated by immunoprecipitation assays in A549 cells. ( D ) Phosphorylation levels of IGF1Rβ in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells. ( E ) Fluorescence-activated cell sorting (FACS) analysis of ALDH1 in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells was performed using ALDEFLUOR assay. ( F ) Colony forming, sphere forming and metastatic analysis in c - Src knockdown A549 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Journal: Scientific Reports

    Article Title: Tescalcin/c-Src/IGF1Rβ-mediated STAT3 activation enhances cancer stemness and radioresistant properties through ALDH1

    doi: 10.1038/s41598-018-29142-x

    Figure Lengend Snippet: TESC mediates the activation of the IGF1R/c-Src/STAT3 signaling pathway. ( A ) The activation of c-Src, FAK, and STAT3 was assayed using western blot analysis in A549 cells transfected with the siRNA and H460 cells transfected with TESC -pcDNA3.1 vector. ( B ) The interactions between TESC, c-Src and IGF1Rβ were determined by immunoprecipitation assays in A549 cells. ( C ) The interactions between TESC, JAK2 and FAK were evaluated by immunoprecipitation assays in A549 cells. ( D ) Phosphorylation levels of IGF1Rβ in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells. ( E ) Fluorescence-activated cell sorting (FACS) analysis of ALDH1 in c - Src knockdown A549 cells and c - Src -overexpressing H460 cells was performed using ALDEFLUOR assay. ( F ) Colony forming, sphere forming and metastatic analysis in c - Src knockdown A549 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Article Snippet: Cells were transiently transfected with pcDNA3.1(+) expression vectors using Lipofectamine 2000 (Invitrogen).

    Techniques: Activation Assay, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Fluorescence, FACS, Two Tailed Test

    TESC regulates the CSC-like self-renewal properties in lung cancer cells. ( A ) Clonogenicities of TESC -knockdown A549 cells with siRNA and TESC -overexpressing H460 cells transfected with expression vector ( TESC -pcDNA3.1) were examined in vitro by colony formation assay. ( B ) Cellular levels of TESC and CSC markers including CD44, CD133, Oct3/4, Sox2, and β-catenin in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( C ) RT-PCR analysis of ALDH1 isozymes in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( D ) Changes in sphere-forming capacity in TESC -knockdown A549 cells or TESC -overexpressing H460 cells. ( E ) Changes in cellular ALDH1 levels were examined using ALDEFLUOR assay in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Journal: Scientific Reports

    Article Title: Tescalcin/c-Src/IGF1Rβ-mediated STAT3 activation enhances cancer stemness and radioresistant properties through ALDH1

    doi: 10.1038/s41598-018-29142-x

    Figure Lengend Snippet: TESC regulates the CSC-like self-renewal properties in lung cancer cells. ( A ) Clonogenicities of TESC -knockdown A549 cells with siRNA and TESC -overexpressing H460 cells transfected with expression vector ( TESC -pcDNA3.1) were examined in vitro by colony formation assay. ( B ) Cellular levels of TESC and CSC markers including CD44, CD133, Oct3/4, Sox2, and β-catenin in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( C ) RT-PCR analysis of ALDH1 isozymes in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. ( D ) Changes in sphere-forming capacity in TESC -knockdown A549 cells or TESC -overexpressing H460 cells. ( E ) Changes in cellular ALDH1 levels were examined using ALDEFLUOR assay in TESC -knockdown A549 cells and TESC -overexpressing H460 cells. Data represent mean ± SD of three independent experiments using two-tailed t-test. *P

    Article Snippet: Cells were transiently transfected with pcDNA3.1(+) expression vectors using Lipofectamine 2000 (Invitrogen).

