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    TaKaRa hela tet off cells
    Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) <t>HeLa</t> <t>Tet-off-β-globin-ARE</t> uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.
    Hela Tet Off Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa hela tet on cells
    Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) <t>HeLa</t> <t>Tet-off-β-globin-ARE</t> uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.
    Hela Tet On Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hela tet on cells/product/TaKaRa
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hela tet on cells - by Bioz Stars, 2022-08
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    Image Search Results


    Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

    doi: 10.1128/MCB.23.20.7177-7188.2003

    Figure Lengend Snippet: Overexpression of constitutively active MK2 stabilizes the β-globin-ARE uPA reporter mRNA, which is not affected by p38 MAP kinase inhibition, and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) or cotransfected with 2 μg each of pcDNA3-MK2-EE and pcDNA3-MK2-K76R (MK2-K76R) for 24 h and then treated with 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin (βglo) and GAPDH mRNA signals. The data shown are representative of three independent experiments. (B) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or pcDNA3-MK2-EE (MK2-EE) for 24 h, treated with 10 μM of SB203580 for 1 h or left untreated (−), and then treated with 1 μg of doxycycline per ml for the times indicated, and total RNA was prepared. Northern blots were sequentially probed to detect β-globin and GAPDH mRNA signals. The data shown are representative of two independent experiments. (C) HeLa Tet-off-β-globin-ARE uPA cells were transfected with the vectors described in panel A or left untreated (−), and total RNA was isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled DNA probes. The data shown are representative of two independent experiments.

    Article Snippet: A specific inhibitor of p38 MAP kinase, SB203580, which strongly destabilizes ARE-mRNAs in MDA-MB-231 cells ( ) and in HeLa Tet-off cells transfected with empty vector ( t 1/2 decreased from 1.5 h [untreated] to 0.5 h [SB203580 treated]) (Fig. ), did not inhibit MK2-EE-mediated mRNA stabilization ( t 1/2 ≈ 3 h in MK2-EE-transfected cells, independent of treatment with SB203580).

    Techniques: Over Expression, Inhibition, Transfection, Plasmid Preparation, Northern Blot, Isolation, Labeling

    Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

    doi: 10.1128/MCB.23.20.7177-7188.2003

    Figure Lengend Snippet: Increased binding of cytoplasmic HuR to the ARE uPA and stabilization of β-globin-ARE uPA mRNA by oxidative stress requires MK2. (A) MK2 was immunoprecipitated from whole-cell lysates of HeLa Tet-off cells treated with 200 μM H 2 O 2 for the times indicated. The cells were serum starved for 4 h before addition of H 2 O 2 . Western blotting was performed with a polyclonal antibody recognizing the phosphorylated Thr 334 of MK2. IP, immunoprecipitation; IB, immunoblotting. (B) HeLa Tet-off cells were transfected with pcDNA3 (vec) or pcDNA3-MK2-K76R (K76R) for 24 h, serum starved for 4 h, treated with 20 μM rottlerin (Rott) for 1 h or left untreated, and then treated with 200 μM H 2 O 2 for 2 h. Preparation of nuclear (N) and cytoplasmic (C) lysates followed immediately after the treatments. WCE, whole-cell extract. Western blotting was performed with antibodies against HuR, cyclin A, and β-tubulin. (C) Cell lysates prepared from cells treated as described in panel B were incubated with radiolabeled ARE uPA RNA, UV cross-linked (except for those in lanes 2 and 13), treated with RNases A and T 1 , and immunoprecipitated with anti-HuR antibodies (except those in lanes 3 and 14). Cross-linked RNA-protein complexes were resolved by SDS-PAGE and analyzed by autoradiography (upper panel). The most intense signal corresponded to a band migrating at ca. 36 kDa, indicated by an arrow and labeled HuR. Before autoradiographic exposure, the gel was stained with Coomassie brilliant blue to assess the equivalence of inputs and anti-HuR immunoprecipitates (lower panel). Data shown in panels A to C are representative of two independent experiments. Ig., immunoglobulin. (D) HeLa Tet-off-β-globin-ARE uPA cells were transfected with 2 μg of pcDNA3 (vector) or 2 μg of pcDNA3-MK2-K76R (MK2-K76R) for 24 h. The cells were left untreated (−) or treated with 200 μM H 2 O 2 for 2 h before the addition of 1 μg of doxycycline (Dox) per ml for the times indicated, and total RNA was prepared. Northern blot analysis was performed to detect specific β-globin and GAPDH mRNA signals. (E) Quantitation of the Northern blot mRNA signals described in panel D. Data shown represent the means and standard errors from three independent experiments.

