control pcmv6 gfp plasmids Search Results


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  • 92
    OriGene control pcmv6 gfp plasmids
    Determination of transfection efficiency of <t>pCMV6-AC-GFP</t> vector using flowcytometry. Right: detection of transfection efficiency by flowcytometry. Transfection efficiency was maintained at 60%, 48 hr post-transfection. Left: dot blot flowcytometry after 48 hr. High efficiency of transfection with fluorescent GFP (green) in A549 cells was easily identified for 48 hr post-transfection (×100). Green fluorescence intensities (FL1-H) are indicated on the x axis, and cell counts are indicated on the y axis. The cytometric analysis was performed for 2 × 10 4 events
    Control Pcmv6 Gfp Plasmids, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Shanghai GenePharma control plasmid pcmv6 ac gfp
    Determination of transfection efficiency of <t>pCMV6-AC-GFP</t> vector using flowcytometry. Right: detection of transfection efficiency by flowcytometry. Transfection efficiency was maintained at 60%, 48 hr post-transfection. Left: dot blot flowcytometry after 48 hr. High efficiency of transfection with fluorescent GFP (green) in A549 cells was easily identified for 48 hr post-transfection (×100). Green fluorescence intensities (FL1-H) are indicated on the x axis, and cell counts are indicated on the y axis. The cytometric analysis was performed for 2 × 10 4 events
    Control Plasmid Pcmv6 Ac Gfp, supplied by Shanghai GenePharma, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    OriGene control pcmv6 gfp vectors
    excessive PTEN abrogates the oncogenic action of HULC. a Cells growthassay using CCK8. b Cell BrdU staining assay. c Cells colony formation assay. d Tumorigenesis test in vivo. The wet weight of tumor was determined for each mouse. e The appearance time of tumor was determined for each mouse. f PCNA staining (DAB staining, original magnification× 100). g positive rate of PCNA staining. h Cells growth assay using CCK8 in <t>pCMV6-A-GFP</t> control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP-HULC+ 3-Methyladenine (3-MA). i Cells colony formation assay in pCMV6-A-GFP control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP–HULC + 3-Methyladenine (3-MA)
    Control Pcmv6 Gfp Vectors, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    OriGene pcmv6 ac gfp control vector a549 pctl
    excessive PTEN abrogates the oncogenic action of HULC. a Cells growthassay using CCK8. b Cell BrdU staining assay. c Cells colony formation assay. d Tumorigenesis test in vivo. The wet weight of tumor was determined for each mouse. e The appearance time of tumor was determined for each mouse. f PCNA staining (DAB staining, original magnification× 100). g positive rate of PCNA staining. h Cells growth assay using CCK8 in <t>pCMV6-A-GFP</t> control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP-HULC+ 3-Methyladenine (3-MA). i Cells colony formation assay in pCMV6-A-GFP control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP–HULC + 3-Methyladenine (3-MA)
    Pcmv6 Ac Gfp Control Vector A549 Pctl, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    OriGene pcmv6 ac gfp redd1 plasmid dna
    excessive PTEN abrogates the oncogenic action of HULC. a Cells growthassay using CCK8. b Cell BrdU staining assay. c Cells colony formation assay. d Tumorigenesis test in vivo. The wet weight of tumor was determined for each mouse. e The appearance time of tumor was determined for each mouse. f PCNA staining (DAB staining, original magnification× 100). g positive rate of PCNA staining. h Cells growth assay using CCK8 in <t>pCMV6-A-GFP</t> control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP-HULC+ 3-Methyladenine (3-MA). i Cells colony formation assay in pCMV6-A-GFP control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP–HULC + 3-Methyladenine (3-MA)
    Pcmv6 Ac Gfp Redd1 Plasmid Dna, supplied by OriGene, used in various techniques. Bioz Stars score: 85/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Genecopoeia pcmv6 ac gfp tgfp
    PCDHB13 disrupts microtubule dynamics in NSCLC cells. ( A ) Cellular localization of PCDHB13 in A549 cells. Cells were stained with PCDHB13 (green) and α-Tubulin (red). Hoechst33342 was used to stain nuclei (blue). Boxed regions are magnified in the panels to the right. Red arrows indicate the filamentary structure of PCDHB13. White circles indicate the cellular aggregates formed by PCDHB13 and α-Tubulin. Bar: 20 μm. ( B ) PCDHB13 and α-Tubulin are shown in red and green, respectively. Cytoskeletal projections of each protein were simulated by Imaris software. Yellow color indicates colocalization of PCDHB13 and α-Tubulin. Bar: 8 μm. ( C ) A549 cell lysates without (lane 1 and 3) or with <t>tGFP-tagged</t> PCDHB13 overexpression (lane 2 and 4) were harvested for co-immunoprecipitation (Co-IP) assay using the anti-tGFP conjugated magnetic beads. IP eluates were analyzed by Western blot for PCDHB13 and α-Tubulin. ( D , E ) <t>GFP</t> ( D ) or PCDHB13-tGFP vector ( E ) transfected A549 and H1975 cells pretreated with 20 μM nocodazole were stained for PCDHB13 (red) and α-Tubulin (white) at 0, 10, and 30 min after nocodazole washout. Higher magnifications of the areas in the white boxes marked by * or # are shown in the corresponding panel. Hoechst33342 was stained for nuclei (blue). Green arrows indicate MTOC with defective microtubule outgrowth. The spatial correlation of PCDHB13 with microtubules in the indicated region (pink lines mark two opposite ends, indicated by pink arrows) is shown by a line-series analysis. The red and white lines in the right panels represent the PCDHB13 and α-Tubulin fluorescence intensities, respectively. au: arbitrary units. Bar: 20 μm. ( F ) Quantification of MTOC formation in cells transfected by GFP or PCDHB13 after nocodazole washout. g Illustration of the phenomena shown in ( D , E ).
    Pcmv6 Ac Gfp Tgfp, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene control gfp
    PCDHB13 disrupts microtubule dynamics in NSCLC cells. ( A ) Cellular localization of PCDHB13 in A549 cells. Cells were stained with PCDHB13 (green) and α-Tubulin (red). Hoechst33342 was used to stain nuclei (blue). Boxed regions are magnified in the panels to the right. Red arrows indicate the filamentary structure of PCDHB13. White circles indicate the cellular aggregates formed by PCDHB13 and α-Tubulin. Bar: 20 μm. ( B ) PCDHB13 and α-Tubulin are shown in red and green, respectively. Cytoskeletal projections of each protein were simulated by Imaris software. Yellow color indicates colocalization of PCDHB13 and α-Tubulin. Bar: 8 μm. ( C ) A549 cell lysates without (lane 1 and 3) or with <t>tGFP-tagged</t> PCDHB13 overexpression (lane 2 and 4) were harvested for co-immunoprecipitation (Co-IP) assay using the anti-tGFP conjugated magnetic beads. IP eluates were analyzed by Western blot for PCDHB13 and α-Tubulin. ( D , E ) <t>GFP</t> ( D ) or PCDHB13-tGFP vector ( E ) transfected A549 and H1975 cells pretreated with 20 μM nocodazole were stained for PCDHB13 (red) and α-Tubulin (white) at 0, 10, and 30 min after nocodazole washout. Higher magnifications of the areas in the white boxes marked by * or # are shown in the corresponding panel. Hoechst33342 was stained for nuclei (blue). Green arrows indicate MTOC with defective microtubule outgrowth. The spatial correlation of PCDHB13 with microtubules in the indicated region (pink lines mark two opposite ends, indicated by pink arrows) is shown by a line-series analysis. The red and white lines in the right panels represent the PCDHB13 and α-Tubulin fluorescence intensities, respectively. au: arbitrary units. Bar: 20 μm. ( F ) Quantification of MTOC formation in cells transfected by GFP or PCDHB13 after nocodazole washout. g Illustration of the phenomena shown in ( D , E ).
    Control Gfp, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    OriGene prfp v rs pcmv6 a gfp
    CUDR combined cyclinD1 or PTEN depletion collectly increases telomerase activity through H19 in human liver cancer stem cell A . RT-PCR analysis of TERC mRNA and Western blotting with anti-cyclinD1, anti-PTEN expression in stable liver cancer stem cells transfected with <t>pCMV6-A-GFP,</t> pCMV6-A-GFP-CUDR, pCMV6-A- GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively (indicated in the left ). β-actin as internal control. B. RNA Immunoprecipitation(RIP) with anti-TERT followed by RT-PCR with TERC mRNA primers in contro or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG RIP as negative controlTERC mRNA as INPUT. C. RT-PCR analysis of TERRA mRNA in control or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN, respectively. D. RNA Immunoprecipitation(RIP) with anti-TERT followed by RT-PCR with TERRA mRNA primers in contro or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG RIP as negative control TERC mRNA as INPUT. E. Super-EMSA(gel-shift) with biotin- TERRA cRNA probe and anti-TERT antibody. The intensity of the band was examined by Western blotting with anti-Biotin. F. Telomerase activity assay with TRAP method mRNA in control or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. Each value was presented as mean ± standard error of the mean (SEM). ** P
    Prfp V Rs Pcmv6 A Gfp, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    OriGene pcmv6 ac gfp catalogue ps100010 vector
    CUDR combined cyclinD1 or PTEN depletion collectly increases telomerase activity through H19 in human liver cancer stem cell A . RT-PCR analysis of TERC mRNA and Western blotting with anti-cyclinD1, anti-PTEN expression in stable liver cancer stem cells transfected with <t>pCMV6-A-GFP,</t> pCMV6-A-GFP-CUDR, pCMV6-A- GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively (indicated in the left ). β-actin as internal control. B. RNA Immunoprecipitation(RIP) with anti-TERT followed by RT-PCR with TERC mRNA primers in contro or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG RIP as negative controlTERC mRNA as INPUT. C. RT-PCR analysis of TERRA mRNA in control or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN, respectively. D. RNA Immunoprecipitation(RIP) with anti-TERT followed by RT-PCR with TERRA mRNA primers in contro or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG RIP as negative control TERC mRNA as INPUT. E. Super-EMSA(gel-shift) with biotin- TERRA cRNA probe and anti-TERT antibody. The intensity of the band was examined by Western blotting with anti-Biotin. F. Telomerase activity assay with TRAP method mRNA in control or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. Each value was presented as mean ± standard error of the mean (SEM). ** P
    Pcmv6 Ac Gfp Catalogue Ps100010 Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene l521f mutant pcmv6 ac gfp vector
    Expression of CLCN5 constructs in human podocyte cell lines. Western blot (WB) showed a single band consistent with the short 83-kDa CLCN5 proteoform. Neph1 expression, which was used a positive control, was confirmed using an anti-Neph1 antibody (Ab; a). Endogenous expression of CLCN5 was also determined by immunofluorescence in cultured human podocytes (b). The upper panel shows human podocytes transfected with green fluorescent protein <t>(GFP)–tagged</t> wild-type (WT) CLCN5 protein demonstrated predominantly cell surface distribution and co-localization with cell surface marker ZO-1. The lower panel shows cultured human podocytes transfected with GFP-tagged <t>L521F</t> mutant CLCN5 protein, which demonstrated predominantly intracellular distribution (c). DAPI, 4′,6-diamidino-2-phenylindole.
    L521f Mutant Pcmv6 Ac Gfp Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    OriGene olfm4 expression plasmid
    Relationship between <t>OLFM4</t> expression and prognosis. (a) Immunohistochemical staining for OLFM4 in pancreatic cancer tissues (magnification: 100×). The left and right figures are the same sample tissue blocks and correspond to staining intensity. Left: HE staining. Right: immunohistochemical staining for OLFM4. (b) Criteria for determination of OLFM4 expression levels. OLFM4 expression levels for immunostaining were determined based on the intensity of staining and percentage of stained cells. Staining intensity and staining percentage criteria are shown. (c) Kaplan-Meier survival analysis in patients with pancreatic cancer (n = 80), showing overall survival according to OLFM4 protein expression. Red line: high expression group (n = 52); blue line: low expression group (n = 28).
    Olfm4 Expression Plasmid, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    OriGene cmv promoter
    Relationship between <t>OLFM4</t> expression and prognosis. (a) Immunohistochemical staining for OLFM4 in pancreatic cancer tissues (magnification: 100×). The left and right figures are the same sample tissue blocks and correspond to staining intensity. Left: HE staining. Right: immunohistochemical staining for OLFM4. (b) Criteria for determination of OLFM4 expression levels. OLFM4 expression levels for immunostaining were determined based on the intensity of staining and percentage of stained cells. Staining intensity and staining percentage criteria are shown. (c) Kaplan-Meier survival analysis in patients with pancreatic cancer (n = 80), showing overall survival according to OLFM4 protein expression. Red line: high expression group (n = 52); blue line: low expression group (n = 28).
    Cmv Promoter, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 458 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene trim21
    <t>Trim21</t> and LFG expression. (A) Results of real-time PCR-analysis of Trim21-expression after 24 h under different culture conditions. Mock, Untreated MDA-MB-231 as negative control, Trim21, 2.0 μl recombinant human Trim21 protein added to culture medium; siControl, adenoviral transfection of MDA-MB-231 with empty vector; siLFG, adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG. (B) Expression analysis of LFG protein under different culture conditions by western blot analysis using 25 μg of total protein. a, Adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG (24 h). b, Adenoviral transfection of MDA-MB-231 with empty vector. c, Untreated MDA-MB-231 as negative control. d, MDA-MB231 cultivated with 2.0 μg recombinant Trim21-protein in culture medium.
    Trim21, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Determination of transfection efficiency of pCMV6-AC-GFP vector using flowcytometry. Right: detection of transfection efficiency by flowcytometry. Transfection efficiency was maintained at 60%, 48 hr post-transfection. Left: dot blot flowcytometry after 48 hr. High efficiency of transfection with fluorescent GFP (green) in A549 cells was easily identified for 48 hr post-transfection (×100). Green fluorescence intensities (FL1-H) are indicated on the x axis, and cell counts are indicated on the y axis. The cytometric analysis was performed for 2 × 10 4 events

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: N-myc downstream regulated gene 2 overexpression reduces matrix metalloproteinase-2 and -9 activities and cell invasion of A549 lung cancer cell line in vitro

    doi:

    Figure Lengend Snippet: Determination of transfection efficiency of pCMV6-AC-GFP vector using flowcytometry. Right: detection of transfection efficiency by flowcytometry. Transfection efficiency was maintained at 60%, 48 hr post-transfection. Left: dot blot flowcytometry after 48 hr. High efficiency of transfection with fluorescent GFP (green) in A549 cells was easily identified for 48 hr post-transfection (×100). Green fluorescence intensities (FL1-H) are indicated on the x axis, and cell counts are indicated on the y axis. The cytometric analysis was performed for 2 × 10 4 events

    Article Snippet: Plasmid amplification and purification A plasmid encoding C-terminal green fluorescent protein (GFP)-tagged NDRG2 (pCMV6–AC–GFP-NDRG2) and a negative control pCMV6–AC-GFP plasmid without NDRG2 (mock plasmid) were purchased from OriGene (OriGene, USA).