    Techniques: Transfection, Expressing, Plasmid Preparation, In Vitro, Colony Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

    Co-culture of NSCs with Notch1-expressing cells or Dll1-expressing cells. (a) NSCs were cultured for 2 days on monolayers of Dll1 (2) or Notch1 (3) over-expressing COS7 cells and immunostained with Tuj1, a marker of neurons (red) and the NSC marker Nestin (green). (1): Control experiment with pCDNA-transfected COS7 cells. (b) NSCs were co-cultured for two (black bar: 2 days diff.) or four (white bar: 4 days diff.) days as described above. After immunostaining, Tuj1-positive neurons and Nestin-positive NSCs were counted. Values are means ± S.D. for 10 independent fields.*** P

    Journal: Nucleic Acids Research

    Article Title: The Delta intracellular domain mediates TGF-?/Activin signaling through binding to Smads and has an important bi-directional function in the Notch-Delta signaling pathway

    doi: 10.1093/nar/gkl1128

    Figure Lengend Snippet: Co-culture of NSCs with Notch1-expressing cells or Dll1-expressing cells. (a) NSCs were cultured for 2 days on monolayers of Dll1 (2) or Notch1 (3) over-expressing COS7 cells and immunostained with Tuj1, a marker of neurons (red) and the NSC marker Nestin (green). (1): Control experiment with pCDNA-transfected COS7 cells. (b) NSCs were co-cultured for two (black bar: 2 days diff.) or four (white bar: 4 days diff.) days as described above. After immunostaining, Tuj1-positive neurons and Nestin-positive NSCs were counted. Values are means ± S.D. for 10 independent fields.*** P

    Article Snippet: Cells were also transfected with pCDNA (Invitrogen) as a negative control.

    Techniques: Co-Culture Assay, Expressing, Cell Culture, Marker, Transfection, Immunostaining

    LSI NCT 5 could regulate miR-20a-5p to improve the growth and metastasis of OS cells. ( A ) RT-qPCR was used to detect the relative expression of miR-20a-5p in OS cells. ( B ) RT-qPCR was used to detect the relative expression of miR-20a-5p in cells transfected with miR-20a-5p-mimics and miR-20a-inhibit. ( C ) CCK-8 experiment was used to detect the proliferation changes of cells transfected with miR-20a-5p-mimics, miR-20a-inhibit and co-transfected with sh-LSINCT5 and pcDNA-LSINCT5. ( D , E ) Transwell test was used to detect the changes of invasion and migration of cells transfected with miR-20a-5p-mimics, miR-20a-inhibit and co-transfected with sh-LSINCT5 and pcDNA-LSINCT5. ( F ) Flow cytometry was used to detect the induction of apoptosis of cells transfected with miR-20a-5p-mimics, miR-20a-inhibit, and co-transfected with sh-LSINCT5 and pcDNA-LSINCT5. * P

    Journal: OncoTargets and therapy

    Article Title: lncRNA LSINCT5 Regulates miR-20a-5p/XIAP to Inhibit the Growth and Metastasis of Osteosarcoma Cells

    doi: 10.2147/OTT.S251843

    Figure Lengend Snippet: LSI NCT 5 could regulate miR-20a-5p to improve the growth and metastasis of OS cells. ( A ) RT-qPCR was used to detect the relative expression of miR-20a-5p in OS cells. ( B ) RT-qPCR was used to detect the relative expression of miR-20a-5p in cells transfected with miR-20a-5p-mimics and miR-20a-inhibit. ( C ) CCK-8 experiment was used to detect the proliferation changes of cells transfected with miR-20a-5p-mimics, miR-20a-inhibit and co-transfected with sh-LSINCT5 and pcDNA-LSINCT5. ( D , E ) Transwell test was used to detect the changes of invasion and migration of cells transfected with miR-20a-5p-mimics, miR-20a-inhibit and co-transfected with sh-LSINCT5 and pcDNA-LSINCT5. ( F ) Flow cytometry was used to detect the induction of apoptosis of cells transfected with miR-20a-5p-mimics, miR-20a-inhibit, and co-transfected with sh-LSINCT5 and pcDNA-LSINCT5. * P

    Article Snippet: An empty pcDNA vector was used as a negative control. miR-20a-5p mimics (miR-20a-5p-mimics), inhibitors (miR-20a-5p-inhibit), or corresponding perturbation controls (miR-NC) were synthesized by RiboBio (Guangzhou, China).