    Article Snippet: A specific inhibitor of p38 MAP kinase, SB203580, which strongly destabilizes ARE-mRNAs in MDA-MB-231 cells ( ) and in HeLa Tet-off cells transfected with empty vector ( t 1/2 decreased from 1.5 h [untreated] to 0.5 h [SB203580 treated]) (Fig. ), did not inhibit MK2-EE-mediated mRNA stabilization ( t 1/2 ≈ 3 h in MK2-EE-transfected cells, independent of treatment with SB203580).

    Techniques: Binding Assay, Immunoprecipitation, Western Blot, Transfection, Incubation, SDS Page, Autoradiography, Labeling, Staining, Plasmid Preparation, Northern Blot, Quantitation Assay

    The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.

    Journal: Molecular and Cellular Biology

    Article Title: Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

    doi: 10.1128/MCB.23.20.7177-7188.2003

    Figure Lengend Snippet: The ARE uPA is a functional mRNA-destabilizing element in HeLa Tet-off cells. (A) Schematic representation of tetracycline-regulated pTRE-β-globin constructs with the wild-type (wt) or mutant (mut) ARE uPA inserted into the Xho I site of pTRE-βglobin-Xho (pTRE-βglo-Xho). (B) Northern blot analysis of the decay of chimeric β-globin mRNAs in HeLa Tet-off cells. Cells stably transfected with the indicated expression vector were subjected to an 8-h doxycycline (Dox) chase. Total RNA was isolated at the indicated times after addition of doxycycline (1 μg/ml), and 10-μg aliquots of RNA were resolved on a formaldehyde-agarose gel. Specific mRNA signals on the same Northern blot were detected using random-primed radiolabeled cDNA probes corresponding to rabbit β-globin or human GAPDH, which was used as a loading control.

    Article Snippet: A specific inhibitor of p38 MAP kinase, SB203580, which strongly destabilizes ARE-mRNAs in MDA-MB-231 cells ( ) and in HeLa Tet-off cells transfected with empty vector ( t 1/2 decreased from 1.5 h [untreated] to 0.5 h [SB203580 treated]) (Fig. ), did not inhibit MK2-EE-mediated mRNA stabilization ( t 1/2 ≈ 3 h in MK2-EE-transfected cells, independent of treatment with SB203580).

    Techniques: Functional Assay, Construct, Mutagenesis, Northern Blot, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Random Primed

    Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Stabilization of Urokinase and Urokinase Receptor mRNAs by HuR Is Linked to Its Cytoplasmic Accumulation Induced by Activated Mitogen-Activated Protein Kinase-Activated Protein Kinase 2

    doi: 10.1128/MCB.23.20.7177-7188.2003

    Figure Lengend Snippet: Overexpression of HuR in HeLa Tet-off cells stabilizes β-globin-ARE uPA reporter mRNA and endogenous uPA and uPAR mRNAs. (A) HeLa Tet-off-β-globin-ARE uPA cells were transfected without (−) or with 2 μg of pcDNA-HA (vector) or pcDNA-HA-HuR (HA-HuR) for 24 h before being lysed and checked for the expression of HA-tagged HuR by Western blotting using anti-HA and anti-β-tubulin (loading control) antibodies. (B) Total RNA from HeLa Tet-off-β-globin-ARE uPA or HeLa Tet-off-β-globin-ΔARE uPA cells transfected as described in panel A were isolated at the times indicated after addition of 1 μg of doxycycline (Dox) per ml and resolved on a 1% formaldehyde-agarose gel. Specific mRNA signals were produced and analyzed as described in Materials and Methods and plotted on a logarithmic scale (lower panel). The β-globin-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the graphic data shown represent the means and standard errors from three independent experiments. (C) Total RNA from HeLa Tet-off-β-globin-ARE uPA cells transfected as described in panel A were isolated at the times indicated after the addition of 20 μg of DRB per ml and subjected to Northern blot analysis. The same blot was sequentially hybridized with human uPA, uPAR, and GAPDH random primer-labeled cDNA probes. The lower panel shows quantitation and graphic representation of uPA and uPAR mRNA decay. The uPA-to-GAPDH or uPAR-to-GAPDH ratio at time 0 h was arbitrarily adjusted to 100%, and the data shown represent the means and standard errors from two independent experiments.