    Techniques: Transfection, Plasmid Preparation, Dot Blot, Fluorescence

    GLI1 regulates VEGFR2 expression. ( A ) Western blot analysis of protein expression in MDA-MB-468 and HUVEC cells treated with NVP-LDE225 (2.5 μ M ). ( B ) Relative expression of sVEGFR2 mRNA in MDA-MB-468 and HUVEC cells treated with NVP-LDE225 (2.5 μ M ), as performed by real-time RT–PCR (qRT-PCR) analysis. Data were calculated with mean cycle threshold (CT) values, normalised to endogenous control. Data represent the mean (±s.d.) of three independent experiments, each performed in triplicate. ( C ) Western blot analysis of protein expression in MDA-MB-468 and HUVEC cells, 24 and 48 h after transfection with scramble or GLI1 siRNA pool (50 nmol l −1 ) using DharmaFECT 1 Transfection Reagent in DMEM. ( D ) Western blot analysis of protein expression in MDA-MB-231 cells, 24 h after transfection with either pCMV6-GFP empty vector, pCMV6-GFP GLI1 or pCMV6-GFP tGLI1 plasmids using lipofectamine 2000 in DMEM. ( E ) Relative luciferase units in MDA-MB-468 cells transfected with the empty pGL4 plasmid or the pGL4 plasmid containing 500 bp fragment of VEGFR2 promoter, and treated with NVP-LDE225 5 μ M for 24 h after transfection. Luciferase activity was determined 48 h after transfection. Results were the average of three independent experiments. ( F ) Chromatin immunoprecipitation (ChIP) assay in MDA-MB-468 cells by using a GLI1 antibody and primers specific for the VEGFR2 promoter. Results were reported as fold change compared to negative control (no antibody); results were the average of three independent experiments. Bars, s.d. Asterisks indicate statistical significance, as determined by the Student t -test (** P

    Journal: British Journal of Cancer

    Article Title: Hedgehog signalling pathway orchestrates angiogenesis in triple-negative breast cancers

    doi: 10.1038/bjc.2017.116

    Figure Lengend Snippet: GLI1 regulates VEGFR2 expression. ( A ) Western blot analysis of protein expression in MDA-MB-468 and HUVEC cells treated with NVP-LDE225 (2.5 μ M ). ( B ) Relative expression of sVEGFR2 mRNA in MDA-MB-468 and HUVEC cells treated with NVP-LDE225 (2.5 μ M ), as performed by real-time RT–PCR (qRT-PCR) analysis. Data were calculated with mean cycle threshold (CT) values, normalised to endogenous control. Data represent the mean (±s.d.) of three independent experiments, each performed in triplicate. ( C ) Western blot analysis of protein expression in MDA-MB-468 and HUVEC cells, 24 and 48 h after transfection with scramble or GLI1 siRNA pool (50 nmol l −1 ) using DharmaFECT 1 Transfection Reagent in DMEM. ( D ) Western blot analysis of protein expression in MDA-MB-231 cells, 24 h after transfection with either pCMV6-GFP empty vector, pCMV6-GFP GLI1 or pCMV6-GFP tGLI1 plasmids using lipofectamine 2000 in DMEM. ( E ) Relative luciferase units in MDA-MB-468 cells transfected with the empty pGL4 plasmid or the pGL4 plasmid containing 500 bp fragment of VEGFR2 promoter, and treated with NVP-LDE225 5 μ M for 24 h after transfection. Luciferase activity was determined 48 h after transfection. Results were the average of three independent experiments. ( F ) Chromatin immunoprecipitation (ChIP) assay in MDA-MB-468 cells by using a GLI1 antibody and primers specific for the VEGFR2 promoter. Results were reported as fold change compared to negative control (no antibody); results were the average of three independent experiments. Bars, s.d. Asterisks indicate statistical significance, as determined by the Student t -test (** P

    Article Snippet: For transfection, 5 μ g of either pCMV6-GFP GLI1, pCMV6-GFP tGLI1, pCMV6-GFP empty vector plasmid (Origene, Rockville, MD, USA) were added together with lipofectamine 2000 Reagent (Invitrogen), following the manufacturer’s instructions.

    Techniques: Expressing, Western Blot, Multiple Displacement Amplification, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Negative Control

    excessive PTEN abrogates the oncogenic action of HULC. a Cells growthassay using CCK8. b Cell BrdU staining assay. c Cells colony formation assay. d Tumorigenesis test in vivo. The wet weight of tumor was determined for each mouse. e The appearance time of tumor was determined for each mouse. f PCNA staining (DAB staining, original magnification× 100). g positive rate of PCNA staining. h Cells growth assay using CCK8 in pCMV6-A-GFP control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP-HULC+ 3-Methyladenine (3-MA). i Cells colony formation assay in pCMV6-A-GFP control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP–HULC + 3-Methyladenine (3-MA)

    Journal: Molecular Cancer

    Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

    doi: 10.1186/s12943-018-0843-8

    Figure Lengend Snippet: excessive PTEN abrogates the oncogenic action of HULC. a Cells growthassay using CCK8. b Cell BrdU staining assay. c Cells colony formation assay. d Tumorigenesis test in vivo. The wet weight of tumor was determined for each mouse. e The appearance time of tumor was determined for each mouse. f PCNA staining (DAB staining, original magnification× 100). g positive rate of PCNA staining. h Cells growth assay using CCK8 in pCMV6-A-GFP control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP-HULC+ 3-Methyladenine (3-MA). i Cells colony formation assay in pCMV6-A-GFP control group, pCMV6-A-GFP-HULC group, and pCMV6-A-GFP–HULC + 3-Methyladenine (3-MA)

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-A-GFP, pCMV6-XL5-PTEN, pGFP-V-RS-PTEN, pGFP-V-RS-Sirt1, pMiR-Target were purchased from Origene (Rockville, MD 20850, USA). pEZX-MT01-PTEN-3’UTR was purchased from GeneCopeia (Rockville, MD, USA).pCMV6-AC-GFP-HULC [the HULC sequence (NR_004855) was synthesized and cloned into the cloning site ofpCMV6-AC-GFP plasmid (Origene)], pcDNA3-sirt1[the Sirt1 sequence in the Flag-SIRT1 (Plasmid #1791,Addigene) was digested and subcloned into the cloning site of pcDNA3], pcDNA3-sirt mutant [the mutant Sirt1 (H363Y) sequence in the Flag-SIRT1 H363Y (Plasmid #1792, addigene) was digested and sub-cloned into the pcDNA3] were constructed by our lab.

    Techniques: BrdU Staining, Colony Assay, In Vivo, Staining, Growth Assay

    HULC activates AKT-PI3K-mTOR pathway via PTEN reduction in liver cancer cells. a Western blotting with anti-PTEN, anti-pPTEN, anti-EGFR (Tyr845),anti-AKT,anti-pAKT,anti-TmTOR,anti-PI3K,anti-pPI3Kanti-mTOR,anti-pmTOR, anti-JUN, anti-Survivin and RT-PCR with HULC primer in Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-HULC,and pCMV6-A-GFP -HULC plus pCMV6-XL5-PTEN,respectively. b (left) CHIP with anti-JUN followed by PCR with H-Ras promoter primers in Hep3B cell line. IgG CHIP as negative control. H-Ras promoter as INPUT. ( right ) quantitative CHIP. c The assay of H-Ras promoter luciferase activity in Hep3B. d (left) The RT-PCR with H-Ras primers in Hep3B cell line. (right) The real-time RT-PCR. e (left) Western blotting with anti-H-Ras in Hep3B cell line. (right) The density analysis of band

    Journal: Molecular Cancer

    Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

    doi: 10.1186/s12943-018-0843-8

    Figure Lengend Snippet: HULC activates AKT-PI3K-mTOR pathway via PTEN reduction in liver cancer cells. a Western blotting with anti-PTEN, anti-pPTEN, anti-EGFR (Tyr845),anti-AKT,anti-pAKT,anti-TmTOR,anti-PI3K,anti-pPI3Kanti-mTOR,anti-pmTOR, anti-JUN, anti-Survivin and RT-PCR with HULC primer in Hep3B cell lines transfected with pCMV6-A-GFP, pCMV6-A-GFP-HULC,and pCMV6-A-GFP -HULC plus pCMV6-XL5-PTEN,respectively. b (left) CHIP with anti-JUN followed by PCR with H-Ras promoter primers in Hep3B cell line. IgG CHIP as negative control. H-Ras promoter as INPUT. ( right ) quantitative CHIP. c The assay of H-Ras promoter luciferase activity in Hep3B. d (left) The RT-PCR with H-Ras primers in Hep3B cell line. (right) The real-time RT-PCR. e (left) Western blotting with anti-H-Ras in Hep3B cell line. (right) The density analysis of band

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-A-GFP, pCMV6-XL5-PTEN, pGFP-V-RS-PTEN, pGFP-V-RS-Sirt1, pMiR-Target were purchased from Origene (Rockville, MD 20850, USA). pEZX-MT01-PTEN-3’UTR was purchased from GeneCopeia (Rockville, MD, USA).pCMV6-AC-GFP-HULC [the HULC sequence (NR_004855) was synthesized and cloned into the cloning site ofpCMV6-AC-GFP plasmid (Origene)], pcDNA3-sirt1[the Sirt1 sequence in the Flag-SIRT1 (Plasmid #1791,Addigene) was digested and subcloned into the cloning site of pcDNA3], pcDNA3-sirt mutant [the mutant Sirt1 (H363Y) sequence in the Flag-SIRT1 H363Y (Plasmid #1792, addigene) was digested and sub-cloned into the pcDNA3] were constructed by our lab.

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Luciferase, Activity Assay, Quantitative RT-PCR

    HULC alters gene expression via autophagy. a Co-Immunoprecipitation (IP) with anti-ATG3 followed by Western blotting with anti-LC3 in Hep3B cell line. IgG IP as negative control. INPUT refers to Western blotting with. Anti-LC3. b Western blotting with anti-becline-1 in Hep3B cell line. c Observation for autophagy (LC3-RFP) in liver cancer cells Hep3B cell line. Scale bars, 100 μm. d Western blotting with anti-SAPK/JaK, anti-PKM2, anti-CDK2, anti-Notch1, anti-C-Jun,anti-PTEN, anti-β-catenin in Hep3B cell lines under starvation or transfected with pCMV6-A-GFP, pCMV6-A-GFP-HULC or pCMV6-A-GFP- HULC plus 3-methyladenine (3-MA),respectively

    Journal: Molecular Cancer

    Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

    doi: 10.1186/s12943-018-0843-8

    Figure Lengend Snippet: HULC alters gene expression via autophagy. a Co-Immunoprecipitation (IP) with anti-ATG3 followed by Western blotting with anti-LC3 in Hep3B cell line. IgG IP as negative control. INPUT refers to Western blotting with. Anti-LC3. b Western blotting with anti-becline-1 in Hep3B cell line. c Observation for autophagy (LC3-RFP) in liver cancer cells Hep3B cell line. Scale bars, 100 μm. d Western blotting with anti-SAPK/JaK, anti-PKM2, anti-CDK2, anti-Notch1, anti-C-Jun,anti-PTEN, anti-β-catenin in Hep3B cell lines under starvation or transfected with pCMV6-A-GFP, pCMV6-A-GFP-HULC or pCMV6-A-GFP- HULC plus 3-methyladenine (3-MA),respectively

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-A-GFP, pCMV6-XL5-PTEN, pGFP-V-RS-PTEN, pGFP-V-RS-Sirt1, pMiR-Target were purchased from Origene (Rockville, MD 20850, USA). pEZX-MT01-PTEN-3’UTR was purchased from GeneCopeia (Rockville, MD, USA).pCMV6-AC-GFP-HULC [the HULC sequence (NR_004855) was synthesized and cloned into the cloning site ofpCMV6-AC-GFP plasmid (Origene)], pcDNA3-sirt1[the Sirt1 sequence in the Flag-SIRT1 (Plasmid #1791,Addigene) was digested and subcloned into the cloning site of pcDNA3], pcDNA3-sirt mutant [the mutant Sirt1 (H363Y) sequence in the Flag-SIRT1 H363Y (Plasmid #1792, addigene) was digested and sub-cloned into the pcDNA3] were constructed by our lab.