    Techniques: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Migration, Flow Cytometry

    Specific binding of LSINCT5 to miR-20a-5p. ( A ) Binding locus of miR-20a-5p and LSINCT5. ( B ) Double luciferase report determined the binding relationship between LSINCT5 and miR-20a-5p. ( C ) RIP experiment revealed the combination of LSINCT5 and miR-20a-5p. ( D ) The combination of LSINCT5 and miR-20a-5p was detected by RNA pull-down. ( E ) RT-qPCR was used to detect the relative expression of miR-20a-5p in cells transfected with sh-LSINCT5 and pcDNA-LSINCT5. * P

    Journal: OncoTargets and therapy

    Article Title: lncRNA LSINCT5 Regulates miR-20a-5p/XIAP to Inhibit the Growth and Metastasis of Osteosarcoma Cells

    doi: 10.2147/OTT.S251843

    Figure Lengend Snippet: Specific binding of LSINCT5 to miR-20a-5p. ( A ) Binding locus of miR-20a-5p and LSINCT5. ( B ) Double luciferase report determined the binding relationship between LSINCT5 and miR-20a-5p. ( C ) RIP experiment revealed the combination of LSINCT5 and miR-20a-5p. ( D ) The combination of LSINCT5 and miR-20a-5p was detected by RNA pull-down. ( E ) RT-qPCR was used to detect the relative expression of miR-20a-5p in cells transfected with sh-LSINCT5 and pcDNA-LSINCT5. * P

    Article Snippet: An empty pcDNA vector was used as a negative control. miR-20a-5p mimics (miR-20a-5p-mimics), inhibitors (miR-20a-5p-inhibit), or corresponding perturbation controls (miR-NC) were synthesized by RiboBio (Guangzhou, China).

    Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, Expressing, Transfection

    miR-20a-5p regulated XIAP to inhibit the growth and metastasis of OS cell. ( A ) RT-qPCR was used to detect the relative expression of XIAP mRNA and protein in cells transfected with pcDNA-XIAP and si-XIAP. ( B ) CCK-8 experiment was used to detect the proliferation changes of cells transfected with pcDNA-XIAP, si-XIAP, and co-transfected with miR-20a-5p-mimics and miR-20a-inhibit. ( C , D ) Transwell test was used to detect the changes of invasion and migration ability of cells transfected with pcDNA-XIAP and si-XIAP and co-transfected with miR-20a-5p-mimics and miR-20a-inhibit. ( E ) Flow cytometry was used to detect the induction of apoptosis of cells transfected with pcDNA-XIAP, si-XIAP and co-transfected with miR-20a-5p-mimics and miR-20a-inhibit. * P

    Journal: OncoTargets and therapy

    Article Title: lncRNA LSINCT5 Regulates miR-20a-5p/XIAP to Inhibit the Growth and Metastasis of Osteosarcoma Cells

    doi: 10.2147/OTT.S251843

    Figure Lengend Snippet: miR-20a-5p regulated XIAP to inhibit the growth and metastasis of OS cell. ( A ) RT-qPCR was used to detect the relative expression of XIAP mRNA and protein in cells transfected with pcDNA-XIAP and si-XIAP. ( B ) CCK-8 experiment was used to detect the proliferation changes of cells transfected with pcDNA-XIAP, si-XIAP, and co-transfected with miR-20a-5p-mimics and miR-20a-inhibit. ( C , D ) Transwell test was used to detect the changes of invasion and migration ability of cells transfected with pcDNA-XIAP and si-XIAP and co-transfected with miR-20a-5p-mimics and miR-20a-inhibit. ( E ) Flow cytometry was used to detect the induction of apoptosis of cells transfected with pcDNA-XIAP, si-XIAP and co-transfected with miR-20a-5p-mimics and miR-20a-inhibit. * P

    Article Snippet: An empty pcDNA vector was used as a negative control. miR-20a-5p mimics (miR-20a-5p-mimics), inhibitors (miR-20a-5p-inhibit), or corresponding perturbation controls (miR-NC) were synthesized by RiboBio (Guangzhou, China).