    Article Snippet: A specific inhibitor of p38 MAP kinase, SB203580, which strongly destabilizes ARE-mRNAs in MDA-MB-231 cells ( ) and in HeLa Tet-off cells transfected with empty vector ( t 1/2 decreased from 1.5 h [untreated] to 0.5 h [SB203580 treated]) (Fig. ), did not inhibit MK2-EE-mediated mRNA stabilization ( t 1/2 ≈ 3 h in MK2-EE-transfected cells, independent of treatment with SB203580).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Isolation, Agarose Gel Electrophoresis, Produced, Northern Blot, Labeling, Quantitation Assay

    Effect on transactivation of NFkB, p53, NFAT, MYC and AP1 in HeLa TET-off (grey) and HeLa MDR-Off (dark grey) cells by luciferase activity assay. Each bar represents the ratio of luciferase activity of cells treated with doxycycline over the luciferase activity of the same cells without doxycycline. (B) Analysis of TRAIL localization. Confocal microscope image of TRAIL localization in KB-C1 Pgp knockdown cells and control cells transfected with scrambled shRNA. DAPI - Nuclear staining (blue) and anti-TRAIL staining (green). (C) Cell surface TRAIL expression analysis of HeLa TET-Off and HeLa MDR-Off cells with and without doxycycline THP-1 cells was used as a positive control (D) Quantitative analysis of soluble TRAIL through ELISA in HeLa TET-Off cells, HeLa MDR-Off and KB-C1 Pgp knockdown cells.

    Journal: Experimental cell research

    Article Title: Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL

    doi: 10.1016/j.yexcr.2015.06.005

    Figure Lengend Snippet: Effect on transactivation of NFkB, p53, NFAT, MYC and AP1 in HeLa TET-off (grey) and HeLa MDR-Off (dark grey) cells by luciferase activity assay. Each bar represents the ratio of luciferase activity of cells treated with doxycycline over the luciferase activity of the same cells without doxycycline. (B) Analysis of TRAIL localization. Confocal microscope image of TRAIL localization in KB-C1 Pgp knockdown cells and control cells transfected with scrambled shRNA. DAPI - Nuclear staining (blue) and anti-TRAIL staining (green). (C) Cell surface TRAIL expression analysis of HeLa TET-Off and HeLa MDR-Off cells with and without doxycycline THP-1 cells was used as a positive control (D) Quantitative analysis of soluble TRAIL through ELISA in HeLa TET-Off cells, HeLa MDR-Off and KB-C1 Pgp knockdown cells.

    Article Snippet: Human cervical cancer HeLa MDR-Off [ ], HeLa TET-off (Clontech Laboratories, Mountain View, CA) and human embryonic kidney 293T cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) in high glucose (4.5 g/l), supplemented with 2 mM L-glutamine, 100 I.U./mL penicillin, 100 μg/mL streptomycin and 10% tetracycline approved FBS (Clontech).

    Techniques: Luciferase, Activity Assay, Microscopy, Transfection, shRNA, Staining, Expressing, Positive Control, Enzyme-linked Immunosorbent Assay

    Comparison of the effects of tetracycline and doxycycline on P-glycoprotein expression in HeLa MDR-Off cells. Minimal doses of doxycycline induced repression of (A) ABCB1 mRNA by rt-RCR, (B) Pgp in total protein lysates by Western blot, and (C) Pgp expression on the cell surface by immunolabeling. (D) FACS histograms showing accumulation of rhodamine 123 in the presence (black) and absence (red) of cyclosporin A in MDR-Off cells. (E) Difference in doxycycline and tetracycline sensitivity between HeLa TET-Off and HeLa MDR-Off cells (F) Colchicine toxicity observed in both HeLa TET-Off and HeLa MDR-Off cells. The relative ABCB 1 mRNA level was normalized to GAPDH mRNA. For comparison of Pgp protein, Na +/ K + ATPase expression was used as a loading control.

    Journal: Experimental cell research

    Article Title: Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL

    doi: 10.1016/j.yexcr.2015.06.005

    Figure Lengend Snippet: Comparison of the effects of tetracycline and doxycycline on P-glycoprotein expression in HeLa MDR-Off cells. Minimal doses of doxycycline induced repression of (A) ABCB1 mRNA by rt-RCR, (B) Pgp in total protein lysates by Western blot, and (C) Pgp expression on the cell surface by immunolabeling. (D) FACS histograms showing accumulation of rhodamine 123 in the presence (black) and absence (red) of cyclosporin A in MDR-Off cells. (E) Difference in doxycycline and tetracycline sensitivity between HeLa TET-Off and HeLa MDR-Off cells (F) Colchicine toxicity observed in both HeLa TET-Off and HeLa MDR-Off cells. The relative ABCB 1 mRNA level was normalized to GAPDH mRNA. For comparison of Pgp protein, Na +/ K + ATPase expression was used as a loading control.