    Techniques: Expressing, Immunoprecipitation, Western Blot, Negative Control, Transfection

    HULC inhibits PTEN through ubiquitin–proteasome system mediated by autophagy-P62. a Western blotting with anti-PTEN and RT-PCR with PTEN primer in Hep3B cell line. β-actin as internal control. b Western blotting with anti-PTEN and RT-PCR with PTEN primer in Hep3B cell lines transfected with pCMV6-A-GFP plus pCMV6-XL5-PTEN (FL), pCMV6-A-GFP-HULC plus pCMV6-XL5-PTEN (FL),respectively. c (left) Co-Immunoprecipitation (IP) with anti-62 followed by Western blotting with anti-PTEN in Hep3B cell lines transfected with pCMV6 -A-GFP plus pCMV6 -XL5-PTEN (FL), pCMV6-A-GFP-HULC plus pCMV6 -XL5-PTEN (FL), pCMV6-A-GFP-HULC plus pGFP-V-RS--Sirt1 plus pCMV6-XL5-PTEN (FL), and pCMV6-A-GFP-HULC plus pLV-miR15a plus pCMV6-XL5-PTEN (FL), respectively. ( right) density analysis of band. d (left) Co-Immunoprecipitation (IP) with anti-HA followed by Western blotting with anti-PTEN in the Hep3B cell line. INPUT refers to Western blotting with. Anti-PTEN. ( right) density analysis of band. e (left) Co-Immunoprecipitation (IP) with anti-HA followed by Western blotting with anti-PTEN in the Hep3B cell line. ( right) density analysis of band. f (left) Cells were incubated with 50 μM MG132 (Sigma) for 6 h at proper after transfection. Western blotting with anti-PTEN in Hep3B cell lines transfected with pCMV6-A-GFP plus pCMV6-XL5-PTEN (FL). g . The assay of PTEN 3'-UTR luciferase activity in Hep3B cells infected with pCMV6-A-GFP or pCMV6-A-GFP-HULC

    Journal: Molecular Cancer

    Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

    doi: 10.1186/s12943-018-0843-8

    Figure Lengend Snippet: HULC inhibits PTEN through ubiquitin–proteasome system mediated by autophagy-P62. a Western blotting with anti-PTEN and RT-PCR with PTEN primer in Hep3B cell line. β-actin as internal control. b Western blotting with anti-PTEN and RT-PCR with PTEN primer in Hep3B cell lines transfected with pCMV6-A-GFP plus pCMV6-XL5-PTEN (FL), pCMV6-A-GFP-HULC plus pCMV6-XL5-PTEN (FL),respectively. c (left) Co-Immunoprecipitation (IP) with anti-62 followed by Western blotting with anti-PTEN in Hep3B cell lines transfected with pCMV6 -A-GFP plus pCMV6 -XL5-PTEN (FL), pCMV6-A-GFP-HULC plus pCMV6 -XL5-PTEN (FL), pCMV6-A-GFP-HULC plus pGFP-V-RS--Sirt1 plus pCMV6-XL5-PTEN (FL), and pCMV6-A-GFP-HULC plus pLV-miR15a plus pCMV6-XL5-PTEN (FL), respectively. ( right) density analysis of band. d (left) Co-Immunoprecipitation (IP) with anti-HA followed by Western blotting with anti-PTEN in the Hep3B cell line. INPUT refers to Western blotting with. Anti-PTEN. ( right) density analysis of band. e (left) Co-Immunoprecipitation (IP) with anti-HA followed by Western blotting with anti-PTEN in the Hep3B cell line. ( right) density analysis of band. f (left) Cells were incubated with 50 μM MG132 (Sigma) for 6 h at proper after transfection. Western blotting with anti-PTEN in Hep3B cell lines transfected with pCMV6-A-GFP plus pCMV6-XL5-PTEN (FL). g . The assay of PTEN 3'-UTR luciferase activity in Hep3B cells infected with pCMV6-A-GFP or pCMV6-A-GFP-HULC

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-A-GFP, pCMV6-XL5-PTEN, pGFP-V-RS-PTEN, pGFP-V-RS-Sirt1, pMiR-Target were purchased from Origene (Rockville, MD 20850, USA). pEZX-MT01-PTEN-3’UTR was purchased from GeneCopeia (Rockville, MD, USA).pCMV6-AC-GFP-HULC [the HULC sequence (NR_004855) was synthesized and cloned into the cloning site ofpCMV6-AC-GFP plasmid (Origene)], pcDNA3-sirt1[the Sirt1 sequence in the Flag-SIRT1 (Plasmid #1791,Addigene) was digested and subcloned into the cloning site of pcDNA3], pcDNA3-sirt mutant [the mutant Sirt1 (H363Y) sequence in the Flag-SIRT1 H363Y (Plasmid #1792, addigene) was digested and sub-cloned into the pcDNA3] were constructed by our lab.

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunoprecipitation, Incubation, Luciferase, Activity Assay, Infection

    HULC promotes liver cancer cell growth in vitro. a The photography of the Hep3B cell lines transfected with pCMV6-A-GFP or pCMV6-A-GFP-HULC. b RT-PCR for HULC in HULC overexpressed control Hep3B stable cell lines; β-actin as internal control. c Cell proliferation assay was performed in 96-well format using the CCK8 cells proliferation kit to determine the cell viability as described by the manufacturer. Data are means of value from three independent experiments, bar±SEM. **, P

    Journal: Molecular Cancer

    Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

    doi: 10.1186/s12943-018-0843-8

    Figure Lengend Snippet: HULC promotes liver cancer cell growth in vitro. a The photography of the Hep3B cell lines transfected with pCMV6-A-GFP or pCMV6-A-GFP-HULC. b RT-PCR for HULC in HULC overexpressed control Hep3B stable cell lines; β-actin as internal control. c Cell proliferation assay was performed in 96-well format using the CCK8 cells proliferation kit to determine the cell viability as described by the manufacturer. Data are means of value from three independent experiments, bar±SEM. **, P

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-A-GFP, pCMV6-XL5-PTEN, pGFP-V-RS-PTEN, pGFP-V-RS-Sirt1, pMiR-Target were purchased from Origene (Rockville, MD 20850, USA). pEZX-MT01-PTEN-3’UTR was purchased from GeneCopeia (Rockville, MD, USA).pCMV6-AC-GFP-HULC [the HULC sequence (NR_004855) was synthesized and cloned into the cloning site ofpCMV6-AC-GFP plasmid (Origene)], pcDNA3-sirt1[the Sirt1 sequence in the Flag-SIRT1 (Plasmid #1791,Addigene) was digested and subcloned into the cloning site of pcDNA3], pcDNA3-sirt mutant [the mutant Sirt1 (H363Y) sequence in the Flag-SIRT1 H363Y (Plasmid #1792, addigene) was digested and sub-cloned into the pcDNA3] were constructed by our lab.

    Techniques: In Vitro, Transfection, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Proliferation Assay

    HULC promotes liver cancer cell growth in vivo . a The photography of xenograft tumors from Balb/C null mouse injected with Hep3B cells transfected with pCMV6-A-GFP,pCMV6-A-GFP-HULC subcutaneously at armpit. b The xenograft tumors weight (gram) in the two groups indicated in left. Data were means of value from nine Balb/c mice, mean ± SEM, n = 7,*, P

    Journal: Molecular Cancer

    Article Title: Long noncoding RNA HULC accelerates liver cancer by inhibiting PTEN via autophagy cooperation to miR15a

    doi: 10.1186/s12943-018-0843-8

    Figure Lengend Snippet: HULC promotes liver cancer cell growth in vivo . a The photography of xenograft tumors from Balb/C null mouse injected with Hep3B cells transfected with pCMV6-A-GFP,pCMV6-A-GFP-HULC subcutaneously at armpit. b The xenograft tumors weight (gram) in the two groups indicated in left. Data were means of value from nine Balb/c mice, mean ± SEM, n = 7,*, P

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-A-GFP, pCMV6-XL5-PTEN, pGFP-V-RS-PTEN, pGFP-V-RS-Sirt1, pMiR-Target were purchased from Origene (Rockville, MD 20850, USA). pEZX-MT01-PTEN-3’UTR was purchased from GeneCopeia (Rockville, MD, USA).pCMV6-AC-GFP-HULC [the HULC sequence (NR_004855) was synthesized and cloned into the cloning site ofpCMV6-AC-GFP plasmid (Origene)], pcDNA3-sirt1[the Sirt1 sequence in the Flag-SIRT1 (Plasmid #1791,Addigene) was digested and subcloned into the cloning site of pcDNA3], pcDNA3-sirt mutant [the mutant Sirt1 (H363Y) sequence in the Flag-SIRT1 H363Y (Plasmid #1792, addigene) was digested and sub-cloned into the pcDNA3] were constructed by our lab.

    Techniques: In Vivo, Injection, Transfection, Mouse Assay

    The P62 Enhanced the METTL3 Occupancy on IGFII mRNA 3′ UTR in the Mesenchymal Stem Cells with TNF-α Treatment (A) RIP with anti-METTL3 followed by RT-PCR with CUDR mRNA primers in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi). IgG RIP as negative control is shown. CUDR mRNA as INPUT is shown. (B) Anti-CTCF co-IP followed by western blotting with anti-METTL3 primers in the mesenchymal stem cells treated with TNF-α or CUDR knockdown and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. IgG IP as negative control is shown. INPUT refers to western blotting with anti-CTCF. (C) Biotin-CUDR cDNA pull-down followed by western blotting with anti-MELLT3 and anti-CTCF primers in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. Biotin as INPUT and histone as internal control are shown. (D) Western blotting with anti-METTL3 and anti-FTO in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. β-actin as internal control is shown. (E) RIP with anti-METTL3 followed by RT-PCR with IGFII mRNA primers in the mesenchymal stem cells treated with TNF-α or CUDR knockdown and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. IgG RIP as negative control is shown. IGFII mRNA as INPUT is shown. (F) Super-EMSA (gel-shift) with biotin-IGFII cRNA probe and anti-METTL3 antibody in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. The intensity of the band was examined by western blotting with anti-biotin. (G) The super-METTL3 bands were quantified by calculating the band gray density value (n = 3). Each value was presented as mean ± SEM. **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Inflammatory-Related P62 Triggers Malignant Transformation of Mesenchymal Stem Cells through the Cascade of CUDR-CTCF-IGFII-RAS Signaling

    doi: 10.1016/j.omtn.2018.03.002

    Figure Lengend Snippet: The P62 Enhanced the METTL3 Occupancy on IGFII mRNA 3′ UTR in the Mesenchymal Stem Cells with TNF-α Treatment (A) RIP with anti-METTL3 followed by RT-PCR with CUDR mRNA primers in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi). IgG RIP as negative control is shown. CUDR mRNA as INPUT is shown. (B) Anti-CTCF co-IP followed by western blotting with anti-METTL3 primers in the mesenchymal stem cells treated with TNF-α or CUDR knockdown and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. IgG IP as negative control is shown. INPUT refers to western blotting with anti-CTCF. (C) Biotin-CUDR cDNA pull-down followed by western blotting with anti-MELLT3 and anti-CTCF primers in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. Biotin as INPUT and histone as internal control are shown. (D) Western blotting with anti-METTL3 and anti-FTO in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. β-actin as internal control is shown. (E) RIP with anti-METTL3 followed by RT-PCR with IGFII mRNA primers in the mesenchymal stem cells treated with TNF-α or CUDR knockdown and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. IgG RIP as negative control is shown. IGFII mRNA as INPUT is shown. (F) Super-EMSA (gel-shift) with biotin-IGFII cRNA probe and anti-METTL3 antibody in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. The intensity of the band was examined by western blotting with anti-biotin. (G) The super-METTL3 bands were quantified by calculating the band gray density value (n = 3). Each value was presented as mean ± SEM. **p

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-AC-GFP, pMiR-Target, and pCMV-miR were purchased from Origene (Rockville, MD 20850, USA). pCMV-miR122, pGFP-V-RS-P62, pGFP-V-RS-CUDR, pGFP-V-RS-H-Ras, pCMV6-AC-GFP-P62, and pMiR-target-IGFII 3′ UTR were prepared by the authors.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control, Co-Immunoprecipitation Assay, Western Blot, Electrophoretic Mobility Shift Assay