    Techniques: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Migration, Flow Cytometry

    Expression of APP in wild-type or mutant APP -transfected cells. (A) The distribution of APP was similar in APP -transfected cells. The scale bar represents 50 μm. (B) The expression of total APP and β-actin in cell lysates (5 μg protein/lane) was detected by immunoblotting. Full-sized images are available in Supplementary Material. The summed density of the two bands (arrows) was quantified as the amount of total APP and demonstrated in (C). Nc: negative control, Vec: pcDNA5/FRT/TO vector-transfected cells, Wt: wild-type, Sw: Swedish, Du: Dutch, Lo: London-type mutant-transfected cells, respectively, a.u.: arbitrary unit.

    Journal: Heliyon

    Article Title: Mutations in the β-amyloid precursor protein in familial Alzheimer’s disease increase Aβ oligomer production in cellular models

    doi: 10.1016/j.heliyon.2018.e00511

    Figure Lengend Snippet: Expression of APP in wild-type or mutant APP -transfected cells. (A) The distribution of APP was similar in APP -transfected cells. The scale bar represents 50 μm. (B) The expression of total APP and β-actin in cell lysates (5 μg protein/lane) was detected by immunoblotting. Full-sized images are available in Supplementary Material. The summed density of the two bands (arrows) was quantified as the amount of total APP and demonstrated in (C). Nc: negative control, Vec: pcDNA5/FRT/TO vector-transfected cells, Wt: wild-type, Sw: Swedish, Du: Dutch, Lo: London-type mutant-transfected cells, respectively, a.u.: arbitrary unit.

    Article Snippet: In order to introduce human wild-type APP 695 cDNA and Swedish-mutant APP 695 cDNA (K670NM671L) into a pcDNA5/FRT/TO vector (Invitrogen), pEF-BOS vectors harboring wild- or Swedish-type APP (gifted from Dementia and Higher Brain Function Research, Tokyo Metropolitan Institute of Medical Science) were used as templates to amplify APP cDNA with or without mutations by PCR.

    Techniques: Expressing, Mutagenesis, Transfection, Negative Control, Plasmid Preparation

    ORF 57 increases ORF 59/58 mRNA and protein levels. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 0.1 μg of pcDNA 3.1 ORF 57. A ds DNA probe specific for ORF 59 was used; bottom, the blot was reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. The ORF 59 protein was detected with a KSHV MAb, and then with a secondary goat anti-mouse conjugated to horseradish peroxidase and ECL (Amersham). Equal amounts (10 μg) of extracts as determined by Bradford assay from each transfection were loaded onto the gel.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities †

    doi:

    Figure Lengend Snippet: ORF 57 increases ORF 59/58 mRNA and protein levels. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 0.1 μg of pcDNA 3.1 ORF 57. A ds DNA probe specific for ORF 59 was used; bottom, the blot was reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 59/58 in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. The ORF 59 protein was detected with a KSHV MAb, and then with a secondary goat anti-mouse conjugated to horseradish peroxidase and ECL (Amersham). Equal amounts (10 μg) of extracts as determined by Bradford assay from each transfection were loaded onto the gel.

    Article Snippet: The resulting fragment was cloned into the Hin dIII and Eco RV sites of pcDNA 3 (Invitrogen) to create pcDNA 3 nut-1.

    Techniques: Northern Blot, Transfection, Western Blot, Bradford Assay

    ). Forty-eight hours posttransfection, luciferase activity was measured and values were plotted that represented fold activation relative to that of ORF 50 alone, which was set to 1. (C) Mutations in the nut-1 promoter abolish synergy between ORF 50 and ORF 57. CV-1 cells were transfected with 2 μg of pcDNA 3 ORF 50, increasing amounts of ORF 57, and either 1 μg of the wild-type nut-1-706 reporter or the nut-1-706 mutant reporter. Forty-eight hours posttransfection, luciferase activity was measured and values were plotted that represent fold activation relative to that of ORF 50 alone, which was set to 1.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities †

    doi:

    Figure Lengend Snippet: ). Forty-eight hours posttransfection, luciferase activity was measured and values were plotted that represented fold activation relative to that of ORF 50 alone, which was set to 1. (C) Mutations in the nut-1 promoter abolish synergy between ORF 50 and ORF 57. CV-1 cells were transfected with 2 μg of pcDNA 3 ORF 50, increasing amounts of ORF 57, and either 1 μg of the wild-type nut-1-706 reporter or the nut-1-706 mutant reporter. Forty-eight hours posttransfection, luciferase activity was measured and values were plotted that represent fold activation relative to that of ORF 50 alone, which was set to 1.