    Article Snippet: Human cervical cancer HeLa MDR-Off [ ], HeLa TET-off (Clontech Laboratories, Mountain View, CA) and human embryonic kidney 293T cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) in high glucose (4.5 g/l), supplemented with 2 mM L-glutamine, 100 I.U./mL penicillin, 100 μg/mL streptomycin and 10% tetracycline approved FBS (Clontech).

    Techniques: Expressing, Western Blot, Immunolabeling, FACS

    Representative Venn diagram illustrating the commonly and exclusively apoptosis-related genes expressed in Pgp-expressing cells (Pgp/ABCB1). HeLa TET-Off and HeLa MDROff cells in the presence or absence of doxycycline (A) and Pgp-expressing drug-resistant cell lines (KB-8-5, KB-8-5-11 and KB-C1) (B) were analyzed by TLDA analysis.

    Journal: Experimental cell research

    Article Title: Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL

    doi: 10.1016/j.yexcr.2015.06.005

    Figure Lengend Snippet: Representative Venn diagram illustrating the commonly and exclusively apoptosis-related genes expressed in Pgp-expressing cells (Pgp/ABCB1). HeLa TET-Off and HeLa MDROff cells in the presence or absence of doxycycline (A) and Pgp-expressing drug-resistant cell lines (KB-8-5, KB-8-5-11 and KB-C1) (B) were analyzed by TLDA analysis.

    Article Snippet: Human cervical cancer HeLa MDR-Off [ ], HeLa TET-off (Clontech Laboratories, Mountain View, CA) and human embryonic kidney 293T cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) in high glucose (4.5 g/l), supplemented with 2 mM L-glutamine, 100 I.U./mL penicillin, 100 μg/mL streptomycin and 10% tetracycline approved FBS (Clontech).

    Techniques: Expressing, TLDA Assay

    Analysis of P-glycoprotein ( ABCB 1) and TRAIL/ TNFSF10 expression in MDR-positive cells. Protein and mRNA analysis of Pgp and TRAIL expression in HeLa MDR-Off and HeLa TET-Off cells in the presence or absence of doxycycline (Dox) (A). Confocal microscope image of TRAIL localization in HeLa MDR-Off in the presence and absence of doxycycline. DAPI - Nuclear staining (blue) and anti-TRAIL staining (green). Time-dependent analysis of Pgp and TRAIL expression in HeLa MDR-Off cells in the presence of Dox (C). Pgp expression analysis in HeLa MDR-Off cells under transient overexpression of TRAIL (D). TRAIL and Pgp expression analysis in KB-8-5-11 and KB-C1 Pgp knockdown cell lines (E), and comparison of Pgp expression in KB-8-5-11 and KB-C1 Pgp knockdown cell lines under transient overexpression of TRAIL (F). The mRNA expression level was normalized to GAPDH. Na +/ K + ATPase was used as a loading control for Western blots.

    Journal: Experimental cell research

    Article Title: Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL

    doi: 10.1016/j.yexcr.2015.06.005

    Figure Lengend Snippet: Analysis of P-glycoprotein ( ABCB 1) and TRAIL/ TNFSF10 expression in MDR-positive cells. Protein and mRNA analysis of Pgp and TRAIL expression in HeLa MDR-Off and HeLa TET-Off cells in the presence or absence of doxycycline (Dox) (A). Confocal microscope image of TRAIL localization in HeLa MDR-Off in the presence and absence of doxycycline. DAPI - Nuclear staining (blue) and anti-TRAIL staining (green). Time-dependent analysis of Pgp and TRAIL expression in HeLa MDR-Off cells in the presence of Dox (C). Pgp expression analysis in HeLa MDR-Off cells under transient overexpression of TRAIL (D). TRAIL and Pgp expression analysis in KB-8-5-11 and KB-C1 Pgp knockdown cell lines (E), and comparison of Pgp expression in KB-8-5-11 and KB-C1 Pgp knockdown cell lines under transient overexpression of TRAIL (F). The mRNA expression level was normalized to GAPDH. Na +/ K + ATPase was used as a loading control for Western blots.

    Article Snippet: Human cervical cancer HeLa MDR-Off [ ], HeLa TET-off (Clontech Laboratories, Mountain View, CA) and human embryonic kidney 293T cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) in high glucose (4.5 g/l), supplemented with 2 mM L-glutamine, 100 I.U./mL penicillin, 100 μg/mL streptomycin and 10% tetracycline approved FBS (Clontech).