    The P62 Promotes the Methylation on IGFII mRNA and Its Expression in the TNF-α-Treated Mesenchymal Stem Cells Transfected with pCMV6-AC-GFP (GFP-Control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi Control), or pGFP-V-RS-P62 (P62 RNAi) (A) RIP with anti-N6Ame followed by RT-PCR with IFGII mRNA primers. IgG RIP as negative control is shown. IGFII mRNA was used as INPUT. (B) Super-EMSA (gel-shift) with biotin-IGFII cRNA probe and anti-N6Ame antibody. The intensity of the band was examined by western blotting with anti-biotin. (C) (a) IGFII rRNA biotin-probe pull-down followed run on assay with anti-N6Ame. Biotin as internal control is shown. (b) IGFII rRNA biotin-probe pull-down followed northern-western blotting assay with anti-N6Ame is shown. Histone as internal control is shown. (D) (a) Western blotting with anti-IGFII in the TNF-α-treated mesenchymal stem cells transfected with pCMV-mir (control), pCMV-miR122, pCMV6-AC-GFP-P62 (GFP-P62), or pCMV-miR122 plus pCMV6-AC-GFP-P62 (GFP-P62). β-actin was used as an internal control. (b) The IGFII 3′ UTR luciferase activity assay in the TNF-α-treated mesenchymal stem cells transfected with pCMV-mir, pCMV-miR122, pCMV6-AC-GFP-P62, and pCMV-miR122 plus pCMV6-AC-GFP-P62 is shown. (E) RT-PCR with IGFII mRNA primers in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), or pGFP-V-RS-P62 (P62 RNAi). β-actin served as internal control. (F) Western blotting with anti-IGFII and anti-pIGFII in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. β-actin was used as an internal control. **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Inflammatory-Related P62 Triggers Malignant Transformation of Mesenchymal Stem Cells through the Cascade of CUDR-CTCF-IGFII-RAS Signaling

    doi: 10.1016/j.omtn.2018.03.002

    Figure Lengend Snippet: The P62 Promotes the Methylation on IGFII mRNA and Its Expression in the TNF-α-Treated Mesenchymal Stem Cells Transfected with pCMV6-AC-GFP (GFP-Control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi Control), or pGFP-V-RS-P62 (P62 RNAi) (A) RIP with anti-N6Ame followed by RT-PCR with IFGII mRNA primers. IgG RIP as negative control is shown. IGFII mRNA was used as INPUT. (B) Super-EMSA (gel-shift) with biotin-IGFII cRNA probe and anti-N6Ame antibody. The intensity of the band was examined by western blotting with anti-biotin. (C) (a) IGFII rRNA biotin-probe pull-down followed run on assay with anti-N6Ame. Biotin as internal control is shown. (b) IGFII rRNA biotin-probe pull-down followed northern-western blotting assay with anti-N6Ame is shown. Histone as internal control is shown. (D) (a) Western blotting with anti-IGFII in the TNF-α-treated mesenchymal stem cells transfected with pCMV-mir (control), pCMV-miR122, pCMV6-AC-GFP-P62 (GFP-P62), or pCMV-miR122 plus pCMV6-AC-GFP-P62 (GFP-P62). β-actin was used as an internal control. (b) The IGFII 3′ UTR luciferase activity assay in the TNF-α-treated mesenchymal stem cells transfected with pCMV-mir, pCMV-miR122, pCMV6-AC-GFP-P62, and pCMV-miR122 plus pCMV6-AC-GFP-P62 is shown. (E) RT-PCR with IGFII mRNA primers in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), or pGFP-V-RS-P62 (P62 RNAi). β-actin served as internal control. (F) Western blotting with anti-IGFII and anti-pIGFII in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. β-actin was used as an internal control. **p

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-AC-GFP, pMiR-Target, and pCMV-miR were purchased from Origene (Rockville, MD 20850, USA). pCMV-miR122, pGFP-V-RS-P62, pGFP-V-RS-CUDR, pGFP-V-RS-H-Ras, pCMV6-AC-GFP-P62, and pMiR-target-IGFII 3′ UTR were prepared by the authors.

    Techniques: Methylation, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Negative Control, Electrophoretic Mobility Shift Assay, Western Blot, Northern Blot, Luciferase, Activity Assay

    P62 Overexpression Combined with TNF-α Triggers Malignant Transformation of Human Mesenchymal Stem Cells In Vitro and In Vivo (A) The schematic illustrates that human mesenchymal stem cells stably transfected with P62 overexpression or knockdown plasmids were transformed into cancer cells that were then treated with TNF-α or control, including groups of pCMV6-AC-GFP (GFP control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), pGFP-V-RS–P62 (P62 RNAi), pCMV6-AC-GFP plus TNF-α, pCMV6-AC-GFP-P62 plus TNF-α, pGFP-V-RS plus TNF-α, and pGFP-V-RS–P62 plus TNF-α. (B) The western blotting analysis with anti-P62, anti-NF-κB, and anti-CLYD in these human mesenchymal stem cells indicated in upper. β-actin and GAPDH as internal control are shown. (C) Cells soft-agar colony formation assay. (D) Cells sphere-formation ability. (E) In vivo test in these human mesenchymal stem cells. The mice were stratified, and the tumors were recovered. (F) The wet weight of each tumor was determined for each mouse. Each value was presented as mean ± SEM (Student’s t test). **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Inflammatory-Related P62 Triggers Malignant Transformation of Mesenchymal Stem Cells through the Cascade of CUDR-CTCF-IGFII-RAS Signaling

    doi: 10.1016/j.omtn.2018.03.002

    Figure Lengend Snippet: P62 Overexpression Combined with TNF-α Triggers Malignant Transformation of Human Mesenchymal Stem Cells In Vitro and In Vivo (A) The schematic illustrates that human mesenchymal stem cells stably transfected with P62 overexpression or knockdown plasmids were transformed into cancer cells that were then treated with TNF-α or control, including groups of pCMV6-AC-GFP (GFP control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), pGFP-V-RS–P62 (P62 RNAi), pCMV6-AC-GFP plus TNF-α, pCMV6-AC-GFP-P62 plus TNF-α, pGFP-V-RS plus TNF-α, and pGFP-V-RS–P62 plus TNF-α. (B) The western blotting analysis with anti-P62, anti-NF-κB, and anti-CLYD in these human mesenchymal stem cells indicated in upper. β-actin and GAPDH as internal control are shown. (C) Cells soft-agar colony formation assay. (D) Cells sphere-formation ability. (E) In vivo test in these human mesenchymal stem cells. The mice were stratified, and the tumors were recovered. (F) The wet weight of each tumor was determined for each mouse. Each value was presented as mean ± SEM (Student’s t test). **p

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-AC-GFP, pMiR-Target, and pCMV-miR were purchased from Origene (Rockville, MD 20850, USA). pCMV-miR122, pGFP-V-RS-P62, pGFP-V-RS-CUDR, pGFP-V-RS-H-Ras, pCMV6-AC-GFP-P62, and pMiR-target-IGFII 3′ UTR were prepared by the authors.

    Techniques: Over Expression, Transformation Assay, In Vitro, In Vivo, Stable Transfection, Transfection, Western Blot, Soft Agar Assay, Mouse Assay

    The Activated H-Ras Induced Some Gene Abnormal Expression of Specific Genes in the P62-Overexpressing or Knockdown Mesenchymal Stem Cells Treated with TNF-α (A) GST pull-down using GST-Raf1-RBD system followed by western blotting anti-GTP Ras in the mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi). INPUT refers to western blotting with anti-H-Ras. (B) Western blotting with anti-pRaf, anti-Raf, anti-pMEKK1, anti-MEKK1, anti-pERK, anti-ERK, anti-pMEKK4, anti-MEKK4, anti-pMEK, anti-MEK, anti-pJak, anti-Jak, anti-pEIK, anti-EIK, anti-P38, anti-pPI3K, anti-PI3K, anti-pmTOR, or anti-pAKT in the mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi). β-actin served as an internal control.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Inflammatory-Related P62 Triggers Malignant Transformation of Mesenchymal Stem Cells through the Cascade of CUDR-CTCF-IGFII-RAS Signaling

    doi: 10.1016/j.omtn.2018.03.002

    Figure Lengend Snippet: The Activated H-Ras Induced Some Gene Abnormal Expression of Specific Genes in the P62-Overexpressing or Knockdown Mesenchymal Stem Cells Treated with TNF-α (A) GST pull-down using GST-Raf1-RBD system followed by western blotting anti-GTP Ras in the mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi). INPUT refers to western blotting with anti-H-Ras. (B) Western blotting with anti-pRaf, anti-Raf, anti-pMEKK1, anti-MEKK1, anti-pERK, anti-ERK, anti-pMEKK4, anti-MEKK4, anti-pMEK, anti-MEK, anti-pJak, anti-Jak, anti-pEIK, anti-EIK, anti-P38, anti-pPI3K, anti-PI3K, anti-pmTOR, or anti-pAKT in the mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi). β-actin served as an internal control.

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-AC-GFP, pMiR-Target, and pCMV-miR were purchased from Origene (Rockville, MD 20850, USA). pCMV-miR122, pGFP-V-RS-P62, pGFP-V-RS-CUDR, pGFP-V-RS-H-Ras, pCMV6-AC-GFP-P62, and pMiR-target-IGFII 3′ UTR were prepared by the authors.

    Techniques: Expressing, Western Blot, Transfection

    The Rescued Experiment of Carcinogenesis Effect of the P62 in TNF-α-Treated Mesenchymal Stem Cells Transfected with pCMV6-AC-P62 (GFP-P62), pCMV6-AC-GFP-P62 (GFP-P62) plus pcDNA3.1-H-Ras (Ras), pCMV6-AC-P62 (GFP-P62) plus TNF-α, and pCMV6-AC-P62 (GFP-P62) plus TNF-α plus pGFP-V-RS-H-Ras (Rasi) (A) Western blotting analysis with anti-P62 and anti-H-Ras in mesenchymal stem cells. β-actin served as internal control. (B) Cells growth assay using CCK8. Each value was presented as mean ± SEM. (C) Cells soft-agar colony formation assay. (D) Tumorigenesis test in vivo in these mesenchymal stem cells. (a) The mice were stratified, and the tumors were recovered. (b) The wet weight of each tumor was determined for each mouse. Each value was presented as mean ± SEM. **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Inflammatory-Related P62 Triggers Malignant Transformation of Mesenchymal Stem Cells through the Cascade of CUDR-CTCF-IGFII-RAS Signaling

    doi: 10.1016/j.omtn.2018.03.002

    Figure Lengend Snippet: The Rescued Experiment of Carcinogenesis Effect of the P62 in TNF-α-Treated Mesenchymal Stem Cells Transfected with pCMV6-AC-P62 (GFP-P62), pCMV6-AC-GFP-P62 (GFP-P62) plus pcDNA3.1-H-Ras (Ras), pCMV6-AC-P62 (GFP-P62) plus TNF-α, and pCMV6-AC-P62 (GFP-P62) plus TNF-α plus pGFP-V-RS-H-Ras (Rasi) (A) Western blotting analysis with anti-P62 and anti-H-Ras in mesenchymal stem cells. β-actin served as internal control. (B) Cells growth assay using CCK8. Each value was presented as mean ± SEM. (C) Cells soft-agar colony formation assay. (D) Tumorigenesis test in vivo in these mesenchymal stem cells. (a) The mice were stratified, and the tumors were recovered. (b) The wet weight of each tumor was determined for each mouse. Each value was presented as mean ± SEM. **p

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-AC-GFP, pMiR-Target, and pCMV-miR were purchased from Origene (Rockville, MD 20850, USA). pCMV-miR122, pGFP-V-RS-P62, pGFP-V-RS-CUDR, pGFP-V-RS-H-Ras, pCMV6-AC-GFP-P62, and pMiR-target-IGFII 3′ UTR were prepared by the authors.

    Techniques: Transfection, Western Blot, Growth Assay, Soft Agar Assay, In Vivo, Mouse Assay

    P62 Regulates IGFII Transcriptional Activity in the Mesenchymal Stem Cells with TNF-α Treatment (A) RNA immunoprecipitation (RIP) with anti-CTCF followed by RT-PCR with CUDR mRNA primers in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS–P62 (P62 RNAi). IgG RIP was used as negative control. CUDR RNA as INPUT is shown. (B) Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP) with anti-CTCF and anti-RNA polII in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. The PCR analysis is applied for detecting IGFII H19ICR-IGFII DMR2 coupling product using H19ICR and IGFII DMR2 primers. The H19ICR and IGFII DMR2 as INPUT are shown. (C) 3C-ChIP with anti-CTCF and anti-RNA polII in the CUDR knockdown mesenchymal stem cells transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. The PCR analysis is applied for detecting IGFII H19ICR-IGFII DMR2 coupling product using H19ICR and IGFII DMR2 primers. The H19ICR and IGFII DMR2 as INPUT are shown. (D) IGFII promoter luciferase activity assay in in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. Each value was presented as mean ± SEM. **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Inflammatory-Related P62 Triggers Malignant Transformation of Mesenchymal Stem Cells through the Cascade of CUDR-CTCF-IGFII-RAS Signaling

    doi: 10.1016/j.omtn.2018.03.002

    Figure Lengend Snippet: P62 Regulates IGFII Transcriptional Activity in the Mesenchymal Stem Cells with TNF-α Treatment (A) RNA immunoprecipitation (RIP) with anti-CTCF followed by RT-PCR with CUDR mRNA primers in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS–P62 (P62 RNAi). IgG RIP was used as negative control. CUDR RNA as INPUT is shown. (B) Chromosome conformation capture (3C)-chromatin immunoprecipitation (ChIP) with anti-CTCF and anti-RNA polII in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. The PCR analysis is applied for detecting IGFII H19ICR-IGFII DMR2 coupling product using H19ICR and IGFII DMR2 primers. The H19ICR and IGFII DMR2 as INPUT are shown. (C) 3C-ChIP with anti-CTCF and anti-RNA polII in the CUDR knockdown mesenchymal stem cells transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. The PCR analysis is applied for detecting IGFII H19ICR-IGFII DMR2 coupling product using H19ICR and IGFII DMR2 primers. The H19ICR and IGFII DMR2 as INPUT are shown. (D) IGFII promoter luciferase activity assay in in the mesenchymal stem cells treated with TNF-α and transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. Each value was presented as mean ± SEM. **p

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-AC-GFP, pMiR-Target, and pCMV-miR were purchased from Origene (Rockville, MD 20850, USA). pCMV-miR122, pGFP-V-RS-P62, pGFP-V-RS-CUDR, pGFP-V-RS-H-Ras, pCMV6-AC-GFP-P62, and pMiR-target-IGFII 3′ UTR were prepared by the authors.