    Article Snippet: The resulting fragment was cloned into the Hin dIII and Eco RV sites of pcDNA 3 (Invitrogen) to create pcDNA 3 nut-1.

    Techniques: Luciferase, Activity Assay, Activation Assay, Transfection, Mutagenesis

    The C-terminally tagged version of ORF 57 is localized to the nucleus. The panels show results of immunofluorescence staining of CV-1 cells 48 h after transfection with either empty pcDNA 3.1-V5 vector (left panel) or pcDNA 3.1-V5 ORF 57 (right panel). Reactivity to anti-V5 antibody (Invitrogen) was detected with a TRITC-conjugated rabbit anti-mouse antibody.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities †

    doi:

    Figure Lengend Snippet: The C-terminally tagged version of ORF 57 is localized to the nucleus. The panels show results of immunofluorescence staining of CV-1 cells 48 h after transfection with either empty pcDNA 3.1-V5 vector (left panel) or pcDNA 3.1-V5 ORF 57 (right panel). Reactivity to anti-V5 antibody (Invitrogen) was detected with a TRITC-conjugated rabbit anti-mouse antibody.

    Article Snippet: The resulting fragment was cloned into the Hin dIII and Eco RV sites of pcDNA 3 (Invitrogen) to create pcDNA 3 nut-1.

    Techniques: Immunofluorescence, Staining, Transfection, Plasmid Preparation

    ORF 50 mRNA and protein levels are not significantly increased by ORF 57 expression. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3 ORF 50 and the following amounts of pcDNA 3.1 ORF 57: 0 μg (lane 1), 0.75 μg (lane 2), 2.5 μg (lane 3), and 7.5 μg (lane 4). An antisense riboprobe for ORF 50 was hybridized to the blot. Bottom, the blot was reprobed with a GAPDH-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 50 and the following amounts of pcDNA 3.1 ORF 57: 0 μg (lane 1), 0.5 μg (lane 2), 2.5 μg (lane 3), and 5 μg (lane 4). The ORF 50 protein was detected with a polyclonal antibody and then with a secondary rabbit anti-mouse antibody conjugated to horseradish peroxidase and ECL (Amersham). Equal amount of extracts as determined by Bradford assay from each transfection were loaded onto the gel.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities †

    doi:

    Figure Lengend Snippet: ORF 50 mRNA and protein levels are not significantly increased by ORF 57 expression. (A) Top, autoradiogram of a Northern blot containing total RNA from CV-1 cells transfected with a fixed amount of pcDNA 3 ORF 50 and the following amounts of pcDNA 3.1 ORF 57: 0 μg (lane 1), 0.75 μg (lane 2), 2.5 μg (lane 3), and 7.5 μg (lane 4). An antisense riboprobe for ORF 50 was hybridized to the blot. Bottom, the blot was reprobed with a GAPDH-specific probe as a loading control. (B) Western blot analysis of protein extracts from CV-1 cells transfected with a fixed amount of pcDNA 3.1 ORF 50 and the following amounts of pcDNA 3.1 ORF 57: 0 μg (lane 1), 0.5 μg (lane 2), 2.5 μg (lane 3), and 5 μg (lane 4). The ORF 50 protein was detected with a polyclonal antibody and then with a secondary rabbit anti-mouse antibody conjugated to horseradish peroxidase and ECL (Amersham). Equal amount of extracts as determined by Bradford assay from each transfection were loaded onto the gel.

    Article Snippet: The resulting fragment was cloned into the Hin dIII and Eco RV sites of pcDNA 3 (Invitrogen) to create pcDNA 3 nut-1.