    Techniques: Expressing, Microscopy, Staining, Over Expression, Western Blot

    Effect of TRAIL ligand (sTRAIL) on inducing apoptosis in Pgp-expressing and –non-expressing cells. (A) Analysis of apoptosis induced by sTRAIL in HeLa TET-Off and HeLa MDR-Off cells. (B) Analysis of apoptosis induced by sTRAIL in KB-3-1 (parental cells) and KB-C1. (C) Analysis of apoptosis induced by sTRAIL in KB-C1 Pgp knockdown cells. GAPDH was used as a loading control for Western blots. (D) MTT assay showing difference in sensitivity to sTRAIL among KB-C1 shNS (-◆-), KB-C1 shABCB1 (-•-) and KB-3-1 (dotted line) cells. Data points showed statistical significance between KB-C1 shNS and KB-C1 shABCB1 cells were marked with asterisk (*). GAPDH was used as a loading control for Western blots.

    Journal: Experimental cell research

    Article Title: Expression of the multidrug transporter P-glycoprotein is inversely related to that of apoptosis-associated endogenous TRAIL

    doi: 10.1016/j.yexcr.2015.06.005

    Figure Lengend Snippet: Effect of TRAIL ligand (sTRAIL) on inducing apoptosis in Pgp-expressing and –non-expressing cells. (A) Analysis of apoptosis induced by sTRAIL in HeLa TET-Off and HeLa MDR-Off cells. (B) Analysis of apoptosis induced by sTRAIL in KB-3-1 (parental cells) and KB-C1. (C) Analysis of apoptosis induced by sTRAIL in KB-C1 Pgp knockdown cells. GAPDH was used as a loading control for Western blots. (D) MTT assay showing difference in sensitivity to sTRAIL among KB-C1 shNS (-◆-), KB-C1 shABCB1 (-•-) and KB-3-1 (dotted line) cells. Data points showed statistical significance between KB-C1 shNS and KB-C1 shABCB1 cells were marked with asterisk (*). GAPDH was used as a loading control for Western blots.

    Article Snippet: Human cervical cancer HeLa MDR-Off [ ], HeLa TET-off (Clontech Laboratories, Mountain View, CA) and human embryonic kidney 293T cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM) in high glucose (4.5 g/l), supplemented with 2 mM L-glutamine, 100 I.U./mL penicillin, 100 μg/mL streptomycin and 10% tetracycline approved FBS (Clontech).

    Techniques: Expressing, Western Blot, MTT Assay

    Observation of mitophagy using mito-Keima cells. ( A ) Tet-off mito-Keima-expressing HeLa cells (mito-Keima cells) were cultured in complete medium or Krebs-Henseleit buffer (starvation) with or without 10 mM 3-MA for 18 h. Mito-Keima localized within the mitochondria was excited by 440-nm light (Keima [mitochondria]; red) and mito-Keima delivered within lysosomes by mitophagy was excited by 590-nm light (Keima [autolysosomes]; green) (emission, 624 nm). Bar = 10 μm. The number of acidic puncta per cell was counted. Data are expressed as means ± SD of at least 30 cells from 3 separate experiments. ( B ) Mito-Keima cells were transfected with control siRNA or PIK3C3 -specific siRNA, and then cultured in complete medium or Krebs-Henseleit buffer for 18 h. Bar = 10 μm. The number of acidic puncta per cell was counted. Data are expressed as means ± SD of at least 30 cells from 3 separate experiments.

    Journal: Autophagy

    Article Title: Mitophagy is primarily due to alternative autophagy and requires the MAPK1 and MAPK14 signaling pathways

    doi: 10.1080/15548627.2015.1023047

    Figure Lengend Snippet: Observation of mitophagy using mito-Keima cells. ( A ) Tet-off mito-Keima-expressing HeLa cells (mito-Keima cells) were cultured in complete medium or Krebs-Henseleit buffer (starvation) with or without 10 mM 3-MA for 18 h. Mito-Keima localized within the mitochondria was excited by 440-nm light (Keima [mitochondria]; red) and mito-Keima delivered within lysosomes by mitophagy was excited by 590-nm light (Keima [autolysosomes]; green) (emission, 624 nm). Bar = 10 μm. The number of acidic puncta per cell was counted. Data are expressed as means ± SD of at least 30 cells from 3 separate experiments. ( B ) Mito-Keima cells were transfected with control siRNA or PIK3C3 -specific siRNA, and then cultured in complete medium or Krebs-Henseleit buffer for 18 h. Bar = 10 μm. The number of acidic puncta per cell was counted. Data are expressed as means ± SD of at least 30 cells from 3 separate experiments.

    Article Snippet: The HeLa Tet-off cell line was obtained from Clontech (631156).

    Techniques: Expressing, Cell Culture, Transfection