    Techniques: Activity Assay, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Luciferase

    Human Mesenchymal Stem Cells Were Subjected to Transformation in Mouse Liver Overexpressing P62 (A) Mouse athymic Balb/C mouse (a severe combined immunodeficiency mouse) liver in vivo transfection with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, and pGFP-V-RS-P62 plasmids. The western blotting analysis with anti-P62 in mice liver tissue is shown. β-actin as internal control is shown. (B) The schematic illustrates that mouse mesenchymal stem cells, in which P62 was overexpressed or knocked down, were injected into the athymic Balb/C mouse liver capsule under the B ultrasound guide. Mice were fed with carbon tetrachloride (CCL 4 ) for three months. (C) The mice were stratified, the tumors recovered, and xenograft tumor photographed in the six groups as indicated in left. (D) The wet weight of each tumor for each mouse in pCMV6-AC-GFP-P62 plus CCL4 group. (E) The wet weight of each tumor was determined for each mouse. Each value was presented as mean ± SEM; **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Inflammatory-Related P62 Triggers Malignant Transformation of Mesenchymal Stem Cells through the Cascade of CUDR-CTCF-IGFII-RAS Signaling

    doi: 10.1016/j.omtn.2018.03.002

    Figure Lengend Snippet: Human Mesenchymal Stem Cells Were Subjected to Transformation in Mouse Liver Overexpressing P62 (A) Mouse athymic Balb/C mouse (a severe combined immunodeficiency mouse) liver in vivo transfection with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, and pGFP-V-RS-P62 plasmids. The western blotting analysis with anti-P62 in mice liver tissue is shown. β-actin as internal control is shown. (B) The schematic illustrates that mouse mesenchymal stem cells, in which P62 was overexpressed or knocked down, were injected into the athymic Balb/C mouse liver capsule under the B ultrasound guide. Mice were fed with carbon tetrachloride (CCL 4 ) for three months. (C) The mice were stratified, the tumors recovered, and xenograft tumor photographed in the six groups as indicated in left. (D) The wet weight of each tumor for each mouse in pCMV6-AC-GFP-P62 plus CCL4 group. (E) The wet weight of each tumor was determined for each mouse. Each value was presented as mean ± SEM; **p

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-AC-GFP, pMiR-Target, and pCMV-miR were purchased from Origene (Rockville, MD 20850, USA). pCMV-miR122, pGFP-V-RS-P62, pGFP-V-RS-CUDR, pGFP-V-RS-H-Ras, pCMV6-AC-GFP-P62, and pMiR-target-IGFII 3′ UTR were prepared by the authors.

    Techniques: Transformation Assay, In Vivo, Transfection, Western Blot, Mouse Assay, Injection

    IFGII Induced Overexpression of H-Ras in the Mesenchymal Stem Cells (A) Anti-IGFII co- IP followed by western blotting with anti-P62,anti-TNFR1, anti-CLYD, anti-EGR1, anti-NFκB, anti-TLR4, and anti-PPARγ in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, pGFP-V-RS-P62, and IgG IP as negative control. INPUT refers to western blotting with anti-IGFII. (B) CHIP with anti-IGFII, anti-P62, anti-TNFR1, anti-CLYD, anti-NFκB, or anti-PPARγ followed by PCR with H-Ras promoter primer in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi). IgG CHIP was used as negative control. H-Ras promoter as INPUT is shown. (C) The H-Ras promoter luciferase activity assay in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), or pGFP-V-RS-P62 (P62 RNAi). (D) RT-PCR with H-Ras primers in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi), respectively. β-actin as internal control is shown. (E) Western blotting with anti-H-Ras and anti-pH-Ras in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6- AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi), respectively. β-actin was used as an internal control. **p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Inflammatory-Related P62 Triggers Malignant Transformation of Mesenchymal Stem Cells through the Cascade of CUDR-CTCF-IGFII-RAS Signaling

    doi: 10.1016/j.omtn.2018.03.002

    Figure Lengend Snippet: IFGII Induced Overexpression of H-Ras in the Mesenchymal Stem Cells (A) Anti-IGFII co- IP followed by western blotting with anti-P62,anti-TNFR1, anti-CLYD, anti-EGR1, anti-NFκB, anti-TLR4, and anti-PPARγ in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, pGFP-V-RS-P62, and IgG IP as negative control. INPUT refers to western blotting with anti-IGFII. (B) CHIP with anti-IGFII, anti-P62, anti-TNFR1, anti-CLYD, anti-NFκB, or anti-PPARγ followed by PCR with H-Ras promoter primer in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi). IgG CHIP was used as negative control. H-Ras promoter as INPUT is shown. (C) The H-Ras promoter luciferase activity assay in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), or pGFP-V-RS-P62 (P62 RNAi). (D) RT-PCR with H-Ras primers in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi), respectively. β-actin as internal control is shown. (E) Western blotting with anti-H-Ras and anti-pH-Ras in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6- AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), and pGFP-V-RS-P62 (P62 RNAi), respectively. β-actin was used as an internal control. **p

    Article Snippet: Plasmid pGFP-V-RS, pCMV6-AC-GFP, pMiR-Target, and pCMV-miR were purchased from Origene (Rockville, MD 20850, USA). pCMV-miR122, pGFP-V-RS-P62, pGFP-V-RS-CUDR, pGFP-V-RS-H-Ras, pCMV6-AC-GFP-P62, and pMiR-target-IGFII 3′ UTR were prepared by the authors.

    Techniques: Over Expression, Co-Immunoprecipitation Assay, Western Blot, Transfection, Negative Control, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction

    PCDHB13 disrupts microtubule dynamics in NSCLC cells. ( A ) Cellular localization of PCDHB13 in A549 cells. Cells were stained with PCDHB13 (green) and α-Tubulin (red). Hoechst33342 was used to stain nuclei (blue). Boxed regions are magnified in the panels to the right. Red arrows indicate the filamentary structure of PCDHB13. White circles indicate the cellular aggregates formed by PCDHB13 and α-Tubulin. Bar: 20 μm. ( B ) PCDHB13 and α-Tubulin are shown in red and green, respectively. Cytoskeletal projections of each protein were simulated by Imaris software. Yellow color indicates colocalization of PCDHB13 and α-Tubulin. Bar: 8 μm. ( C ) A549 cell lysates without (lane 1 and 3) or with tGFP-tagged PCDHB13 overexpression (lane 2 and 4) were harvested for co-immunoprecipitation (Co-IP) assay using the anti-tGFP conjugated magnetic beads. IP eluates were analyzed by Western blot for PCDHB13 and α-Tubulin. ( D , E ) GFP ( D ) or PCDHB13-tGFP vector ( E ) transfected A549 and H1975 cells pretreated with 20 μM nocodazole were stained for PCDHB13 (red) and α-Tubulin (white) at 0, 10, and 30 min after nocodazole washout. Higher magnifications of the areas in the white boxes marked by * or # are shown in the corresponding panel. Hoechst33342 was stained for nuclei (blue). Green arrows indicate MTOC with defective microtubule outgrowth. The spatial correlation of PCDHB13 with microtubules in the indicated region (pink lines mark two opposite ends, indicated by pink arrows) is shown by a line-series analysis. The red and white lines in the right panels represent the PCDHB13 and α-Tubulin fluorescence intensities, respectively. au: arbitrary units. Bar: 20 μm. ( F ) Quantification of MTOC formation in cells transfected by GFP or PCDHB13 after nocodazole washout. g Illustration of the phenomena shown in ( D , E ).

    Journal: Cancers

    Article Title: FOSB–PCDHB13 Axis Disrupts the Microtubule Network in Non-Small Cell Lung Cancer

    doi: 10.3390/cancers11010107

    Figure Lengend Snippet: PCDHB13 disrupts microtubule dynamics in NSCLC cells. ( A ) Cellular localization of PCDHB13 in A549 cells. Cells were stained with PCDHB13 (green) and α-Tubulin (red). Hoechst33342 was used to stain nuclei (blue). Boxed regions are magnified in the panels to the right. Red arrows indicate the filamentary structure of PCDHB13. White circles indicate the cellular aggregates formed by PCDHB13 and α-Tubulin. Bar: 20 μm. ( B ) PCDHB13 and α-Tubulin are shown in red and green, respectively. Cytoskeletal projections of each protein were simulated by Imaris software. Yellow color indicates colocalization of PCDHB13 and α-Tubulin. Bar: 8 μm. ( C ) A549 cell lysates without (lane 1 and 3) or with tGFP-tagged PCDHB13 overexpression (lane 2 and 4) were harvested for co-immunoprecipitation (Co-IP) assay using the anti-tGFP conjugated magnetic beads. IP eluates were analyzed by Western blot for PCDHB13 and α-Tubulin. ( D , E ) GFP ( D ) or PCDHB13-tGFP vector ( E ) transfected A549 and H1975 cells pretreated with 20 μM nocodazole were stained for PCDHB13 (red) and α-Tubulin (white) at 0, 10, and 30 min after nocodazole washout. Higher magnifications of the areas in the white boxes marked by * or # are shown in the corresponding panel. Hoechst33342 was stained for nuclei (blue). Green arrows indicate MTOC with defective microtubule outgrowth. The spatial correlation of PCDHB13 with microtubules in the indicated region (pink lines mark two opposite ends, indicated by pink arrows) is shown by a line-series analysis. The red and white lines in the right panels represent the PCDHB13 and α-Tubulin fluorescence intensities, respectively. au: arbitrary units. Bar: 20 μm. ( F ) Quantification of MTOC formation in cells transfected by GFP or PCDHB13 after nocodazole washout. g Illustration of the phenomena shown in ( D , E ).

    Article Snippet: The PCDHB13 promoter-driven mCherry reporter construct, tGFP-tagged FOSB/PCDHB13, monomeric GFP (mGFP)-tagged FOSΔB, and pCMV6-ac-GFP (tGFP) were purchased from GeneCopoeia, Inc. (Rockville, MD, USA).

    Techniques: Staining, Software, Over Expression, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Plasmid Preparation, Transfection, Fluorescence

    Loss of cytoskeletal integrity upon FOSB induction. ( A , B ) Gene ontology (GO) analyses of dysregulated genes revealed three distinct functional categories ( A ). Twelve out of twenty-two annotation terms were assigned to the cellular component ontology, including genes that are involved in the regulation of cytoskeleton and membrane ( B ). ( C , D ) Total lysates from A549 cells transfected with EGFP or FOSB-tGFP plasmid were analyzed by Western blot using antibodies against GFP, GAPDH, FOSB, EMT markers, PCDHB13, and Stathmin. ( E , F ) Total lysates from A549 cells without (Co.) or with TP4 (T) were analyzed by Western blot using antibody against GAPDH, EMT markers, PCDHB13, and Stathmin. ( G , H ) Total lysates from A549 cells transfected with control (Neg-si) or FOSB siRNAs (FOSB-si-1+2) with or without TP4 treatment were analyzed by Western blot using antibodies against GAPDH, EMT markers, and PCDHB13. Quantitative measurements of protein levels were normalized to GAPDH in ( D , F , H ). Quantitative results represent the mean ± SD ( n = 3, two-tailed t -test in ( D , G , H ): * p

    Journal: Cancers

    Article Title: FOSB–PCDHB13 Axis Disrupts the Microtubule Network in Non-Small Cell Lung Cancer

    doi: 10.3390/cancers11010107

    Figure Lengend Snippet: Loss of cytoskeletal integrity upon FOSB induction. ( A , B ) Gene ontology (GO) analyses of dysregulated genes revealed three distinct functional categories ( A ). Twelve out of twenty-two annotation terms were assigned to the cellular component ontology, including genes that are involved in the regulation of cytoskeleton and membrane ( B ). ( C , D ) Total lysates from A549 cells transfected with EGFP or FOSB-tGFP plasmid were analyzed by Western blot using antibodies against GFP, GAPDH, FOSB, EMT markers, PCDHB13, and Stathmin. ( E , F ) Total lysates from A549 cells without (Co.) or with TP4 (T) were analyzed by Western blot using antibody against GAPDH, EMT markers, PCDHB13, and Stathmin. ( G , H ) Total lysates from A549 cells transfected with control (Neg-si) or FOSB siRNAs (FOSB-si-1+2) with or without TP4 treatment were analyzed by Western blot using antibodies against GAPDH, EMT markers, and PCDHB13. Quantitative measurements of protein levels were normalized to GAPDH in ( D , F , H ). Quantitative results represent the mean ± SD ( n = 3, two-tailed t -test in ( D , G , H ): * p

    Article Snippet: The PCDHB13 promoter-driven mCherry reporter construct, tGFP-tagged FOSB/PCDHB13, monomeric GFP (mGFP)-tagged FOSΔB, and pCMV6-ac-GFP (tGFP) were purchased from GeneCopoeia, Inc. (Rockville, MD, USA).