    Techniques: Expressing, Northern Blot, Transfection, Western Blot, Bradford Assay

    Effect of introns on synergistic activation of different promoters by the ORF 50-ORF 57 combination. CV-1 cells were cotransfected with fixed amounts of the indicated KSHV promoter constructs with or without introns (Nut-1, TK, and Kaposin), a fixed amount of the transcriptional activator, pcDNA 3 ORF 50, and increasing amounts of pcDNA 3.1 ORF 57 (0, 0.3, 1, and 3 μg). Luciferase activity was measured 48 h after transfection. (A) Log scale graph displaying the effects on the Nut-1 promoter with and without an intron. (B) Graph displaying the effects on the Kaposin and TK promoters with and without an intron. Values are plotted as fold luciferase activity over that of ORF 50 alone, which was set to 1. Error bars representing standard deviations are from two experiments performed in duplicate. Note that activation is plotted on an arithmetic scale in B and on a log scale in A.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities †

    doi:

    Figure Lengend Snippet: Effect of introns on synergistic activation of different promoters by the ORF 50-ORF 57 combination. CV-1 cells were cotransfected with fixed amounts of the indicated KSHV promoter constructs with or without introns (Nut-1, TK, and Kaposin), a fixed amount of the transcriptional activator, pcDNA 3 ORF 50, and increasing amounts of pcDNA 3.1 ORF 57 (0, 0.3, 1, and 3 μg). Luciferase activity was measured 48 h after transfection. (A) Log scale graph displaying the effects on the Nut-1 promoter with and without an intron. (B) Graph displaying the effects on the Kaposin and TK promoters with and without an intron. Values are plotted as fold luciferase activity over that of ORF 50 alone, which was set to 1. Error bars representing standard deviations are from two experiments performed in duplicate. Note that activation is plotted on an arithmetic scale in B and on a log scale in A.

    Article Snippet: The resulting fragment was cloned into the Hin dIII and Eco RV sites of pcDNA 3 (Invitrogen) to create pcDNA 3 nut-1.

    Techniques: Activation Assay, Construct, Luciferase, Activity Assay, Transfection

    Effect of ORF 57 expression on viral promoter constructs with and without introns. CV-1 cells were cotransfected with increasing amounts of pcDNA 3.1 ORF 57 (0, 0.3, 1, and 3 μg) and fixed amounts of the following KSHV promoter luciferase constructs with or without an intron: DNA Pol, Kaposin, Nut-1, and TK. Luciferase activity was measured 48 h posttransfection. Graphs are representative examples of experiments performed in duplicate. Error bars represent standard deviations.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities †

    doi:

    Figure Lengend Snippet: Effect of ORF 57 expression on viral promoter constructs with and without introns. CV-1 cells were cotransfected with increasing amounts of pcDNA 3.1 ORF 57 (0, 0.3, 1, and 3 μg) and fixed amounts of the following KSHV promoter luciferase constructs with or without an intron: DNA Pol, Kaposin, Nut-1, and TK. Luciferase activity was measured 48 h posttransfection. Graphs are representative examples of experiments performed in duplicate. Error bars represent standard deviations.

    Article Snippet: The resulting fragment was cloned into the Hin dIII and Eco RV sites of pcDNA 3 (Invitrogen) to create pcDNA 3 nut-1.

    Techniques: Expressing, Construct, Luciferase, Activity Assay

    Upregulation of nut-1 RNA accumulation by ORF 57 expression. The indicated constructs (bottom) were transfected into CV-1 cells in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. Forty-eight hours later, total RNA was prepared from whole-cell lysates, electrophoresed through 1% agarose-formaldehyde gels, transferred to Hybond-N+ membranes, and hybridized to probes specific for nut-1 (A) or ORF 74/GCR (B, left panel) or ORF K5 (B, right panel). Below each panel is shown rRNA bands in ethidium bromide-stained lanes as a loading control.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities †

    doi:

    Figure Lengend Snippet: Upregulation of nut-1 RNA accumulation by ORF 57 expression. The indicated constructs (bottom) were transfected into CV-1 cells in the presence (+) or absence (−) of 1 μg of pcDNA 3.1 ORF 57. Forty-eight hours later, total RNA was prepared from whole-cell lysates, electrophoresed through 1% agarose-formaldehyde gels, transferred to Hybond-N+ membranes, and hybridized to probes specific for nut-1 (A) or ORF 74/GCR (B, left panel) or ORF K5 (B, right panel). Below each panel is shown rRNA bands in ethidium bromide-stained lanes as a loading control.