    Techniques: Functional Assay, Transfection, Plasmid Preparation, Western Blot, Two Tailed Test

    Elevated expression of PCDHB13 inhibits cell invasion and triggers cell death. ( A,C ) The underside of Matrigel-coated polycarbonate membranes used for cell invasion assays on EGFP-, PCDHB13-, and FOSB-transfected cells ( A ) or cells incubated with 1.68 and 3.35 μM TP4 ( C ). Cells were stained by Hoechst33342 shown in blue ( A ) or white ( C ). Bar: 200 μm. ( B , D ) Quantification of the cells that migrated across the membrane. Data were calculated by normalizing GFP/tGFP-positive to the total cell counts ( n = 463 in EGFP transfected groups, n = 300 in PCDHB13-tGFP transfected groups, and n = 297 in FOSB-tGFP transfected group) in ( B ) or by counting Hoechst-dye stained cells in ( D ). ( E ) Schematic of the A549 cell xenotransplantation procedure. Cells were used for RO injection at 24 h post-transfection. Cell-transplanted zebrafish were cultured for 4 d. ( F ) Transmitted light and fluorescent images of A549 cells without or with transfection. Bar: 100 μm. ( G ) Quantification of fluorescent signal in dissected organs ( n = 5 in the nontransfected/NT group and n = 9 in the transfected groups). au: arbitrary unit. ( H ) Tissue extracts from tGFP-, PCDHB13-, and FOSB-transfected A549-transplanted zebrafish were analyzed by Western blot using antibodies against N-Cadherin, α/β-Tubulin, and tGFP. P: Lysates from tGFP/PCDHB13/FOSB-transfected A549 cells. N: Lysate from nontransfected A549 cell. ( I ) Quantification of the EGFP/tGFP levels from Western blots ( n = 9). ( J ) Cell viability was determined by the ATP assay for A549 and NCI-H1975 cells after transfection with PCDHB13. Ten replicate wells were analyzed for each dose ( n = 3). ( K ) LDH release in A549 and NCI-H1975 cultures were determined 24 h after FOSB and PCDHB13 transfection. Triton-X was used as a positive control. Each independent replicate was measured at least in triplicate ( n = 3). ( L ) Total lysates from A549 cells transfected with control (Neg.) or PCDHB13 siRNAs were analyzed by Western blot using antibodies against GAPDH and PCDHB13. Bottom, PCDHB13 levels were quantified and normalized to GAPDH. ( M ) Cell viability was determined by the ATP assay for A549 cells transfected with siRNAs targeting PCDHB13. Eight replicate wells were analyzed for each condition ( n = 3). Quantitative results represent the mean ± SD (One-way ANOVA test in ( K ); two tailed t -test in ( M ): ns, not significant; * p

    Journal: Cancers

    Article Title: FOSB–PCDHB13 Axis Disrupts the Microtubule Network in Non-Small Cell Lung Cancer

    doi: 10.3390/cancers11010107

    Figure Lengend Snippet: Elevated expression of PCDHB13 inhibits cell invasion and triggers cell death. ( A,C ) The underside of Matrigel-coated polycarbonate membranes used for cell invasion assays on EGFP-, PCDHB13-, and FOSB-transfected cells ( A ) or cells incubated with 1.68 and 3.35 μM TP4 ( C ). Cells were stained by Hoechst33342 shown in blue ( A ) or white ( C ). Bar: 200 μm. ( B , D ) Quantification of the cells that migrated across the membrane. Data were calculated by normalizing GFP/tGFP-positive to the total cell counts ( n = 463 in EGFP transfected groups, n = 300 in PCDHB13-tGFP transfected groups, and n = 297 in FOSB-tGFP transfected group) in ( B ) or by counting Hoechst-dye stained cells in ( D ). ( E ) Schematic of the A549 cell xenotransplantation procedure. Cells were used for RO injection at 24 h post-transfection. Cell-transplanted zebrafish were cultured for 4 d. ( F ) Transmitted light and fluorescent images of A549 cells without or with transfection. Bar: 100 μm. ( G ) Quantification of fluorescent signal in dissected organs ( n = 5 in the nontransfected/NT group and n = 9 in the transfected groups). au: arbitrary unit. ( H ) Tissue extracts from tGFP-, PCDHB13-, and FOSB-transfected A549-transplanted zebrafish were analyzed by Western blot using antibodies against N-Cadherin, α/β-Tubulin, and tGFP. P: Lysates from tGFP/PCDHB13/FOSB-transfected A549 cells. N: Lysate from nontransfected A549 cell. ( I ) Quantification of the EGFP/tGFP levels from Western blots ( n = 9). ( J ) Cell viability was determined by the ATP assay for A549 and NCI-H1975 cells after transfection with PCDHB13. Ten replicate wells were analyzed for each dose ( n = 3). ( K ) LDH release in A549 and NCI-H1975 cultures were determined 24 h after FOSB and PCDHB13 transfection. Triton-X was used as a positive control. Each independent replicate was measured at least in triplicate ( n = 3). ( L ) Total lysates from A549 cells transfected with control (Neg.) or PCDHB13 siRNAs were analyzed by Western blot using antibodies against GAPDH and PCDHB13. Bottom, PCDHB13 levels were quantified and normalized to GAPDH. ( M ) Cell viability was determined by the ATP assay for A549 cells transfected with siRNAs targeting PCDHB13. Eight replicate wells were analyzed for each condition ( n = 3). Quantitative results represent the mean ± SD (One-way ANOVA test in ( K ); two tailed t -test in ( M ): ns, not significant; * p

    Article Snippet: The PCDHB13 promoter-driven mCherry reporter construct, tGFP-tagged FOSB/PCDHB13, monomeric GFP (mGFP)-tagged FOSΔB, and pCMV6-ac-GFP (tGFP) were purchased from GeneCopoeia, Inc. (Rockville, MD, USA).

    Techniques: Expressing, Transfection, Incubation, Staining, Injection, Cell Culture, Western Blot, ATP Assay, Positive Control, Two Tailed Test

    CUDR combined cyclinD1 or PTEN depletion collectly increases telomerase activity through H19 in human liver cancer stem cell A . RT-PCR analysis of TERC mRNA and Western blotting with anti-cyclinD1, anti-PTEN expression in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A- GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively (indicated in the left ). β-actin as internal control. B. RNA Immunoprecipitation(RIP) with anti-TERT followed by RT-PCR with TERC mRNA primers in contro or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG RIP as negative controlTERC mRNA as INPUT. C. RT-PCR analysis of TERRA mRNA in control or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN, respectively. D. RNA Immunoprecipitation(RIP) with anti-TERT followed by RT-PCR with TERRA mRNA primers in contro or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG RIP as negative control TERC mRNA as INPUT. E. Super-EMSA(gel-shift) with biotin- TERRA cRNA probe and anti-TERT antibody. The intensity of the band was examined by Western blotting with anti-Biotin. F. Telomerase activity assay with TRAP method mRNA in control or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. Each value was presented as mean ± standard error of the mean (SEM). ** P

    Journal: Oncotarget

    Article Title: CUDR promotes liver cancer stem cell growth through upregulating TERT and C-Myc

    doi:

    Figure Lengend Snippet: CUDR combined cyclinD1 or PTEN depletion collectly increases telomerase activity through H19 in human liver cancer stem cell A . RT-PCR analysis of TERC mRNA and Western blotting with anti-cyclinD1, anti-PTEN expression in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A- GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively (indicated in the left ). β-actin as internal control. B. RNA Immunoprecipitation(RIP) with anti-TERT followed by RT-PCR with TERC mRNA primers in contro or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG RIP as negative controlTERC mRNA as INPUT. C. RT-PCR analysis of TERRA mRNA in control or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN, respectively. D. RNA Immunoprecipitation(RIP) with anti-TERT followed by RT-PCR with TERRA mRNA primers in contro or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG RIP as negative control TERC mRNA as INPUT. E. Super-EMSA(gel-shift) with biotin- TERRA cRNA probe and anti-TERT antibody. The intensity of the band was examined by Western blotting with anti-Biotin. F. Telomerase activity assay with TRAP method mRNA in control or H19 knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. Each value was presented as mean ± standard error of the mean (SEM). ** P

    Article Snippet: Plasmid pGFP-V-RS, pRFP-V-RS pCMV6-A-GFP, were purchased from Origene (Rockville, MD 20850, USA).

    Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Immunoprecipitation, Negative Control, Telomerase Activity Assay

    The rescued experiment of carcinogenesis effect of the synergetic effect of CUDR, CyclinD1 and PTEN kn ockdown C-myc knockdown or TERT knockdown abrogated the oncogenic function of CUDR combined with CyclinD1 or PTEN knockdown in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, pCMV6-A-CUDR plus pGFP-V-RS-C-myc, pCMV6-A-CUDR plus pcDNA3.1-CyclinD1 plus pGFP-V-RS-C-myc, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN plus pGFP-V-RS-C-myc, pCMV6-A-GFP-CUDR plus pGFP-V-RS-TERT, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1 plus pGFP-V-RS-TERT, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN plus pGFP-V-RS- TERT, A. The RT-PCR of CUDR mRNA and the western blotting analysis with anti-CyclinD1, anti-PTEN, anti-C-myc and anti-TERT. β-actin as internal control. B. Cells growth assay. Each value was presented as mean ± standard error of the mean (SEM). C. Cells soft agar colony formation assay. Each value was presented as mean ± standard error of the mean (SEM). ** P

    Journal: Oncotarget

    Article Title: CUDR promotes liver cancer stem cell growth through upregulating TERT and C-Myc

    doi:

    Figure Lengend Snippet: The rescued experiment of carcinogenesis effect of the synergetic effect of CUDR, CyclinD1 and PTEN kn ockdown C-myc knockdown or TERT knockdown abrogated the oncogenic function of CUDR combined with CyclinD1 or PTEN knockdown in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, pCMV6-A-CUDR plus pGFP-V-RS-C-myc, pCMV6-A-CUDR plus pcDNA3.1-CyclinD1 plus pGFP-V-RS-C-myc, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN plus pGFP-V-RS-C-myc, pCMV6-A-GFP-CUDR plus pGFP-V-RS-TERT, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1 plus pGFP-V-RS-TERT, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN plus pGFP-V-RS- TERT, A. The RT-PCR of CUDR mRNA and the western blotting analysis with anti-CyclinD1, anti-PTEN, anti-C-myc and anti-TERT. β-actin as internal control. B. Cells growth assay. Each value was presented as mean ± standard error of the mean (SEM). C. Cells soft agar colony formation assay. Each value was presented as mean ± standard error of the mean (SEM). ** P

    Article Snippet: Plasmid pGFP-V-RS, pRFP-V-RS pCMV6-A-GFP, were purchased from Origene (Rockville, MD 20850, USA).

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Growth Assay, Soft Agar Assay

    CUDR overexpression cooperated with cyclinD1 overexpression or PTEN depletion accerlates the liver cancer stem cell proliferation A. The growth and colony formation ability in the stable human liver cancer stem cell(HLCSC) lines and non-HLCSC transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN, respectively. a . RT-PCR analysis of CUDR mRNA and Western bloting with anti-cyclinD1, anti-PTEN expression in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS-PTEN, respectively (indicated in the left ). β-actin as internalcontrol. b . Cell proliferation assay in vitro in liver cancer stem cells and unstemic liver cancer. Data are means of value from three independent experiment, bar ± SEM. ** P

    Journal: Oncotarget

    Article Title: CUDR promotes liver cancer stem cell growth through upregulating TERT and C-Myc

    doi:

    Figure Lengend Snippet: CUDR overexpression cooperated with cyclinD1 overexpression or PTEN depletion accerlates the liver cancer stem cell proliferation A. The growth and colony formation ability in the stable human liver cancer stem cell(HLCSC) lines and non-HLCSC transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN, respectively. a . RT-PCR analysis of CUDR mRNA and Western bloting with anti-cyclinD1, anti-PTEN expression in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS-PTEN, respectively (indicated in the left ). β-actin as internalcontrol. b . Cell proliferation assay in vitro in liver cancer stem cells and unstemic liver cancer. Data are means of value from three independent experiment, bar ± SEM. ** P

    Article Snippet: Plasmid pGFP-V-RS, pRFP-V-RS pCMV6-A-GFP, were purchased from Origene (Rockville, MD 20850, USA).

    Techniques: Over Expression, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Proliferation Assay, In Vitro

    CUDR, cyclinD1 and PTEN synergistically alters induced hepatocyte-like cells growth in vitro and in vivo A. Induction and identification of hepatocyte-like cells. a . The schematic digram illustrates a model of liver stem cells induction from human embryic stem cells MEL-1. b . Western bloting with anti- Oct3, anti-SSEA3, anti-Sox2, anti-Sox17, anti-HNF4α, anti-Albumin, anti-AFP in liver stem cell and embryic stem cell MEL-1. β-actin as internal control. B. a . RT-PCR analysis of CUDR mRNA and Western bloting with anti-cyclinD1, anti-PTEN expression in stable hepatocyte-like cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively (indicated in the left ). β-actin as internal control. b . Cell proliferation assay in vitro . Data are means of value from three independent experiment, bar ± SEM. ** P

    Journal: Oncotarget

    Article Title: CUDR promotes liver cancer stem cell growth through upregulating TERT and C-Myc

    doi:

    Figure Lengend Snippet: CUDR, cyclinD1 and PTEN synergistically alters induced hepatocyte-like cells growth in vitro and in vivo A. Induction and identification of hepatocyte-like cells. a . The schematic digram illustrates a model of liver stem cells induction from human embryic stem cells MEL-1. b . Western bloting with anti- Oct3, anti-SSEA3, anti-Sox2, anti-Sox17, anti-HNF4α, anti-Albumin, anti-AFP in liver stem cell and embryic stem cell MEL-1. β-actin as internal control. B. a . RT-PCR analysis of CUDR mRNA and Western bloting with anti-cyclinD1, anti-PTEN expression in stable hepatocyte-like cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively (indicated in the left ). β-actin as internal control. b . Cell proliferation assay in vitro . Data are means of value from three independent experiment, bar ± SEM. ** P

    Article Snippet: Plasmid pGFP-V-RS, pRFP-V-RS pCMV6-A-GFP, were purchased from Origene (Rockville, MD 20850, USA).

    Techniques: In Vitro, In Vivo, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Proliferation Assay

    CUDR overexperssion cyclinD1 overexpression PTEN depletion synergistically enhances H19 expression on liver cancer stem cells A. H19 promoter methylation analysis by Methylated DNA Immunoprecipitation (MeDIP)-Dot blot-western blotting with anti-5-Methylcytosine (5-mC) in expression in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A- GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS-PTEN, respectively (indicated in the upper ). B. H19 promoter methylation analysis by MspI plus BamHI digestion in CREPT knockdown or control stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1- CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN, respectively PTEN, respectively (indicated in the upper and lower ). C. H19 promoter luciferase activity assay in in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. Each value was presented as mean ± standard error of the mean (SEM). ** P

    Journal: Oncotarget

    Article Title: CUDR promotes liver cancer stem cell growth through upregulating TERT and C-Myc

    doi:

    Figure Lengend Snippet: CUDR overexperssion cyclinD1 overexpression PTEN depletion synergistically enhances H19 expression on liver cancer stem cells A. H19 promoter methylation analysis by Methylated DNA Immunoprecipitation (MeDIP)-Dot blot-western blotting with anti-5-Methylcytosine (5-mC) in expression in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A- GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS-PTEN, respectively (indicated in the upper ). B. H19 promoter methylation analysis by MspI plus BamHI digestion in CREPT knockdown or control stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1- CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS-PTEN, respectively PTEN, respectively (indicated in the upper and lower ). C. H19 promoter luciferase activity assay in in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A- GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A- GFP-CUDR plus pGFP-V-RS PTEN, respectively. Each value was presented as mean ± standard error of the mean (SEM). ** P

    Article Snippet: Plasmid pGFP-V-RS, pRFP-V-RS pCMV6-A-GFP, were purchased from Origene (Rockville, MD 20850, USA).

    Techniques: Over Expression, Expressing, Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Dot Blot, Western Blot, Transfection, Luciferase, Activity Assay

    CUDR combined cyclinD1 or PTEN depletion collectly increases C-myc expression dependent on CTCF A. anti-CTCF or anti-P300 Co-Immunoprecipitation(IP) followed by Western blotting with anti-RNApolII, anti-P300, anti-CTCF expression in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG IP as negative control. INPUT refers to Western blotting with anti-RNApolII, anti-P300, anti-CTCF. B. Chromatin Immunoprecipitation(CHIP) with anti-RNA PolII followed by PCR with C-myc promoter primer in control or CTCF knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG CHIP as negative control. C-myc promoter DNA as INPUT. C. Chromosome conformation capture (3C) -chromatin immunoprecipitation (ChIP) with anti-CTCF, anti-RNA polII in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. The chromatin is cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin is immunoprecipitated using an antibody (anti-CTCF, anti-RNA polII)against the protein of interest. Thereafter, the cross-links are reversed, and the DNA is purified further. The PCR anlysis is applied for detecting c-myc promoter-enhancer coupling product using C-myc promoter and enhancer primers. The C-myc promoter and enhancer as INPUT. D. C-myc promoter luciferase activity assay in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. Each value was presented as mean ± standard error of the mean (SEM). ** P

    Journal: Oncotarget

    Article Title: CUDR promotes liver cancer stem cell growth through upregulating TERT and C-Myc

    doi:

    Figure Lengend Snippet: CUDR combined cyclinD1 or PTEN depletion collectly increases C-myc expression dependent on CTCF A. anti-CTCF or anti-P300 Co-Immunoprecipitation(IP) followed by Western blotting with anti-RNApolII, anti-P300, anti-CTCF expression in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG IP as negative control. INPUT refers to Western blotting with anti-RNApolII, anti-P300, anti-CTCF. B. Chromatin Immunoprecipitation(CHIP) with anti-RNA PolII followed by PCR with C-myc promoter primer in control or CTCF knocked-down stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. IgG CHIP as negative control. C-myc promoter DNA as INPUT. C. Chromosome conformation capture (3C) -chromatin immunoprecipitation (ChIP) with anti-CTCF, anti-RNA polII in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. The chromatin is cross-linked, digested with restriction enzymes, and ligated under conditions that favor intramolecular ligation. Immediately after ligation, the chromatin is immunoprecipitated using an antibody (anti-CTCF, anti-RNA polII)against the protein of interest. Thereafter, the cross-links are reversed, and the DNA is purified further. The PCR anlysis is applied for detecting c-myc promoter-enhancer coupling product using C-myc promoter and enhancer primers. The C-myc promoter and enhancer as INPUT. D. C-myc promoter luciferase activity assay in stable liver cancer stem cells transfected with pCMV6-A-GFP, pCMV6-A-GFP-CUDR, pCMV6-A-GFP-CUDR plus pcDNA3.1-CyclinD1, pCMV6-A-GFP-CUDR plus pGFP-V-RS PTEN, respectively. Each value was presented as mean ± standard error of the mean (SEM). ** P

    Article Snippet: Plasmid pGFP-V-RS, pRFP-V-RS pCMV6-A-GFP, were purchased from Origene (Rockville, MD 20850, USA).

    Techniques: Expressing, Immunoprecipitation, Western Blot, Transfection, Negative Control, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Ligation, Purification, Luciferase, Activity Assay

    Expression of CLCN5 constructs in human podocyte cell lines. Western blot (WB) showed a single band consistent with the short 83-kDa CLCN5 proteoform. Neph1 expression, which was used a positive control, was confirmed using an anti-Neph1 antibody (Ab; a). Endogenous expression of CLCN5 was also determined by immunofluorescence in cultured human podocytes (b). The upper panel shows human podocytes transfected with green fluorescent protein (GFP)–tagged wild-type (WT) CLCN5 protein demonstrated predominantly cell surface distribution and co-localization with cell surface marker ZO-1. The lower panel shows cultured human podocytes transfected with GFP-tagged L521F mutant CLCN5 protein, which demonstrated predominantly intracellular distribution (c). DAPI, 4′,6-diamidino-2-phenylindole.

    Journal: Kidney International Reports

    Article Title: A Novel CLCN5 Mutation Associated With Focal Segmental Glomerulosclerosis and Podocyte Injury

    doi: 10.1016/j.ekir.2018.06.003

    Figure Lengend Snippet: Expression of CLCN5 constructs in human podocyte cell lines. Western blot (WB) showed a single band consistent with the short 83-kDa CLCN5 proteoform. Neph1 expression, which was used a positive control, was confirmed using an anti-Neph1 antibody (Ab; a). Endogenous expression of CLCN5 was also determined by immunofluorescence in cultured human podocytes (b). The upper panel shows human podocytes transfected with green fluorescent protein (GFP)–tagged wild-type (WT) CLCN5 protein demonstrated predominantly cell surface distribution and co-localization with cell surface marker ZO-1. The lower panel shows cultured human podocytes transfected with GFP-tagged L521F mutant CLCN5 protein, which demonstrated predominantly intracellular distribution (c). DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: The pCMV6-AC-GFP vector carrying the wild-type CLCN5 gene (WT_CLCN5) and L521F mutant pCMV6-AC-GFP vector (L521F_CLCN5) were purchased from Origene (Rockville, MD).

    Techniques: Expressing, Construct, Western Blot, Positive Control, Immunofluorescence, Cell Culture, Transfection, Marker, Mutagenesis

    Relationship between OLFM4 expression and prognosis. (a) Immunohistochemical staining for OLFM4 in pancreatic cancer tissues (magnification: 100×). The left and right figures are the same sample tissue blocks and correspond to staining intensity. Left: HE staining. Right: immunohistochemical staining for OLFM4. (b) Criteria for determination of OLFM4 expression levels. OLFM4 expression levels for immunostaining were determined based on the intensity of staining and percentage of stained cells. Staining intensity and staining percentage criteria are shown. (c) Kaplan-Meier survival analysis in patients with pancreatic cancer (n = 80), showing overall survival according to OLFM4 protein expression. Red line: high expression group (n = 52); blue line: low expression group (n = 28).

    Journal: PLoS ONE

    Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

    doi: 10.1371/journal.pone.0226707

    Figure Lengend Snippet: Relationship between OLFM4 expression and prognosis. (a) Immunohistochemical staining for OLFM4 in pancreatic cancer tissues (magnification: 100×). The left and right figures are the same sample tissue blocks and correspond to staining intensity. Left: HE staining. Right: immunohistochemical staining for OLFM4. (b) Criteria for determination of OLFM4 expression levels. OLFM4 expression levels for immunostaining were determined based on the intensity of staining and percentage of stained cells. Staining intensity and staining percentage criteria are shown. (c) Kaplan-Meier survival analysis in patients with pancreatic cancer (n = 80), showing overall survival according to OLFM4 protein expression. Red line: high expression group (n = 52); blue line: low expression group (n = 28).

    Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

    Techniques: Expressing, Immunohistochemistry, Staining, Immunostaining

    Kaplan-Meier plots summarizing the results from analysis of the correlations between OLFM4 mRNA expression and patient survival in TCGA pancreatic cancer database (n = 176). Red line: high expression (n = 138), blue line: low expression (n = 38).

    Journal: PLoS ONE

    Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

    doi: 10.1371/journal.pone.0226707

    Figure Lengend Snippet: Kaplan-Meier plots summarizing the results from analysis of the correlations between OLFM4 mRNA expression and patient survival in TCGA pancreatic cancer database (n = 176). Red line: high expression (n = 138), blue line: low expression (n = 38).

    Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

    Techniques: Expressing

    Identification of chemotherapy resistance molecules. (a) Schematic of the procedure for NGS analysis. (b, c) NGS analysis for the GEM administration and GEM + nab-PTX administration groups. Treatment resistance score was defined as the NE value ratio (treated group / untreated group) × NE value difference (treated group–untreated group). (d, e) The NE value of OLFM4 mRNA. The ratio of NE values for treated and control groups were greater than 1.0 for all lines of PDXs. GEM, gemcitabine. nab-PTX, nab-paclitaxel.

    Journal: PLoS ONE

    Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

    doi: 10.1371/journal.pone.0226707

    Figure Lengend Snippet: Identification of chemotherapy resistance molecules. (a) Schematic of the procedure for NGS analysis. (b, c) NGS analysis for the GEM administration and GEM + nab-PTX administration groups. Treatment resistance score was defined as the NE value ratio (treated group / untreated group) × NE value difference (treated group–untreated group). (d, e) The NE value of OLFM4 mRNA. The ratio of NE values for treated and control groups were greater than 1.0 for all lines of PDXs. GEM, gemcitabine. nab-PTX, nab-paclitaxel.

    Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

    Techniques: Next-Generation Sequencing

    Strong OLFM4 immunostaining was detected in chemotherapy-administered PDXs. (a) Immunostaining for OLFM4 in PDXs. Control and chemotherapy-administered PDXs are shown at 200× each. (b) Analysis of the number of pixels of OLFM4-stained cells using Image J.

    Journal: PLoS ONE

    Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

    doi: 10.1371/journal.pone.0226707

    Figure Lengend Snippet: Strong OLFM4 immunostaining was detected in chemotherapy-administered PDXs. (a) Immunostaining for OLFM4 in PDXs. Control and chemotherapy-administered PDXs are shown at 200× each. (b) Analysis of the number of pixels of OLFM4-stained cells using Image J.

    Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

    Techniques: Immunostaining, Staining

    Cell viability assay using cancer cell lines. (a) Schematic representation of the procedure. Expression of the control vector and OLFM4 was induced in indicated cell lines. After 24 h (day 1), GEM was added at various concentrations. Cell viability assays were performed 48 h after GEM administration (day 3). (b and c) Rate of change of each measured OD value of the control vector and OLFM4-expressing HeLa cells (b) and MIA Paca2 cells (c) is shown. (d) Rate of change of each measured OD value of siRNA negative control and specific siRNA targeting OLFM4 induced in SUIT-2 cells is shown.

    Journal: PLoS ONE

    Article Title: High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer

    doi: 10.1371/journal.pone.0226707

    Figure Lengend Snippet: Cell viability assay using cancer cell lines. (a) Schematic representation of the procedure. Expression of the control vector and OLFM4 was induced in indicated cell lines. After 24 h (day 1), GEM was added at various concentrations. Cell viability assays were performed 48 h after GEM administration (day 3). (b and c) Rate of change of each measured OD value of the control vector and OLFM4-expressing HeLa cells (b) and MIA Paca2 cells (c) is shown. (d) Rate of change of each measured OD value of siRNA negative control and specific siRNA targeting OLFM4 induced in SUIT-2 cells is shown.