    Article Snippet: The resulting fragment was cloned into the Hin dIII and Eco RV sites of pcDNA 3 (Invitrogen) to create pcDNA 3 nut-1.

    Techniques: Expressing, Construct, Transfection, Staining

    Cloning of amiRNA into pcDNA™6.2-GW/EmGFP-miR vector. (A) Schematic representations of the JEV 3 ′ UTR structure. (B) Schematic illustrations of the three amiRNA designed against the target sites cloned into pcDNA6.2-GW/EmGFP-miR vector. amiRNA, artificial microRNA; JEV, Japanese encephalitis virus; UTR, untranslated region. Color images available online at www.liebertpub.com/nat

    Journal: Nucleic Acid Therapeutics

    Article Title: Artificial MicroRNA-Mediated Inhibition of Japanese Encephalitis Virus Replication in Neuronal Cells

    doi: 10.1089/nat.2018.0743

    Figure Lengend Snippet: Cloning of amiRNA into pcDNA™6.2-GW/EmGFP-miR vector. (A) Schematic representations of the JEV 3 ′ UTR structure. (B) Schematic illustrations of the three amiRNA designed against the target sites cloned into pcDNA6.2-GW/EmGFP-miR vector. amiRNA, artificial microRNA; JEV, Japanese encephalitis virus; UTR, untranslated region. Color images available online at www.liebertpub.com/nat

    Article Snippet: We selected top three high scoring amiRNA sequences and the oligos were synthesized (Sigma) along with a control sequence, which was not specific against JEV 3′UTR sequence ( and ). amiRNAs were cloned into pcDNA™6.2-GW/EmGFP-miR vector (Invitrogen, Inc.) according to the manufacturer's protocol ( ).

    Techniques: Clone Assay, Plasmid Preparation

    Calibration EYFP-53BP1 reporter system. ( A ) EYFP-53BP1 primary structures. EYFP coding sequence is joined to full-length 53BP1, with Tudor and BRCT domains indicated. EYFP1-53BP coding sequence was inserted into pcDNA-DEST40 (Invitrogen) for expression under control of the CMV promoter. ( B ) Dose response to calibrated doses of 137 Cs reference radiation. Cells were co-transfected with EYFP-53BP1 WT and H2B-diHcRed. Live-cell images were collected 30 min post-irradiation. ( C ) Quantification of dose response. Foci were scored using 8–24 individual nuclei at each dose point, and linear regression was performed to obtain the slope of the dose-response curve.

    Journal: Nucleic Acids Research

    Article Title: Use of a microscope stage-mounted Nickel-63 microirradiator for real-time observation of the DNA double-strand break response

    doi: 10.1093/nar/gkq409

    Figure Lengend Snippet: Calibration EYFP-53BP1 reporter system. ( A ) EYFP-53BP1 primary structures. EYFP coding sequence is joined to full-length 53BP1, with Tudor and BRCT domains indicated. EYFP1-53BP coding sequence was inserted into pcDNA-DEST40 (Invitrogen) for expression under control of the CMV promoter. ( B ) Dose response to calibrated doses of 137 Cs reference radiation. Cells were co-transfected with EYFP-53BP1 WT and H2B-diHcRed. Live-cell images were collected 30 min post-irradiation. ( C ) Quantification of dose response. Foci were scored using 8–24 individual nuclei at each dose point, and linear regression was performed to obtain the slope of the dose-response curve.

    Article Snippet: The resulting construct was transferred using phage lambda recombinase into pcDNA-DEST40 (Invitrogen) for mammalian expression under the control of the CMV promoter/enhancer.

    Techniques: Sequencing, Expressing, Transfection, Irradiation