    Article Snippet: OLFM4 expression plasmid (pCMV6-Ac-OLFM4-GFP tag plasmids, NM_006418) was purchased from OriGene Technologies, Inc. (Rockville, MD, USA) and an empty vector was used as a control.

    Techniques: Viability Assay, Expressing, Plasmid Preparation, Negative Control

    Trim21 and LFG expression. (A) Results of real-time PCR-analysis of Trim21-expression after 24 h under different culture conditions. Mock, Untreated MDA-MB-231 as negative control, Trim21, 2.0 μl recombinant human Trim21 protein added to culture medium; siControl, adenoviral transfection of MDA-MB-231 with empty vector; siLFG, adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG. (B) Expression analysis of LFG protein under different culture conditions by western blot analysis using 25 μg of total protein. a, Adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG (24 h). b, Adenoviral transfection of MDA-MB-231 with empty vector. c, Untreated MDA-MB-231 as negative control. d, MDA-MB231 cultivated with 2.0 μg recombinant Trim21-protein in culture medium.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Trim21 and LFG expression. (A) Results of real-time PCR-analysis of Trim21-expression after 24 h under different culture conditions. Mock, Untreated MDA-MB-231 as negative control, Trim21, 2.0 μl recombinant human Trim21 protein added to culture medium; siControl, adenoviral transfection of MDA-MB-231 with empty vector; siLFG, adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG. (B) Expression analysis of LFG protein under different culture conditions by western blot analysis using 25 μg of total protein. a, Adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG (24 h). b, Adenoviral transfection of MDA-MB-231 with empty vector. c, Untreated MDA-MB-231 as negative control. d, MDA-MB231 cultivated with 2.0 μg recombinant Trim21-protein in culture medium.

    Article Snippet: Co-immunoprecipitation MDA-MB-231 were seeded in 100 mm culture plates and transfected with vectors coding for proteins LFG (pTriEx-1.1-FAIM2, NM-0112306; GeneArt, Regensburg, DE, USA) and TRIM21 (pCMV6-AC-GFP-Trim21, NM_003141; OriGene) by using FuGENE 6 (Promega, Madison, WI, USA) according to manufacturer's instructions.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Negative Control, Recombinant, Transfection, Plasmid Preparation, Western Blot

    TRIM21 domain. (A) (Left) Vector map of the used pMK-RQ vector (Life Technologies), (right) amino acid sequence of TRIM21 and the sequences of an individual domains. (B) Western blot analysis after native 12.5% polyacrylamide gel. Detection of TRIM21 with Li-Cor 800 anti-goat, detection of LFG with Li-Cor-680 anti-rabbit. Sample 1a (PRY domain), sample 1b (SPRY domain), sample 2 (coiled-coil domain), sample 3 (B-box domain), sample 4 (RING domain), marker (M). The samples 2–4 show clearly visible bands of ~80 and 60 kDa. The bands at 80 kDa are larger but slightly less visible as the bands at the height of 60 kDa. In samples 1a and 1b no bands are visible.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: TRIM21 domain. (A) (Left) Vector map of the used pMK-RQ vector (Life Technologies), (right) amino acid sequence of TRIM21 and the sequences of an individual domains. (B) Western blot analysis after native 12.5% polyacrylamide gel. Detection of TRIM21 with Li-Cor 800 anti-goat, detection of LFG with Li-Cor-680 anti-rabbit. Sample 1a (PRY domain), sample 1b (SPRY domain), sample 2 (coiled-coil domain), sample 3 (B-box domain), sample 4 (RING domain), marker (M). The samples 2–4 show clearly visible bands of ~80 and 60 kDa. The bands at 80 kDa are larger but slightly less visible as the bands at the height of 60 kDa. In samples 1a and 1b no bands are visible.

    Article Snippet: Co-immunoprecipitation MDA-MB-231 were seeded in 100 mm culture plates and transfected with vectors coding for proteins LFG (pTriEx-1.1-FAIM2, NM-0112306; GeneArt, Regensburg, DE, USA) and TRIM21 (pCMV6-AC-GFP-Trim21, NM_003141; OriGene) by using FuGENE 6 (Promega, Madison, WI, USA) according to manufacturer's instructions.

    Techniques: Plasmid Preparation, Sequencing, Western Blot, Marker

    Arrays. (A) Tissue samples of breast tumors having different degrees of malignity (US Biomax, Inc.) (a and b). Specific antibody against Trim21 and fluorescence labeled second antibody (Li-Cor-800), and staining of nucleic acid with Syto-60. Detection was carried out using Odyssey (Li-Cor Biosciences). (B) Bar chart of the fluorescence intensity of the tissue samples, degrees of malignity (IIa, IIb, IIIa, IIIb and IV); * P

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Arrays. (A) Tissue samples of breast tumors having different degrees of malignity (US Biomax, Inc.) (a and b). Specific antibody against Trim21 and fluorescence labeled second antibody (Li-Cor-800), and staining of nucleic acid with Syto-60. Detection was carried out using Odyssey (Li-Cor Biosciences). (B) Bar chart of the fluorescence intensity of the tissue samples, degrees of malignity (IIa, IIb, IIIa, IIIb and IV); * P

    Article Snippet: Co-immunoprecipitation MDA-MB-231 were seeded in 100 mm culture plates and transfected with vectors coding for proteins LFG (pTriEx-1.1-FAIM2, NM-0112306; GeneArt, Regensburg, DE, USA) and TRIM21 (pCMV6-AC-GFP-Trim21, NM_003141; OriGene) by using FuGENE 6 (Promega, Madison, WI, USA) according to manufacturer's instructions.

    Techniques: Fluorescence, Labeling, Staining

    NF-κB array. Bar chart of gene expression of common NF-κB regulated genes after real-time PCR-analysis. Trim21, MDA-MB-231 cultivated with 2.0 μg/ml recombinant human Trim21-protein for 24 h. Control, MDA-MB-231 cells cultivated for 24 h under standard conditions.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: NF-κB array. Bar chart of gene expression of common NF-κB regulated genes after real-time PCR-analysis. Trim21, MDA-MB-231 cultivated with 2.0 μg/ml recombinant human Trim21-protein for 24 h. Control, MDA-MB-231 cells cultivated for 24 h under standard conditions.

    Article Snippet: Co-immunoprecipitation MDA-MB-231 were seeded in 100 mm culture plates and transfected with vectors coding for proteins LFG (pTriEx-1.1-FAIM2, NM-0112306; GeneArt, Regensburg, DE, USA) and TRIM21 (pCMV6-AC-GFP-Trim21, NM_003141; OriGene) by using FuGENE 6 (Promega, Madison, WI, USA) according to manufacturer's instructions.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Recombinant

    Co-immunoprecipitation. (A) Native western blot analysis after co-immunoprecipitation of LFG and TRIM21. LFG was specifically isolated from the cell lysate of MDA-MB-231 after transfection with vectors coding for LFG and TRIM21 (24 h) by use of antibody coated magnetic beads (Dynabeads; Invitrogen). Detection of LFG (FAIM2) and TRIM21 (Ssa1/2) (both from Santa Cruz Biotechnology, Inc.) by specific first antibodies and fluorescence labeled second antibodies. LFG is visible as a green signal (Alexa Fluor 546), TRIM21 is visible as a red signal (Alexa Fluor 488), and combined signals are visible as a yellow signal. (B) Analysis of LFG and TRIM21 protein in the MDA-MB-231 breast cancer cells 24 h after transfection with vectors coding for LFG and TRIM21, by specific first and fluorescence labeled second antibodies. a, Green signal for LFG; b, red signal for TRIM21; c, blue signal for DAPI-stained core; d, overlay of the single signals with yellow signals in places where green and red signals appear in the same spot.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Co-immunoprecipitation. (A) Native western blot analysis after co-immunoprecipitation of LFG and TRIM21. LFG was specifically isolated from the cell lysate of MDA-MB-231 after transfection with vectors coding for LFG and TRIM21 (24 h) by use of antibody coated magnetic beads (Dynabeads; Invitrogen). Detection of LFG (FAIM2) and TRIM21 (Ssa1/2) (both from Santa Cruz Biotechnology, Inc.) by specific first antibodies and fluorescence labeled second antibodies. LFG is visible as a green signal (Alexa Fluor 546), TRIM21 is visible as a red signal (Alexa Fluor 488), and combined signals are visible as a yellow signal. (B) Analysis of LFG and TRIM21 protein in the MDA-MB-231 breast cancer cells 24 h after transfection with vectors coding for LFG and TRIM21, by specific first and fluorescence labeled second antibodies. a, Green signal for LFG; b, red signal for TRIM21; c, blue signal for DAPI-stained core; d, overlay of the single signals with yellow signals in places where green and red signals appear in the same spot.

    Article Snippet: Co-immunoprecipitation MDA-MB-231 were seeded in 100 mm culture plates and transfected with vectors coding for proteins LFG (pTriEx-1.1-FAIM2, NM-0112306; GeneArt, Regensburg, DE, USA) and TRIM21 (pCMV6-AC-GFP-Trim21, NM_003141; OriGene) by using FuGENE 6 (Promega, Madison, WI, USA) according to manufacturer's instructions.

    Techniques: Immunoprecipitation, Western Blot, Isolation, Multiple Displacement Amplification, Transfection, Magnetic Beads, Fluorescence, Labeling, Staining

    Caspase-3-activity and cell cycle-analysis. (A) Analysis of caspase-3-activity in MDA-MB-231 cells. Cultivation for 48 h under different conditions and addition of 100 ng agonistic anti-Fas (clone CH11; Abcam). Measurement of caspase-3-activity after 24 h. TRIM21, addition of 1.0 μg/ml recombinant human TRIM21-protein every 24 h. Control, untreated MDA-MB-231 cultivated for 48 h. Detection of LFG, TRIM21 and actin by specific first antibodies and fluorescence labeled second antibodies. (B) Plots of cell cycle-analysis of MDA-MB-231 after 24 h cultivation in presence of different concentration of recombinant human TRIM21-protein after staining with propidium iodide. a-1, Cultivation of MDA-MB-231 without addition of TRIM21-recombinant protein. b-1, Addition of 0.25 μg/ml TRIM21-recombinant protein to the culture medium. c-1, Addition of 0.5 μg/ml TRIM21-recombinant protein to the culture medium. d-1, Addition of 0.75 μg/ml TRIM21-recombinant protein to the culture medium. e-1, Addition of 1.0 μg/ml TRIM21-recombinant protein to the culture medium.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Caspase-3-activity and cell cycle-analysis. (A) Analysis of caspase-3-activity in MDA-MB-231 cells. Cultivation for 48 h under different conditions and addition of 100 ng agonistic anti-Fas (clone CH11; Abcam). Measurement of caspase-3-activity after 24 h. TRIM21, addition of 1.0 μg/ml recombinant human TRIM21-protein every 24 h. Control, untreated MDA-MB-231 cultivated for 48 h. Detection of LFG, TRIM21 and actin by specific first antibodies and fluorescence labeled second antibodies. (B) Plots of cell cycle-analysis of MDA-MB-231 after 24 h cultivation in presence of different concentration of recombinant human TRIM21-protein after staining with propidium iodide. a-1, Cultivation of MDA-MB-231 without addition of TRIM21-recombinant protein. b-1, Addition of 0.25 μg/ml TRIM21-recombinant protein to the culture medium. c-1, Addition of 0.5 μg/ml TRIM21-recombinant protein to the culture medium. d-1, Addition of 0.75 μg/ml TRIM21-recombinant protein to the culture medium. e-1, Addition of 1.0 μg/ml TRIM21-recombinant protein to the culture medium.

    Article Snippet: Co-immunoprecipitation MDA-MB-231 were seeded in 100 mm culture plates and transfected with vectors coding for proteins LFG (pTriEx-1.1-FAIM2, NM-0112306; GeneArt, Regensburg, DE, USA) and TRIM21 (pCMV6-AC-GFP-Trim21, NM_003141; OriGene) by using FuGENE 6 (Promega, Madison, WI, USA) according to manufacturer's instructions.

    Techniques: Activity Assay, Cell Cycle Assay, Multiple Displacement Amplification, Recombinant, Fluorescence, Labeling, Concentration Assay, Staining

    Analysis of the interaction. Array analysis of the interaction of over 9,000 different proteins with LFG. Recombinant LFG protein (10 μM) was used and detected by specific first antibody (FAIM2; Santa Cruz Biotechnology, Inc.) and fluorescence labeled second antibody (Alexa Fluor 546). The display window shows the signal stimulated by the interaction of LFG with TRIM21 (white signal).

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Analysis of the interaction. Array analysis of the interaction of over 9,000 different proteins with LFG. Recombinant LFG protein (10 μM) was used and detected by specific first antibody (FAIM2; Santa Cruz Biotechnology, Inc.) and fluorescence labeled second antibody (Alexa Fluor 546). The display window shows the signal stimulated by the interaction of LFG with TRIM21 (white signal).

    Article Snippet: Co-immunoprecipitation MDA-MB-231 were seeded in 100 mm culture plates and transfected with vectors coding for proteins LFG (pTriEx-1.1-FAIM2, NM-0112306; GeneArt, Regensburg, DE, USA) and TRIM21 (pCMV6-AC-GFP-Trim21, NM_003141; OriGene) by using FuGENE 6 (Promega, Madison, WI, USA) according to manufacturer's instructions.

    Techniques: Recombinant, Fluorescence, Labeling